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1310 Cards in this Set
- Front
- Back
What is the main difference between a bacteria and a virus? |
Bacteria: Cellular organisms (single/clusters of cells), living Viruses: Not cellular, not "living" |
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What are viruses? |
Essentially nucleic acid (DNA or RNA) wrapped in a protein lipid coat
Do not have a "metabolism"
Require help of cells to replicate their genome and make progeny |
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Do bacteria exhibit social and multi-cellular characteristics? |
Vast majority do |
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Very briefly, describe a bacteria's metabolism: |
Bacteria take up chemicals from environment and convert them into biomass or energy They excrete waste and products Bacterial cells contain lipids, proteins, sugars and nucleic acids (RNA and DNA) |
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How do bacteria reproduce? |
Autonomously |
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How do bacteria reproduce autonomously? |
Bacteria take simple chemicals from the environment to generate more complex ones These are used to create new cells under the direction of genetic information contained within the pre-existing cells |
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Can bacteria differentiate? |
Yes they can but they do not always do so |
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What does it mean by "bacteria can differentiate"? |
Many bacteria can generate new cells that are strikingly different than the parental cells They don't look the same during different stages of growth |
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How do bacteria communicate and coordinate with one another? |
By means of chemical signaling Sensing and adapting to their environment |
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How do bacteria move? |
Autonomously |
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What does it mean by "bacteria can move autonomously"? |
Many bacteria possess flagella or other cell structures to help them move
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How do bacteria responding when they are sensing their environment? |
Either directly or by changing the genes they are expressing |
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Do bacteria evolve? |
Yes Bacteria change over time to both gain new properties and lose old ones Their genomes are highly plastic |
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Briefly, what is the life cycle of Myxococcus xanthus? |
A) Spherical structure that contains ~1*10^5 cells that contains stress-resistant spores. Very tightly packed B-D) Fruiting body receives nutrients, individual spores germinate and cells emerge together as an "instant" swarm E-F) Whey prey/nutrient available, swarm becomes predatory collective that moves and feed cooperatively, pooling extracellular enzymes to lyse and consume prey bacteria G) Wave formation H) Aggregates and mound building |
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What can Staphylococcus aureus cause in humans? |
Nausea Vomiting Abdominal cramping Explosive diarrhea |
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What did Yersinia pestis cause? |
It caused the bubonic plague Lethal (killed 1/3 of the population of Europe and Asia during the bubonic plague) |
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What is unique about Trichonympha? |
It is an anaerobic eukaryotic protist that has the ability to break down wood as a food souce |
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What is cellulose? |
A polymer of glucose very similar to starch except for the presence of a beta(1-4) linkage between every other sugar molecule This enables very tight packing adjacent glycan chains making it hard for enzymes to access easil |
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Describe the Trichonympha's coat: |
Trichonympha carries a coat of bacteria that are essential for it to survive on wood as a primary energy source These bacteria provide organic nitrogen in the form of urea or amino acids that themselves fix from atmospheric nitrogen |
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What does ectosymbionts mean? |
Symbionent relationships where lives on the outside of the bacterial cell |
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What are enveloped viruses? |
Surrounded by lipids Uses host cell machinery to replicate |
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In taxonomy, what is classification? |
Arrangement of organisms into groups (taxa) |
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In taxonomy, what is nomenclature? |
Assignment of names |
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In taxonomy, what is identification? |
Determining to which recognized taxon any given bacterial isolate belongs |
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In taxonomy, what is phylogeny analysis? |
Classification based on evolutionary relationships |
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What is the significance of taxonomy? |
Allows to organize huge amounts of knowledge about organisms because all members of a particular group share many characteristics Allow to make predictions for further research based on knowledge of similar organisms Allow to communicate efficiently in the scientific community Essential for accurate identification of microorganisms |
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What is the ranks/hierarchy of taxnomy? |
1) Domains/Kingdoms 2) Phylum 3) Class 4) Order 5) Family 6) Genus 7) Species (subseven: strains) |
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In taxonomy, what is a genus? |
Collection of species |
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In taxonomy, what is a species? |
A collection of strains that share many stable properties and different significantly from other groups of strains |
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What is a bacterial strain? |
Population of organisms that descended from a single organism or a pure culture isolate that differ form other members of the species in a small way |
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How are strains different from another? How are there strain variation? |
In strains, they will have subtle variations, changes and properties where one can cause disease and the other does not where vast majroity of genes in the genomes, they are most (99%) likely the same |
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What is significant about the E. coli strain O1578:H7? |
Gets you very sick when you eat it in meat |
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What is reproductive isolation? |
Method to see if you can you reproduce with another thing and produce a viable offspring where the cells can reproduce
If can be achieved, you are of the same species |
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What is phenetic classification? |
Classification based on phenotypic characteristics may reveal possible evolutionary relationships They can be: 1) Morphological characteristics 2) Physiological and metabolic characteristics 3) Molecular characteristics (DNA GC-content, lipids, capsule types, etc...) |
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What type of bacteria morphology is this? |
Straight rod or bacillus |
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Give an example of a straight rod/bacillus bacteria? |
Escherichia |
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What type of bacteria morphology is this? |
Club-shaped rod |
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What type of bacteria morphology is this? |
Branching rod |
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What type of bacteria morphology is this? |
Comma form |
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What type of bacteria morphology is this? |
Spore forming rod |
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What type of bacteria morphology is this? |
Spiral form |
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What type of bacteria morphology is this? |
Spherical/Cocci |
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If bacteria grows like this, what type of cell arrangement is it? |
Diplococci (one division plane) |
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If bacteria grows like this, what type of cell arrangement is it? |
Chain (one division plane) |
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If bacteria grows like this, what type of cell arrangement is it? |
Tetrad (two division planes) |
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If bacteria grows like this, what type of cell arrangement is it? |
Cluster (three division planes) |
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What did Hans Christian Gram do? |
Develop a staining procedure that gained wide-spread use as a primary diagnostic test to identify bacteria species |
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If a bacteria is purple/pink after gram staining, what type of gram bacteria are they? |
Gram-negative |
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If a bacteria is dark blue after gram staining, what type of gram bacteria are they? |
Gram-positive |
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Does antibiotic perceptibly widely differ depending on bacteria's gram? |
Yes |
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What is the gram stain procedure? |
1) Flood heat-fixed smear with crystal violet for 1 min (all cells turn purple)
2) Add iodine solution for 3 min
3) Decolorize with alcohol briefly for about 20 sec (gram positive purple/blue, gram negative cell colourless)
4) Counterstrain with safranin for 1-2 minutes (positive blue/purple, negative pink/red) |
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Why would you add iodine solution in a gram stain procedure? |
Interacts with crystal violet and makes insoluble rocks with the dye |
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What does the alcohol in gram staining do in the gram staining procedure? |
Removes crystal violet iodine from the gram negative cells and the slide background |
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Why in gram stain procedure would you use the counterstrain safranin? |
To help make the gram negative bacteria more visible
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Why are there differences in gram staining between gram positive and negative? |
Differences in cell wall structure of gram-positive and gram-negative cells |
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What makes gram-positive cells different from gram-negative cells? |
90% of their cell wall consists of peptidoglycan layers This thick peptidoglycan of gram-positive bacteria traps crystal violet/iodine complexes that form during the gram stain procedure They are more susceptible to antibiotics |
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Why are gram-positive cells more susceptible to antibiotics? |
Because drugs have an easy time getting past peptidoglycan layer as it has a lot of pores in it |
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What makes gram-negative cells different from gram-positive cells? |
Only 10% of the cell wall is peptidoglycan (they only have one layer) Their thin peptidoglycan that fails to trap the crystal violet/iodine during de-staining procedure with alcohol is main different in staining Their outer layer is highly impermeable (lipid bilayer) to a lot of compounds that are bigger than a dissaccharide |
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What does nosocomial infections mean? |
Hospital infections |
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When it comes to treatments, which is more deadly, gram negative or gram positive? |
Gram negative |
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List one possible bacteria that this microscope image shows: |
Staphylococcus |
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List one possible bacteria that the gram negative rods could be: |
E. coli |
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What type of morphology does Escherichia have? |
Straight rod/bacillus |
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What type of morphology does Corynebacterium have? |
Club-shaped rod |
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What type of morphology doe Actinomyces have? |
Branching rod |
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What type of morphology does Vibrio have? |
Comma form |
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What type of morphology does Bacillus have? |
Spore forming rod |
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What type of morphology does Spirochaeta have? |
Spiral form |
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What type of morphology does Staphylococcus have? |
Spherical/Cocci |
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What type of growth cell arrangement does Neisseria have? |
Diplococci |
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What type of growth cell arrangement does Streptococcus have? |
Chain |
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What type of growth cell arrangement does Sarcina have? |
Tetrad |
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What type of growth cell arragement does Staphylcoccus have? |
Cluster |
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Is Staphlycoccus gram positive or negative? |
Gram positive |
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Is Listeria gram positive or negative? |
Gram positive |
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Is Clostridia (C. diff) gram positive or negative? |
Gram positive |
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Is Mycobacterium tuberculosis gram positive or negative? |
Gram positive |
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Is Neisseria gram positive or negative? |
Gram negative |
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What is the morphology of Neisseria? |
Spherical/Cocci |
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What can Neisseria cause in humans? |
Gonorrhea |
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Is Haemophillus gram positive or negative? |
Gram negative |
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What is the morphology of Haemophillus? |
Spherical/cocci |
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What can Haemophillus cause in humans? |
Pneumoniae |
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Is E. coli gram positive or negative? |
Gram negative
|
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Is Salmonella gram positive or negative? |
Gram negative |
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Is Yersinia gram positive or negative? |
Gram negative |
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Is Vibrio cholerae gram positive or negative? |
Gram negative |
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What are the criteria for evolutionary chronometers? |
Molecule (DNA or protein) should be universally distributed in all organisms studied They must be functionally equivalent in each organism Must be able to align the sequence properly in order to identify regions of sequence of homology and sequence heterogeneity The sequence changes of these molecules must reflect the evolutionary distance |
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Which rRNA's make up majority of the ribosomal small subunit? |
16s rRNA of bacteria 18s rRNA of eukaryotes |
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What are Archaea? |
Hybrid ancestors of eukaryotes |
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What are the three fundamental groups of life-forms or domains? |
Bacteria Arhcaea Eukarya |
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The top of the red line is positive, everything below is gram negative. Why? |
They are evolutionary distant as they have to make huge radical change to their surface where they kind of became a whole new 'animal' |
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What is genomics? |
A branch of science/technology that specializes in the systematic study of genomes |
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What were the first two viruse genomes completely sequenced? |
1976 - MS2 phage 1977 - PhiX174 phage |
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What is the Sanger Method? |
Have stand you want to be sequence with a primer that is complimentary Prepare four reaction mixtures in test tubes, each including dNTPs which you use those nucleotides to expand as well as small amounts of dideoxynucleotides with A, C, T, G which terminate growth of the chain Replicate products and separate produce by gel electrophoresis Read sequences as complement of bands containing labeled strands |
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What is the Sanger Method based on? |
It is based on DNA polymerase-dependent synthesis of a complementary DNA strain in presence of natural 2'-deoxynucleotides (dNTPs) and 2',3'-dideoxynucleotides (ddNTPs) that server as nonreversible synthesis terminators The DNA synthesis reaction is randomly terminated whenever a ddNTP is added to growing oligonucleotide chain, resulting in truncated products of varying length with appropriate ddNTP at their 3' terminus |
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What role does the ddNTPs serve in the Sanger Method? |
Nonreversible synthesis terminators They randomly terminate DNA synthesis reaction when added to a growing olgonucleotide chain resulting in a truncated products of varying length with an appropriate ddNTP at their 3' terminus |
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What is the Shotgun-cloning method? |
1) DNA is randomly sheared into small fragments that are then ligated into plasmids 2) Thousands of plasmids are generated, each of which has some random fragment of the genome you want to sequence 3) "Library" of plasmids are put into E. coli so that each may be purified in amounts sufficient for cloning so when grown, each colony of E. coli has a different plasmid 4) Plasmids are isolated from thousands of E. coli clones each harboring a plasmid with different random segment of the genome you are trying to sequence 5) Determine 500-1000bp sequence from each end of cloned piece of DNA (done by using primers complmentary to plasmid area flanking either end of insert) 6) After sequencing 1000s of plasmids this way the sequence can be stitched together by a computer to assemble the full genome |
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Why would you not want to use sonification in DNA sheering for shotgun-cloning? |
If used sonification, you would need to repair ends and make sure size of fragment is 3kbp |
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What are the limitations to whole genome shotgun-cloning method? |
Cloning bias - Some gene segments toxic to E. coli and cannot be cloned resulting in"gaps" in assembled sequence that must be filled Project maintenance issues - Must maintain A LOT of different E. coli isolates Slow and expensive |
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What is Illumina/Solexa sequencing? |
Next-generation sequencing platforms and very commonly used
It uses a Sanger-like sequencing reaction using dye-labeled reversible terminator nucleotides and it amplifies the DNA fragment on surface of a slide or a "flow cell"
Building sequencing with dye-labeled nucleotides and treated with certain wavelength/chemicals where the dye will block the next nucleotide and then remove the dye and do the next step then zap with chemical/wavelength removing the dye and do the next one… etc… etc. … etc… |
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What are the steps to Illumina/Solexe sequencing? |
1) Prepare genome DNA samples 2) Attach DNA to surface 3) Bridge amplification 4) Fragments become double stranded 5) Denature the double-stranded molecules 6) Complete amplification 7) Determine first base 8) Image first base 9) Determine second base 10) Image second chemistry cycle 11) Sequence reads over multiple chemistry cycles 12) Align data |
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In Illumina/Solexe sequencing, what is happening during the first step "Prepare genome DNA sample"? |
Random fragment and sheer it RE or sonification and ligated adapted (ligationis covalently linking known pieces of DNA onto unknown DNA where you can put your primer onto) |
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In Illumina/Solexe sequencing, what is happening during the second step "Attach DNA to surface"? |
Theseare coded and can capture the piece of DNA and do it at such a dilution wherethe DNAs on the slide will fall apart far from one another and bound the DNA tothe surface |
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In Illumina/Solexe sequencing, what is happening during the third step "Bridge amplification"? |
Do PCR amplification on the (the n's) slide using the primers that are attached to the surface of the slide |
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In Illumina/Solexe sequencing, what is happening during the fourth step "Fragments become double stranded"? |
Enzyme incorporates nucleotide to build double-stranded bridges on the solid phase substrate |
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In Illumina/Solexe sequencing, what is the happening during the 5th step "Denature the double-stranded molecules"? |
Denaturation leaves single-stranded templates anchored to the substrate |
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In Illumina/Solexe sequencing, what is happening during the 6th step "Complete amplification"? |
Several million dense clusters of double-stranded DNA are generated in such channel of the flow cell
Unique piece of DNA which amplified to create its own unique piece of DNA which are different from other clusters |
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In Illumina/Solexe sequencing what is happening during the 7th step "Determine the first base"? |
First chemistry cycle to initiate the first sequencing cycle, add all four labeled reversible terminators, primers and DNA polymerase enzyme to the flow cell |
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In Illumina/Solexe sequencing what is happening during the 8th step "Image first base"? |
Do oneround of synthesize where above the slide, there is a camera that looks downonto the DNA where depending on what colour the DNA changes, that is thenucleotide it is It allows you to look at millions of sot where each spot lining up differently This allows the build of millions of sequencing simultaneously |
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In Illumina/Solexe sequencing, what is happening during the 9th step "Determine second base"? |
Second chemistry cycle to initiate the next sequencing cycle, add all four labeled reversible terminators and enzyme to the flow cell |
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In Illumina/Solexe sequencing, what is happening during the 10th step "Image second chemistry cycle"? |
After laser excitation, collect the image data as before. Record the identity of the second base of each cluster |
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In Illumina/Solexe sequencing, what is happening during the 11th step "Sequence reads over multiple chemistry cycles"? |
Repeat cycles of sequencing to determine the sequence of base in a given fragment in single base at a time |
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In Illumina/Solexe sequencing, what is happening during the final step "Align data"? |
Blew apart the DNA (shot gun) with millions and reading and begin to stitch together the entire sequence based on the reads |
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How are the genomes sequenced by Illumina/Solexe or other methods assembled? |
By "stitch" together millions of individual "reads" Looking for overlap between sequences that fill gaps, match other sequences or extend sequences |
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In genome sequence alignment, what do the blue squares mean? |
They are errors where there is mismatch from the rest of them One of the two: 1) Mutation 2) Mistake by sequencing and/or primer/polymerize |
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What are open reading frames? |
Stretches of DNA that code for a protein |
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What does annotating a DNA sequence mean? |
Basically understanding what the sequence means Requires computers to look for patterns and largely automated which can lead to errors |
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What is metagenomics? |
Going to an environment, taking whatever is there and sequencing it Formicrobes this is a huge deal as many microbes are uncultivable and no way ofstudying (95%) of them as we did not know how to culture (and did not even knewthey existed) |
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What are the two strategies for metagenomics? |
1) SEQUENCE EVERYTHING!!!!! 2) Sequence 16S genes in population |
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What is the metagenomics strategy "Sequence everything"? |
Take the total DNA and sequence everything that is there
What you get is protein coding genes, ribosome genes and a whole bunch of a lot of things
It is very hard for the computer to do because there are many genomes and it would be hard for the computer to figure out what and where to overlap |
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What is the metagenomics strategy "Sequence 16S genes in population"? |
If you want to know who is there, you can sequence the 16S rRNA DNA With these you can see how the gut microbiome changes when there is a change in diet You have to PCR the 16S rRNA DNA There are current databases of different microbes and what their different 16S rRNA looks like If you do this procedure properly, you can get good ratios and tell that an specific population increases and/or decreases Do this by counting the number of E. coli/species rRNA reads are on the slide |
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What does 16S rRNA sequencing require? |
PCR of 16S rRNA requires that you use primers against the highly conserved regions of the molecule These primers will amplify about 900 bases of sequences containing enough variability to tell you what you are looking at |
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What is significant about these two pictures? |
These two pictures are stained with nucleic acid dye Nucleotide basically fills up the entire cytoplasm on bacteria while in eukaryotes, the DNA is contained in a membrane enclosed nucleus Eukaryotes is all about enclosing compartments in membranes while during mitosis/mitosis, membrane breaks down but during regular gap phases, its DNA is usually kept separate from cytoplasm |
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Compare Eukaryotic vs Bacteria/Archaeal DNA: |
Eukaryotes have linear chromosomes - Bacterial tend to be circular Eukaryotes have problems with replicating ends (solved by capping with telomeres) - Bacteria have no problem |
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What is a possible explanation for why Protopterus aethiopicus (marbled lungfish) has such a large genome of ~130 billion bp? |
Probablythis because of transpose got in and made a lot of copies/junk DNA |
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At what size do bacterial genomes usually max out at? |
~12 million |
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What is the chromosomes like for bacteria? |
Bacteria usually have one major chromosome that contains all housekeeping functions (not always case) Some has two chromosome Some has three chromosome which may actually be two chromosomes and mega plasmid |
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Why is it significant that the %GC content differ between bacteria? |
GCcontent differ a quite bit between bacteria, usually E. coli land in the 50% Looking at codon table, most AA are coded by first two nucleotides, third base in triplet is the wobble position that gives variability (chooses G or C at third position of triplet w/o effecting protein sequence) |
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Is there a correlation among total number of ORFs in the genome noncoding DNA and genome size for prokaryotic genomes? |
Consistently observe that bacterial genomes encode about 1 open reading frame for every 1000 base pairs of DNA regardless of size
Most bacterial genomes don't have a lot of "junk" DNA, that they are very streamlined |
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What does it mean by "Bacterias are masters of efficiency"? |
As soon as one protein sequence one ends, another begins
Typically, a stop codon leads into a start codon |
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Eukaryotic genomes tend to be loaded with what? |
Junk DNA Silence transposable elements that tend to replicate itself |
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Why is soil a unique niche for bacteria? |
Soil isrich with different microbes and different temperatures and will need manydifferent genes for many different functions and they have different types food They have highly dynamic environments Bacteria can go into arms race and kill each other They need genes to fight against phages, amibos, microbes, bacteria, eating complex carbohydrates, etc... |
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What is the %GC content of Burkholderia vietnamiensis? |
65.7% |
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What is the niche for Burkholderia vietnamiensis? |
Soil bacterium |
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What is the %GC content for Mycobacterium marinum? |
65.7% |
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What is niche for Mycobacterium marinum? |
Free living water bacterium |
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What is the %GC content for Pseudomonas aeruginosa? |
%66.6 |
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What is the niche for Pseudomonas aeruginosa? |
Soil bacterium |
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What are plasmids in bacteria? |
Plasmids are small circles Plasmids carries accessory function (i.e. antibiotic resistance) whereas chromosomes carries essential functions for cell Plasmids are dispensable, chromosomes are not Bacterial chromosomes are not shaped the same way as eukaryotic genome |
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How does amino acid codon create biases in bacteria genomes? |
Little diversity mechanism to protect them from foreign invaders which allows to distinguish self from non-self Basically it's like if a bacteria's genome has a lot of "A" and "T" bases, but then it encounters a gene that has a lot of "G" and "C" bases, then they'd be like oh this isn't my gene, it's probably foreign then (for example, a bacteriophage trying to inject its genome into a bacterial cell) |
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What is a mutation of an existing gene? |
A gene in the genome is altered by recombination or through a mutagenic process to yield a gene with altered function |
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What is a gene duplication? |
A gene is duplicated into two copies One copy keeps the ancestral function The other copy is free to change by mutation |
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What is horizontal (lateral) gene transfer? |
Just steal what you need from another bacteria Bacterial exclusive |
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What plays a major role in prokaryotic evolution? |
Horizontal (lateral) gene transfer |
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What are genomic or pathogenicity islands? |
Large blocks of newly acquired genes are called genomic islands but if they are involved in disease, they are pathogenicity islands |
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Salmonella typhimurium encodes 5 major pathogenicity islands called what? |
SPI-1 through -5 |
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What does the pathogenicity islands of Salmonella typhimurium do? |
Help the bacteria survive and cause inflammation in the host |
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What does Salmonella typhimurium SPI-1 do? |
Encodes a type-III secretion system necessary for invasion into animal cells Invasion |
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What does Salmonella typhimurium SPI-2 do? |
Encodes a type-III secretion system necessary for survival in macrophages Macrophage survival |
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What does Salmonella typhimurium SPI-3 do? |
Genes here work with SPI-2 to ensure survival in macrophages Macrophage survival |
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What does Salmonella typhimurium SPI-4 do? |
Encodes an adhesin and works with SPI-1 to help invasion Epithelial interaction |
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What does Salmonella typhimurium SPI-5 do? |
Necessary for survival in macrophages Inflammation |
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How can you identify genes acquired by horizontal gene transfer? |
Compare region to several other closely related species
Look at GC-content difference (different bacteria prefer to use different codon to code for the same amino acid where if it doesn't make the strain/species, you can guess that it was obtained lately and from whom)
Encoded on phage or transposon |
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What do the red lines mean? |
Red lines connect genes that from the top genome to their identical counterpart in the bottom genome |
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What does the gap (the arrow is pointing to) mean? |
A genomic island
Strain 1 has a gene that strain 2 does not have which could be indication of horizontal acquired islands |
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What factor helps distinguish the Salmonella typhimurium SPI's from rest of genome? |
The SPI's are more AT-rich than rest of genome |
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Where does Mycobacterium tuberculosis live? |
Lives almost exclusively in human long
Not well adapted for outside life |
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What is Mycobacterium leprae niche? |
Lives exclusively in human host and cannot survive on its own |
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What does Mycobacterium leprae cause? |
Leprosy |
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What is the niche of Candidatus carsonella? |
Endosymbiont |
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What does endosymbionts mean? |
Bacterial cells that reside permanently inside another cell |
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True of False: Mitochondria is an ancient endosymbiont |
TRUE |
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Where are majority of the proteins found in the mitochondria from? |
Not encoded by mtDNA but instead encoded in nucleus of host cell Such proteins have to be transported to mitochondria after synthesis in cell cytoplasm |
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What is a reducing agent? |
Takes a compound and takes electrons out to oxidize it |
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What is an oxidizing agent? |
Adds an electron to reduce a compound |
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When "A" is being oxidized, is it gaining or losing electrons? |
Losing |
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When "B" is being reduced, is it gaining or losing electrons? |
Gaining |
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Why are redox reactions important? |
Oxidation-reduction (redox) reactions are important because they are the principal sources of energy on this planet Both neutral, or biological and artifical |
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What does oxidation of molecules usually do (in respects to energy)? |
Oxidation of molecules by removal of hydrogen or combination with oxygen normally liberates large quantities of energy |
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When something is going from reduced state to oxidized state, what is happening to the energy? |
Diminishing availability of energy from oxidative processes |
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What is the oxidized form of K? |
|
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What is the oxidized form of Mg? |
|
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What is the oxidized form of H2? |
|
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What are electron donors? |
In catabolism, electron donor referred to as an energy source Amount of energy released depends on energy donor and acceptor |
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Briefly, what is the electron transport chain? |
Consistsof a series of enzyme that are anchored in a membrane together which carriesout oxidation |
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Where does the ETC take place? |
On the mitochondrial membrane in eukaryotes On cell membrane in bacteria |
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Is ETC an oxidizing or reducing reaction? |
Oxidizing |
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Briefly explain the ETC of bacteria: |
1) Usually starts with NADH converted to NAD+, releasing an electron into NDH-1 2) Two electrons go through NHD-1 pushing 4 protons (H+) from inside to outside of cell where the electrons then with 2 more protons join the Quinone pool 3) Quinone releases 2 electrons and protons to Cyt bo which pushes 2 protons out and water 4) Protons go through ATP synthase to convert protons as an motive force to generate ATP from ADP |
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How many protons have been used moving electrons from NHD-1 to quinone pool? |
6 H+ |
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What does ETC usually start with in bacteria? |
NADH |
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What are ATP synthase of bacteria? |
Present on cytoplasmic membrane Generate ATP form ADP using proton motive force Multi-subunit motor consisting of two parts, F0 (proton-conducting channel in membrane, C units rotate) and F1 (headpiece in cytoplasm, transmits energy to create ATP) |
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Where are ATP synthase found in bacteria? |
Cytoplasmic membrane |
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What do ATP synthase of bacteria use to generate ATP? |
Using proton motive force (PMF) to generate ATP from ADP |
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What is the multi-subunit motor F0 of the ATP synthase in bacteria? |
Proton-conducting channel in membrane C units rotate |
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What is the multi-subunit motor F1 of ATP synthase in bacteria? |
Headpiece in cytoplasm Transmits energy to create ATP |
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Briefly, label the steps of the citric acid cycle: |
1) Pyruvate converts NAD+ + CoA into NADH releasing CO2 becoming Acetyl-CoA 2) Citrate synthase converts Acetyl-CoA into Citrate(3-) releasing CoA 3) Citrate(3-) converted into Aconitate(3-) 4) Aconitate(3-) converted into Isocitrate(3-) 5) With isocitrate(3-), converts NAD(P)+ into CO2 and NAD(P)H and itself into alpha-Ketoglutarate(2-) 6) CoA + NAD+ converted into NADH and CO2 with alpha-Ketoglutrarate(2-) into Succinyl-CoA 7) Using Succinyl-CoA, GDP + Pi into GTP and CoA and Succinate(2-) 8) Succinate(2-) converted into Fumarate(2-) with FADH into FAD 9) Fumarate(2-) into Malate(2-) 10) With Malate(2-), NAD+ into NADH and Oxalacetate(2-) 11) Begin cycle again at step 2) |
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What are the product of the citric acid cycle? |
NAD(P)H and NADH and FADH |
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From glycolysis, how many electrons are carried to ETS (ETC of bacteria) and via what? |
4 electrons carried via 2 NADH + 2H+ |
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What are the beginning and ending products of glycolysis? |
Glucose into 2 Pyruvate |
|
How many ATPs are generated from Glycolysis? |
2 ATP |
|
From the conversion of 2 pyruvate to 2 Acetyl-CoA, how many electrons are carried to ETS and via what? |
4 Electrons carried via 2 NADH + 2 H+ |
|
From the TCA cycle, how many electrons are carried to the ETS and via what? |
16 electrons carried via 6 NADH + 6 H+ and 2 FADH2 |
|
How many ATP's are generated from the TCA cycle? |
2 ATP |
|
How many ATPs are generated from ETS? |
34 ATP |
|
How many ATPs are generated from the complete oxidation of glucose? |
38 ATP |
|
What is heterotrophic nutrition? |
Relying on organic compounds for energy |
|
What are chemolithotrophs? |
Organism that is able to use inorganic reduced compounds as a source of energy Most are autotrophic, obtain carbon from CO2 Their reducing power for biosynthesis comes from directly from inorganic compounds (sufficiently low E0') and reverse electron flow |
|
How much free energy of a energy-rich phosphate bond of ATP has? |
-31.8 kJ/mol |
|
Do anaerobes that use alternate electron acceptors have oxygen as final oxidizer? |
It can but not all which is why they do not need O2 to live |
|
What are sulfur oxidizing bacteria? |
Take in such compounds as sulfur compounds or pure sulfur and turn into energy Final product is H+ and sulphuric acid |
|
What are sulfur granules in sulfur oxidizing bacteria? |
Storage system for intermediates in their metabolism Sulfur can be found in here |
|
What do sulfur oxidizing bacteria do with hydrogen sulfide? |
Convert it into sulfur which gets converted into sulfate |
|
What are the two ways sulfite can be oxidized? |
1) Sulfite oxidase pathway 2) Adenosine phosphosulfate (APS) reductase |
|
What reductant can be generated by reverse electron flow? |
NADH |
|
What is the ETC like in sulfur oxidizing bacteria? |
Usually have different starting enzyme compared to other bacteria HS- gets oxidized by FP which goes into either ETC or Reverse electron flow ETC: 1) Electron from oxidized HS- to Quinone pumping out H+ 2) Quinone to Cyt B to Cyt C which takes of S2O3(2-) or S(0) 3) Pushes electron to Cya aa3 which converts 1/2 O2 into H2O and pushes more H+ out 4) H+ gradient creates ATP |
|
What is the reverse electron flow like in sulfur oxidizing bacteria? |
Usually have different starting enzyme compared to other bacteria HS- gets converted into FP which goes into either ETC or Reverse electron flow Reverse electron flow: 1) FP causes NAD+ to be converted into NADH 2) With CO2 + ATP, NADH goes into Calvin cycle |
|
What is unique about the tubeworm (Rfitia pachyptila) relating to sulfur? |
These bacteria take hydrogen sulfide and produce energy and organic molecules
Convert inorganic substances into organic compounds which tubeworm uses to grow
They live next to underwater vents which pump out hydrothermal fluid which contain H2S |
|
Ferrous iron is insoluble at what pH? What happens to it at that pH? |
Neutral pH Spontaneously oxidizes and precipitates as ferric hydroxide Fe(OH)3 |
|
Where can iron oxidizing bacteria be found? |
Acid, iron-rich environments |
|
What type of energy consuming bacteria is Thiobacillus ferroxidans? |
Iron oxidizing bacteria |
|
What type of energy consuming bacteria is Leptospirrillum ferroxidans? |
Iron oxidizing bacteria |
|
What are iron oxidizing bacteria? |
Uses iron (ferrous into ferric) for their energy source and have a very short route of electron transport to oxygen Must oxidize large amounts of iron in order to grow |
|
What is Rusticyanin? |
Enzyme that mediates the oxidation of ferrous iron Copper-containing protein in periplasm Mediates oxidation of Fe2+ and passes electrons to Cytochrome C Also passes electrons to Cytochrome A which oxidizes oxygen and pumps H+ out |
|
What are the electron donors of inorganic compounds? |
Phosphite
Hydrogen
Sulfide
Sulfur
Ammonium
Nitrate
Ferrous iron |
|
What is the ETC of iron oxidizing bacteria? |
1) Rusticyanin oxidizes Fe2+ into Fe3+, forcing an electron inside membrane to Cyt C 2) Cyt C electron goes to Cyt A pushing protons outside 3) With 2e-, converts 1/2 O2 into H2O and, releasing 2 H+ 4) Proton gradient creates ATP |
|
What is the reverse electron flow of iron oxidizing bacteria? |
1) Rusticyanin oxidizes Fe2+ into Fe3+, forcing an electron inside membrane to Cyt C 2) Cyt C electron causes NAD+ converted into NADH in pH 6 environment 3) With CO2 + ATP, NADH goes into Calvin cycle |
|
Where does the Rusticyanin sit on iron oxidizing bacteria? |
On the outside of the outer membrane |
|
What is the ETC of H2 oxidizing bacteria? |
1) H2 oxidized by H2ase pushing electron to Quinone 2) Quinone pushes protons out of cell and electron to Cyt B 3) To Cyt C 4) To Cyt A 5) Cyt A forces protons out and converts 1/2 O2 to H2O 6) Proton gradient creates ATM |
|
What is the reverse electron flow of H2 oxidizing bacteria? |
1) H2ase oxidizes H2 2) Reduces NAD+ with H+ to NADH 3) With CO2 + ATP, NADH goes into calvin cycle |
|
What is Nitrite Oxidoreductase? |
Oxidize nitrite into nitrate
Oxygen is the primal acceptor |
|
What is anaerobic respiration? |
Terminal electron acceptor other than oxygen Involves electron transport chain |
|
What is assimilative reduction? |
Inorganic compounds reduced for use as nutrient source Only enough reduced for growth needs |
|
What is dissimilative reduction? |
Reduced for energy production Large amount of inorganic compound is reduced Products of reduction are excreted |
|
What is the most common electron acceptor for anaerobics? |
Nitrate |
|
Can methane be used as an energy source? |
Yes |
|
What is the source of energy and electron of iron bacteria (iron oxidizing bacteria)? |
Ferrous iron into ferric iron Fe2+ -> Fe3+ |
|
What is the source of energy and electrons of nitrosifying bacteria? |
Ammonia into nitrite NH3 -> NO2- |
|
What is the source of energy and electrons of nitrifying bacteria? |
Nitrite into nitrate NO2- -> NO3- |
|
What is the source of energy and electrons of purple sulfur bacteria? |
Sulfide into sulfur S2- -> S0 |
|
What is the source of energy and electrons of sulfur-oxidizing bacteria? |
Sulfur into sulfate S0 -> SO4(2-) |
|
What is the source of energy and electrons of aerobic hydrogen bacteria? |
Hydrogen into water H2 -> H2O |
|
What is the source of energy and electrons of anammox bacteria? |
Ammonia into nitrogen NH3 -> N2 |
|
What is the source of energy and electrons of carboxydotrophoic bacteria? |
Carbon monoxide into carbon dioxide
CO -> CO2 |
|
What is autotrophic CO2 fixation? |
Need to incorporate carbon from CO2 for biosynthesis |
|
What are some pathways for atutotrophic CO2 fixation? |
Calvic cycle Reverse citric acid cycle Hydroxypropionate cycle |
|
What are required for calvin cycle? |
NADPH and ATP |
|
What is RubisCO? |
Ribulose biphosphate carboxylase (a key enzyme in the calvin cycle) It is a rate limiting step for CO2 fixation in the Calvin cycle Most abundant protein on earth and source of energy for all heterotrophs Attaches CO2 to a sugar (Ribulose biphosphate) and convert it into Phosphoglyceric acid (PGA) |
|
What does RubisCO convert? |
CO2 + ribulose biphosphate -> Phosphoglyceric acid (PGA) |
|
What are carboxysomes? |
Usually have RubisCO and carbonic anhydrase inside in close interaction which increases efficiency of reaction Sugars like bicarbonates enter carboxysomes |
|
Briefly, what are the steps in the Calvin cycle? |
1) Ribulose 1,5-bis-phosphate converted using 3 CO2 and 3 H2O into 3-Phosphate-Glycerate by RubisCO 2) Using 6 ATPs, 6 NADPHs and 6 H+, 3-Phosphate-Glycerate is reduced to Glyceraldhyde-3-Phosphate 3) After 6 Glyceraldehyde-3-Phosphate produces, 1 enters biosynthesis of glucose, 5 gets phosphorylated into Ribulose-5-Phosphate 4) Ribulose-5-Phosphate undergoes phosphorylation using 3 ATP and restarts cycle |
|
How much energy is required for the Calvin cycle? |
9 ATP |
|
What is a difference between reverse citric acid cycle and citric acid cycle? |
Most enzymes are the same except working backwards Only enzyme difference is citrate synthase replaced with citrate lyase |
|
What are the two ferredoxin-linked reactions in reverse citric acid cycle? |
1) Carboxylation of succinyl-CoA to alpha-ketoglutarate 2) Carboxylation of acetyl-CoA to pyruvate |
|
What is the net reaction of Reverse citric acid cycle? |
3 CO2 + 12 H+ + 5 ATP into Triose-Phosphate |
|
Briefly, what happens in the reverse citric acid cycle? |
1) Oxalacetate with 2H+ gets converted into Malate 2) Gets converted into Fumarate 3) Gets converted into Succinate with 2H+ 4) Gets converted into Succinyl-CoA using ATP 5) Using Ferredoxin red and CO2, gets converted into alpha-Ketoglutarate 6) Using 2H+ and CO2, gets converted into Isocitrate 7) Gets converted into Citrate 8) Using Citrate lyase either gets converted into Oxalacetate and restarts cycle OR using ATP, gets converted into Acetyl-CoA 9) Using Ferredoxin red and CO2, gets converted into Pyruvate 10) Gets converted into Phosphoenolpyruvate using ATP 11) Using ATP and 2 H+, gets converted into Triose-Phosphate 12) Gets converted into Hexose-Phosphate 13) Used for cell amterial |
|
What happens if you take our Ferredoxin red out of the Reverse citric acid cycle? |
It will stop production/conversion of alpha-ketoglutarate and/or pyruvate |
|
From Oxalacetate to Triose-Phosphate, how much ATP is used in the reverse citric acid cycle? |
4 ATP |
|
What is the hydroxypropionate cycle? |
Present only in green nonsulfer phototroph Chloroflexus Two CO2 is reduced to glyoxylate (Acetyl-CoA is carboxylated twice and yields methylmalonyl-CoA rearranged to acetyl-CoA and glyoxylate which is converted to other compounds by serine or glycine intermediate) |
|
Briefly, what happens during the hydroxypropionate cycle? |
1) Acetyl-CoA converted into Hydroxypropionyl-CoA using 2 ATPs, 4 H+ and a CO2 2) Gets converted into Propionyl-CoA using 2 H+ 3) Gets converted in Methylmalonyl-CoA using ATP and CO2 4) Releases 2 H+ and either gets converted to Acetyl-CoA and restarts cycle OR Glyoxylate which is used for cell material |
|
What is the net reaction for the hydroxypropinate cycle? |
2 CO2 + 4 H+ + 3 ATP -> Glyoxylate |
|
Is the Hydroxypropionate cycle found in Eukaryotes? |
No (or at least none are known) |
|
Is the Reductive acetyl-CoA pathway found in Eukaryotes? |
No (or at least none are known) |
|
Is the Reverse (reductive) TCA cycle found in Eukaryotes? |
Yes, anaplerotic reactions fix CO2 to regenerate TCA |
|
What happens during Glycolysis? |
1) Glucose converted into Glucose-6-P using Hexokinases and ATP 2) Using Isomerase, converted into Fructose-6-P 3) Using ATP with Phosphofructokinase, converted into Fructose-6-1,6-P 4) Using Aidolase, converted to 2 Glyceraldehyde-3-P 5) Using a inorganic phosphate and Glyceraldehyde-3-P dehydrogenase while releasing an electron to reduce 2 NAD+ into 2 NADH, converted into 2 1,3-Biphosphoglycerate-P (non-reversible redox reaction) 6) Using Phosphoglycerckinase, converted into 2 3-Phosphoglycerate-P, producing 2 ATP 7) Converted into 2 2-Phosphoglycerate-P 8) Using Enolase, converted into 2 Phosphoenoipyruvate-P 9) Using Pyruvate kinase, converted into 2 Pyruvate, producing 2 ATP |
|
How many ATPs are produced during glycolysis? |
4 ATP |
|
What is another name for Glycolyss? |
Embden-Meyerhof pathway |
|
Does glycolysis occur in the presence or absence of O2? |
Both |
|
In glycolysis, 4 ATP are produced, but only 2 can be used, why? |
2 ATP are used during glycolysis early steps |
|
In glycolysis, how many NADH are produced per glucose? |
2 NADH |
|
What is phototraphy? |
Use of light as energy source by photosynthesis (conversion of light energy to chemical energy) |
|
What are photosystems? |
Systems of proteins and membranes that are found in bacteria and plants that allow energy derived from sunlight (light energy, packages of photons) into chemical energy (excited electrons, like making ATP)
Act like electron transport chains by generating protein gradient across photosynthetic membrane |
|
What are photosynthetic vesicles? |
Membrane invaginations where these photosynthetic complexes are set up Can be found in purple bacteria |
|
What are purple bacteria? |
Produce intracytoplasmic membranes into which pigments are inserted Originated from invaginations of cytoplasmic membrane Allow purple bacteria to increase the amount of pigment and better utilize light |
|
What are green bacteria? |
Another type of photosynthetic bacteria
They have chlorosomes, looks like organelles along plasma membrane (this where photosynthesis happens) |
|
What are Antenna pigments? |
Funnel light energy to reaction center Very important at low light intensity |
|
What is located on top of a reaction center of phototrophic bacteria? |
A cytochrome that transfer electron in |
|
What is the purpose of the cytochrome on top of a reaction center of phototrophic bacteria? |
To transfer electrons in |
|
Is there chlorophyll in a reaction center of phototrophic bacteria? If so, where? |
In the middle of the special pair with pigments going around it to the side |
|
What are the point to pigments in the reaction center of phototrophic bacteria? |
To hold the pigments |
|
What is the "Special pair" of bacteriochlorophyll for a reaction center of phototrophic bacteria? |
Called special pair because there are two cholorphylls together in a special orientation at 7A away which allows them to absorb light |
|
What differs bacteriopheophytin from bacteriochlorophyll? |
Bacteriopheophytin is Bacteriochlorophyll without Mg2+
|
|
Why is Chlorophyll very good at light absorbence? |
Due to its structure and the Mg2+ ion in the middle of it |
|
Briefly, what are the steps of the reaction center of phototrophic bacteria when light interact with it? |
1) Excited electron located in special pair is transferred to bacteriochlorophyll where electron is then quickly transferred to a bacteriopheophytin 2) Electron is then transferred to QA (quinone, when this happens, it becomes ubiquinol) 3) Electron is then slowly transferred to QB via nonheme ferrous iron 4) The electron hole in the special pair is filled by an electron from the heme of the cytochrome c 5) Second exciton is transferred via the same route to the semiquinone to form ubiquinol which is free to diffuse within the membrane |
|
What does the Antenna complex that surrounds the reaction center do? |
Featured in all photosynthetic center and also absorb light at wavelengths that are distinct from chlorophyll which helps get light funnel into reaction center but they cannot make excited electrons but transfer light by excitation transfer (transfer without transfer radiation) |
|
What is exciton transfer? |
Excitation energy transferred by a radiationless process to a neighbouring molecule No electron transfer |
|
What are carotenoids? |
Accessory pigments allow more energy capture Associated with light harvesting pigment which can transfer energy to reaction center Serve protection role to absorb light in blue (high energy) region and quench toxic oxygen species |
|
What is the electron flow and ATP synthesis in purple bacteria like? |
Electron to ubiquinol to cytochrome c2 which donated its electron to reduce radical cation of the special pair For every two electrons transferred to ubiquinol to cytochrome c2, four H+ are released into the periplasmic space, generating a proton gradient This is an infinite cycle |
|
Briefly, what are the steps of electron flow in purple bacteria? |
1) Red or infrared light hits the P870 2) P870 goes from +0.5V to -1.0V 3) Electron goes from P870 to Bph 4) Then to QA then QB then Q/pool where there will be a H+ transport across photosyntethic membra 5) Electron then go to Cyt c2 then P870 |
|
What is a problem with purple bacteria and generating reducing power and its solution? |
Quinone is more positive than NAD -> NADH (NADH more energetic) Solution: Organism must undergo reverse electron flow which require ATP |
|
What is reverse electron flow in purple bacteria? |
1) Red or infrared light hits the P870 2) P870 goes from +0.5V to -1.0V 3) Electron goes from P870 to Bph 4) Then to QA then QB then Q/pool where reverse electron flow begins 5) Using proton gradient, causes NAD(P)+ to be reduced to NAD(P)H using ATP When continuing the electron flow, there is an external electron donor for the Cyt c2 step In reverse electron flow, electrons move from inorganic electron donor to NADP (passed from donor to cytochrome 2on outside, quinone to NAD(P) on inside) |
|
Why does the reverse electron flow in purple bacteria begin at the quinone pool step? |
The first table electron accept is the quinone as it has more positive E0' than NADH |
|
When does reverse electron flow begin for green sulfur bacterium and Helicobacterium? |
Their first stable electron acceptor is FeS as it has more negative E0' than NADH NOTE: These bacterium do not actually have a reverse electron flow as FeS is in an energy state higher than NADH so you can directly to it |
|
What is anoxygenic photosynthesis? |
Photosynethsis without oxygen production Involves only one photosystem Electron transport through a series of electron carries Carriers organized in photosynthetic membranes in series from negative to positive reduction potentials No net input or consumption of electrons |
|
What do photosynthesis of cyanobacteria produce? |
O2
|
|
What are thylakoids? |
These are membranes This is where photosynthesis is taken place Can be found in cyanobacteria |
|
What do chloroplast in plants and algae do? |
Mediates photosynthesis in plants and algae Chloroplast is cyanobacteria that got swallowed by an ancient eukaryote (similar to mitochondria) |
|
What O2 levels in the atmosphere rose and atmospheric CH4 levels reduced, what happened to the earth? |
Loss of this greenhouse gas led to a prolonged ice age (300-400 million years where ice at equator was ~1600 meters thick) |
|
What are stromatolites? |
Bacterial fossils with iron oxide trapped in them |
|
What is ferrodoxin? |
They are iron-sulfur proteins that mediate electron transfer in a range of metabolic reactions
Generated by separate photosystem in cyanobacteria
Reduced from light energy
Used for CO2 fixation or to generated NADH or NADPH |
|
What are quinone? |
Mobile, lipid-soluble carriers that shuttle electrons (and protons) between large, relatively immobile macromolecular complexes embedded in the membrane |
|
In the Z pathway of oxygenic photosynethesis, what is photosystem 1? |
What is like in purple bacteria Uses light energy to generate reduced ferredoxin, a powerful reductant |
|
In the Z pathway of oxygenic photosynthesis, what is photosytem 2? |
Oxygenic photosynthesis When sunlight hits it, it becomes a strong oxidizing agent it strips oxygen off from water generating O2 and protons that can go into electron transport chain |
|
What can plastocyanin (PC) do? |
Cantake electron, pass it to photosystem 1 to generate the reducing compoundneeded to fix carbon |
|
Briefly, what happens in the Z pathway reaction complexes and ATP synthesis? |
Water gets excited, releasing O2, producing protons used for proton motive force and transferring electron to ETC Passing electron to ferrodoxin, then pass it to Fd reductase to make NADPH from NADP |
|
What is Oxygenic photosynthesis? |
Two photosystems (P700 and P680) that are connected by an electron transport chain |
|
Why do oxygenic photosynthesis not need reverse transport flow? |
Electron that is passed onto Fd (from FeS) is higher energy state than NADH and thus it can make NADH without consuming energy for reverse transport and doesn't require an electron donor |
|
What does oxygenic photosynthesis consume? |
Water
|
|
How is ferredoxin reduced from light energy? |
1) Light photoexcites bacteriochloroophyll 2) Drives splitting (photolysis) of 2H+ from electron donors (sulphides and organic molecules) 3) Electrons flow to phylloquinone then ferredoxin |
|
How can ferredoxin be used to generated NADPH? |
Via Ferredoxin NADH Oxidoreducatase (FNR) |
|
What are the most efficient nitrogen fixing bacteria? |
Symbiotic living bacteria |
|
Why does fixing N2 take a lot of energy? |
It requires breaking three bonds in N2 |
|
What are the steps for root hairs of plants host symbiotic relationship with bacteria? |
1) Recognition and attachment (rhicadhesin-mediated) 2) Excretion of nod factors by bacterium causing root hair curling 3) Invasion where Rhizobia penetrate root hair and multiply within an "infection thread" 4) Bacteria in infection thread grow toward root cell 5) Formation of bacteroid state within plant cell 6) Continued plant and bacterial cell division |
|
For symbiotic relationships, bacteria species tend to be quite specific to certain plants, why? |
Because the plant and bacteria are "talking" (chemical signals) to each other and if they don't understand each other, they do not make good nodules Plants will only grow root hairs for bacteria if "talking" well |
|
What is an infection thread? |
Bacterial material to allow it to attract more bacteria into the root All done by signaling between bacteria and plant |
|
After the bacteria has successfully invaded the plant cells and formation of bacteroid state within plant cell is done, what enzyme do the bacteria start producing? |
Nitrogenase |
|
After the bacteria has successfully invaded the plant cells and formation of bacteroid state within plant cell is done, what does the bacteria start doing? |
Start fixing nitrogen while the plant will start sending organic carbon to supply energy for the bacteria |
|
After the bacteria has successfully invaded the plant cells and formation of bacteroid state within plant cell is done, what does the plant start doing for the bacteria? |
Send organic carbon to supply energy for the bacteria to start fixing nitrogen |
|
What do the nodules do for the bacteria in a plant? |
Protect the bacteria from O2 exposure |
|
In nitrogen fixation, for one molecule of nitrogen, how many ATPs do you need? |
16 ATP |
|
In nitrogen fixation from N2 into 2 NH4+, is the flavodoxin being oxidized or reduced? |
Oxidized |
|
What enzyme is used in nitrogen fixation from N2 to 2 NH4+? |
Nitrogenase
|
|
How does nitrogenase work? |
There are actually two enzymes in nitrogenase Nitrogenase ONE reduces ferrodoxin Nitrogenase TWO oxidizes MoFe This process uses 2 ATP The reduced form of MoFe converts N2 + 8H into NH4 + H2 |
|
Do all organisms that fix nitrogen have some version of Nitrogenase? |
Yes |
|
What is Nitrogenase reductase? |
A 60kD homodimer with a single 4Fe-4S cluster that keeps the nitrogenase in a reduced state Very oxygen-sensitive Binds MgATP where 4 ATP required per pair of electron transfer Causes reduction of N2 to 2 NH3 + H2 which requires 4 pairs of electrons (16 ATP per N2) |
|
What is nitrogenase? |
A 220 kD heterotetramer Each molecule of enzyme contains 2 Molybdenum (Mo), 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters) It is slow, uses only three molecules of N2 per second Highly O2 sensitive |
|
What is Leghaemoglobin? |
Made by plants and exported into nodule to bind to excess O2 Bacteria and plant must be present for production of this molecule and only found in nodule |
|
Why would a plant produce Leghaemoglobin? |
Nitrogenase (enzyme used by symbiotic bacteria Rhizobia in nodule) is a highly oxygen sensitive enzyme Leghaemoglobin will bind to any excess oxygen in the nodule |
|
What are the two principal pathways for ammonium assimilation? |
Principal route: GDH/GS in organisms rich in N Secondary route: GS/GOGAT in organisms confronting N limitation |
|
What is GOGAT? |
Glutamate synthase (or Glutamate: oxo-glutarate aminotransferase) Converts alpha-ketoglutarate + glutamine into glutamate |
|
What happens during pathway 1 (secondary route) of ammonia assimilatory cycle? |
1) NH4+ + Glutamate + ATP will be converted into Glutamine + ADP + Pi 2) If enough Nitrogen around, Glutamine converted into amino acids, proteins, purines and pyrimidines. If not enough Nitrogen around: |
|
What happens during pathway 2 (principal route) of ammonia assimilatory cycle? |
1) NH4+ + alpha-ketoglutarate gets convereted into Glutamate by Glutamate Dehydrogenase (GDH) 2) Glutamate gets converted into amino acids and proteins |
|
During the secondary route of ammonia assimilatory cycle, what happens if there is not enough nitrogen? |
Glutamine gets converted back into glutamate by addition of alpha-ketoglutarate by GOGAT |
|
What is denitrification? |
Denitrification converts nitrate (NO3) in the soil to atmospheric nitrogen (N2) Denitrifying bacteria live deep in soil and in aquatic sediments where conditions make it difficult for them to get oxygen (using nitrate as an electron acceptor) Denitrifying bacteria use nitrate as an alternative to oxygen, leaving free nitrogen gas as a byproduct |
|
What enzymes can be found in denitrification? |
Nitrate reductase
Nitrous oxide reductase |
|
What are the two types of nitrogen fixers recognized? |
1) Free-living (non-symbiotic) 2) Mutualistic (symbiotic) |
|
What happens during the sulfur cycle? |
|
|
Why would most bacteria live in communities compared to living on their own? |
Communities allows them to take up products made by other organisms and consume those |
|
In Li-Hung Lin, et. al. research "Long-Term Sustainability of a High-Energy, Low-Diversity Crustal Biome", they found alkaline saline ground water at 2.8 kilometers depth in Archaean metabasalt microbial biome dominated by a single phlyotype. How were the sulfate reducers sustained? |
Sulfate reducers were sustained by geological produced sulfate and hydrogen at concentrations sufficient to maintain activities for millions of years |
|
What do most Mycobacterium have in common? |
Gram positive Waxy coat on surface |
|
What is Mycobacterium marinum? |
Free living bacterium
Lives in water Double the genome size of Mycobacterium leprae |
|
What is Mycobacterium tuberculosis? |
Grows in humans and gets into lungs but can cultivate on a petri dish
Most of the time, it undergoes latent state where people can live with TB infection without knowing it An immunosuppressent can activate TB which can cause really bad pulmonary infection |
|
What is Mycobacterium leprae? |
Genome size ~3.27 million bp GC rich genome Causes leprosy Very slow growing bacteria Can only live within humans and cannot cultivate on lab media (only animals) |
|
What are pseudogenes? |
Genes that should look like genes and should code for proteins but are inactivated |
|
What is Candidatus Carsonella? |
Endosymbionts Symbiotic relationship by living inside another cell Smallest genome size |
|
What is symbiosis characterized by? |
1) Bacteria are restricted to a single inset cell type - "mycetocyte" or "bacteriocyte" - where they are not spread out throughout the body but rather contained within a specialized cell (mutalistic relationship) 2) Bacteria are maternally transmitted 3) Association between the bacteria and the host is required by both partners (host may die if bacteria is taken out) |
|
Endosymbiotic bacteria reside in what type of cells? |
Specialized cells called "bacteriocytes" or "mycetocytes" |
|
The relationship between the whiteflies, devastating agricultural parasites, and their endosymbionts goes back ~180 million years. Why? |
These bacteria can help capture and recycle nitrogen from uric acid, preventing it from being lost from the insects
These insects eat diets that are high in carbon but low in nitrogen |
|
Is inheritance of endosymbionts usually vertical, horizontal, maternal, etc...? |
Vertical and maternal |
|
Can a male with an endosymbionts give inheritances to that endosymbiont? |
No, inherit only directly from mothers |
|
What are the ways that a mother can give inheritance of their endosymbiont? |
Trans-ovarial Smearing of endosymbionts on shell of deposited egg (when egg shell breaks, insect will pick up endosymbionts) Transmission through feed (mother have specialized "sacs" to secrete bacteria into young during feeding) |
|
What happens when an Wolbachia infects an insect such as a worm (filarial nematode)? |
Engage in reproductive parasitism where they affect how the insect reproduce so they can spread Once infects one female insect, it ensures its sreadp to all progeny, often means getting rid of the males |
|
What is cytoplasmic incompatibility of reproductive parasitism? |
Most common reproductive manipulation caused by Wolbachia endosymbionts Embryos derived when infected males mate with uninfected females are inviable Infected females can mate with both infected and uninfected males (population rapidly acquires more and more infected members) |
|
With cytoplasmic incompatibility of reproductive parasitism, what happens when a infected male mates with an uninfected female? |
Eggs will die because there are factors in the sperm that is incompatible with the cytoplasm of the uninfected female |
|
When an insect infected with a reproductive parasitism like Wolbachia, what happens during ferminization? |
During development process, genetic male has genetic material to make a male But Wolbachia endosymbiont suppresses those hormones and female structures are made instead, making functional female (they will reproduce like females) |
|
When an insect infected with a reproductive parasitism like Wolbachia, what is male killing? |
In some insects, Wolbachia infected male embryos fail to develop properly, leading to entire brood of infected females This helps spread Wolbachia in highly resource-limited environments where competition between siblings for resource is fiere |
|
What an insect infected with a reproductive parasitism like Wolbachia, what is parthenogensis (virgin birth)? |
Some insects (notably wasps) have a sex-determination system where unfertilized (haploid) embyros are male and fertilized embryos (diploids) are female Wolbachia infected haploid embryos undergo a second round of RNA replication to become diploid and hence develop into females even though no fertilization occured |
|
Are endosymbiont genomes AT or GC rich? |
Very AT-rich |
|
What is the endosymbiotic theory or "symbiogenesis"? |
States the new forms of life can arise form the merging of two separate cells into a single functioning entity |
|
Who is Lynn Margulis? |
Developed and proposed the Endosymbiotic theory |
|
TRUE or FALSE: Mitochondria and chloroplast (plastids) are ancient endosymbionts |
TRUE |
|
What evidence for the endsymbiotic origins of mitochondria and plastids (chloroplast) do we have? |
They have their own DNA that appears bacterial in origin They encode ribosomes similar to bacterial ribosomes Mitochondria have their own double membrane and includes lipids (cardiolipin) that are similar to bacteria They multiply and divide using binary fission They use N-formylmethionine as the intiating amino acid in protein synthesis Some eukaryotes have chloroplast that have cell walls |
|
The mitochondria have their own what that the cell also has? |
Ribosome tRNA genes NADH dehydrogenase ATP synthase Cytochrome oxidase subunits |
|
What is a serious problem with antibiotics that block bacterial protein synthesis (aminoglycosides and tetracyclines)? |
Mitochondrial ribosomes are similar enough to bacterial ribosomes that the antibiotics block protein synthesis of the mitochondria |
|
How are mitochondria inherited? |
Maternally |
|
When did King Richard III die and what did it signify? |
King Richard III of England was killed August 22, 1485 by the army of Henry VII at the Battle of Bosworth Field His death marked the end of the Plantegenet dynasty which had ruled for over 300 years |
|
What is a cell envelope? |
Shell of membranes and peptidoglycan that surround the bacterial cytoplasm Generic term for what surrounds the cytoplasm, regardless what is in it |
|
Do gram-positive cells have a peptidoglycan? If so where? |
Yes on the outside |
|
How many membranes do gram-negative bacteria have? |
2, outer and cytoplasmic/inner |
|
Is this a gram positive or negative cell? Label its components |
Positive |
|
Is this a gram positive or negative cell? Label its components |
Negative |
|
Do gram negative bacteria have a thick or thin peptidoglycan layer? |
Thin |
|
Do gram positive bacteria have a thick or thin peptidoglycan layer? |
Thick |
|
Do gram negative or positive bacteria have a outer membrane? |
Negative |
|
Do gram negative or positive bacteria have a periplasm? |
Negative |
|
In one sentence, describe what a periplasm is: |
Concentrated gel-like matrix in space between inner cytoplasm membrane and bacterial order membrane |
|
Do gram negative or positive bacteria have lipopolysaccharides (LPS)? |
Negative |
|
Why do gram negative bacteria have LPS and not gram positive? |
The LPS is part is the phospholipid bilayer which extends outwards towards the outside of the cell Due to gram negative bacteria having an outer membrane (and positive only have a peptidoglycan layer on the outside), only gram negative bacteria will have LPS |
|
Do gram negative or positive bacteria have lipoteichoic acid? |
Positive |
|
What is lipoteichoic acid? |
Surface-assocaited adhesion amphiphile Regulator of autolytic wall enzymes Released from bacterial cells mainly after bacteriolysis induced by lysozyme, cationic peptides from leucocytes or beta-lactam antibiotics |
|
Why are lipoteichoic acids only found on gram positive bacteria? |
They are only found on the peptidoglycan layer to interact with the outside environment Due to gram positive bacteria have peptidogycan layer as their outermost layer and negative as middle layer, only positive can have lipoteichoic acid |
|
Do gram positive or negative bacteria have teichoic acid? |
Positive |
|
How are lipid content different between gram positive and negative bacteria? |
Negative has high lipid content, positive is low |
|
Why do gram negative bacteria have high lipid content compared to gram positive? |
They have high lipid content because they have an extra membrane |
|
What is a phospholipid mainly composed? |
A glycerol derivative (of three C's) that are attached to two hydrophobic fatty acids on the non-polar side and a phosphoryl head group attached to another group on the polar/hydrophillic side |
|
True or False? Lipid bilayers spontaneously organize in aqueous environments |
True |
|
What is cardiolipin? |
AKA diphosphatidylglycerol Phosphate group of the lipid attaches onto another phosphate group of another lipid by a glycerol bridge Found in bacteria (like E. coli) and in the mitochondria |
|
Where can you find cardiolipin? |
In bacteria like E. coli and in mitochondria membrane |
|
What functions do bacteria membranes serve? |
Permeability barrier Anchor for surface proteins Energy capture and storage |
|
How does the membrane of the bacteria function as a permeability barrier? |
Prevents leakage and functions as a gateway for transport of nutrients into and out of the cell |
|
How does the membrane of bacteria function as an energy capture and storage? |
Generation and maintenance of ion gradients (e.g. proton motive force) Use proteins on membrane to capture charges on outside of cell and bring them in |
|
What happens to rate of permeability across a bacterial membrane if you add three carbons? |
A reduction of a thousand fold reduction of getting through the lipid bilayer membrane's permeability spontaneously |
|
Where is the periplamic space located? |
In the space between outer membrane and cytoplasmic membrane and includes spaces between strands of peptidolycan (cell wall) of gram negative bacteria |
|
What does it mean that the periplasm is iso-osmotic with the cytoplasm? |
Both periplasm and cytoplasm have same number of molecules and ions per unit volume Keeping same osmotic pressure and concentration of particles on outside and inside |
|
True of False: Periplasm is not a dynamic changeable matrix of material |
FALSE Periplasm is a dynamic changeable matrix of material |
|
Describe the outer membrane of a gram-negative bacteria? |
A asymmetric lipid bilayer consists of phospholipids (inner leaflet), and lipopolysacharides (LPS, in outer leaflet) and protein ~8nm thick Strong permeability barrier to hydrophobic compounds and large hydrophilic agents and is responsible for resistance to various antibiotics and chemotherapeutic agents |
|
Approximately how thick generally is the outer membrane of a gram-negative bacteria? |
~8nm
|
|
Does the outer membrane of gram-negative bacteria play a role in resistance to various antibiotics and chemotherapeutic agents? |
Yes |
|
Can LPS be found in humans? |
No, exclusive to bacteria |
|
What does a human body do when an LPS is found in its blood stream? |
Our immune system has specific receptors to recognize LPS and distinguish self-cells from non-self-cells |
|
Is LPS an antigen? |
It is a potent antigen |
|
What is a main cause/symptom of an immune response to an LPS? |
Sepsis or septic shock |
|
What are serotypes and serotyping? |
Serotypes are groups within a single species of microorganism which share distinctive surface structures (like O and H antigens) Serotyping is using serotypes as pathogenic diagnostics methods |
|
What are LPS composed of? |
Proximal, hydrophobic lipid A region Distal (sticks towards environment), hydrophilic "O-antigen" polysaccharide Core oligosaccharide region that connects the two |
|
What are O-antigens? |
Multiple repeats of short olgiosaccharide
Highly variable between species and even strains within a species These sugars repeat over and over and over again, attached to one another |
|
What are core olgiosaccharides? |
Short olgiosaccharide that links O-antigen to lipid A anchor |
|
What is Lipid A? |
Forms the outer leaflet (or face) of the outer membrane
Inner leaflet or face of the outer membrane is composed of phospholipids The unsaturated fatty acids which are linked to two sugars which have been modified with phosphates and is one thing the human immune system reacts to This region is hydrophobic |
|
What part of the LPS is lipid A? |
The hydrophobic, membrane-anchoring region Attached to the core oligosaccharide through KDO (2-keto-3-deoxyoctonic acid) which is negative charged and only found in bacteria |
|
What does Lipid A consist of? |
Phosphorylated N-acetylglucosamine (NAG) dimer with 4 to 7 saturated fatty acids attached |
|
What connects Lipid A and the core olgiosaccharide in LPS? |
KDO (2-keto-3-deoxyotonic acid) |
|
Can variation happen with Lipid A of LPS? |
Yes |
|
Why does variation happen with Lipid A of LPS?
|
Bacteria can modify their lipid to avoid some antibiotics Bacteriophages (viruses) will bind to LPS, inject their viruses and by scrambling up their LPS, bacteria can avoid these viruses Variation can cause it to be harder for the human immune system to recognize |
|
Do core olgiosaccharides of LPS have variation? |
They do but are more conserved between species so less variations |
|
What do core olgiosaccharides consist of? |
Short chain of sugars |
|
What are the two unusual sugars present in core olgiosaccharides of LPS? |
Heptose and 2-keto-3-deoxyoctonoic (KDO) |
|
What is KDO? |
2-keto-3-deoxyoctonoic acid A unique and invariably present in LPS and an indicator in assays for LPS Connects Lipid A to core olgiosaccharides in LPS |
|
What is the o-polysaccharide of the LPS? |
AKA O-antigen Consists of repeating olgiosaccharide subunits made up of 3-5 sugars where length ranging up to 40 repeat units (much longer than core polysaccharide) and maintains hydrophilic domains of LPS molecules Variation in sugar content contribute to wide variety of antigen types in gram-negative species |
|
Can bacteria produce their own specified and exclusive sugars? |
Yes |
|
Where can you find divalent cations in the LPS? |
Proximal part of core containing KDO and lipid A backbone It binds to the negative charges |
|
What happens when you phosphorylate KDO? |
You make it more negative which attracts divalent cations |
|
Many gram-negative bacteria are naturally resistant to what? |
Hydrophobic antibiotics Detergents Hydrophobic dyes |
|
Which is more resistant to chemotherapeutic agents, gram negative or positive? |
Negative |
|
What is a function of divalent cations in LPS? |
Can neutralize negative charges on membrane lipids This assists in tight packing |
|
What is a chelator? |
Chemicals that "grab" ions like Mg2+ and/or Ca2+ |
|
What are the effects of chelators? |
Brief treatment of E. coli with EDTA released about half of the LPS from cells owing to the removal of divalent cations such as Mg2+ and Ca2+
EDTA-treated cells become hypersusceptible to a wide range of hydrophobic antibiotics, dyes and detergents
The increase in permeability is likely due to filling in of the space, formerly occupied by LPS, by phospholipid molecules, thereby creating phospholipid bilayer domain |
|
What happens to a bacterial cell when you treat it with EDTA? |
Becomes hypersusceptible to wide range of hydrophobic antibiotics, dyes and detergents |
|
What causes the increased permeability when treating bacterial cells with chelators? |
Likely due to filling in of space which was formerly occupied by LPS by phospholipid molecules |
|
Kauffmann-White O-serogrouping system for E. coli uses what as its basis? |
Differences in O-antigens |
|
When an animal generates an antibody against one type of O-antigen, will it react with another type of O-antigen? |
No, once created they are specific for that type of O-antigen
|
|
Of serological classification, what is the O-antigen? |
Outermost portion of bacteria's surface covering O-antigen of the LPS |
|
Of serological classification, what is the H-antigen? |
Slender threadlike structures Ex) Flagella |
|
Of E. coli strain O157:H7, what do the numbers and letters mean? |
The O means O-antigen, the 157 is the 157th identified O-structure and the 7th type of flagella (H) antigen |
|
What is another name for LPS? |
Endotoxin |
|
When infecting purified LPS or Lipid A into animals, what does it cause? |
Wide spectrum of nonspecific pathophysiological reactions such as: Fever Changes in white blood cell count Disseminated intravascular coagulation Tumor necrosis Hypotension Shock Death |
|
When infecting purified LPS or Lipid A into animals, what causes wide spectrum of nonspecific pathophysiological reactions? |
A hyperactive immune response that can lead to septic shock |
|
What is septic shock? |
Serious condition that occurs when a body-wide infection leads to a dangerously low blood pressure |
|
What does Hydrophilic O poly-saccharides do for Lipid A? |
Act as a water-solubilizing carrier for toxic Lipid A |
|
What is peptidolycan? |
AKA PG or murein Chemically unique, rigid structural component of cell wall found in bacteria Human immune system has receptors for these Provide shape of cells and preserve integrity of cytoplasmic membrane from rupture in medium or low osmolarity Contains compounds unique to the microbial world like D-amino acids, diaminopimelic acid (DAP) and N-acetyl muramic acid (NAM) |
|
What gives bacterial cells their shape and help preserve integrity of cytoplasmic membrane from rupture in medium or low osmolarity? |
The peptidoglycan |
|
Will you find DAP or NAM in humans? |
No, exclusive to microbes
|
|
What happens to a bacteria cell when you have a lysozyme digest its walls in a low solute solution? |
Water will enter the cell, causing lysis (cell bursting) |
|
What happens to a bacteria cell when you have lysozyme digest its walls in an isotonic solute solution? |
The internal components (its membrane and everything in it) will be a protoplast outside of the lysised cell wall |
|
In peptidoglycan, what is the wall glycan made up of? |
NAG (N-acetyl-glucosamine) NAM (N-acetyl-muramic acid) |
|
In peptidoglycan, what is the wall peptide made up of? |
5 amino acid peptides L-alanine D-isoglutamate or Di-isoglutamine Lysine or mDAP (not produced in human bodies) D-alanine And another D-alanine |
|
Will you find NAG in humans? |
NAG is found in mammals (yes) |
|
Will you find NAM in humans? |
No NAM is a derivative of NAG that has an extra carboxyl group that can link it to the N-terminus (amino group) of wall peptide, unique to bacteria |
|
NAM is a derivative of what and what makes it different from that? |
Derivative of NAG Has extra carboxyl group that can link it to the N-terminus of the wall peptide |
|
What chirality will you most likely find in human amino acids and bacteria peptidoglycan amino acids? |
L- in humans, D- in bacteria peptidoglycan |
|
Where will you find D-isoglutamate in bacteria? |
In the peptidoglycan of gram negative bacteria |
|
Where will you find D-isoglutamine in bacteria? |
In peptidoglycan of gram positive bacteria |
|
What is transglycoslyation? |
Polymerization of peptidoglycan subunits via their sugars leads to formation of long glycan strands that alternate between NAG and NAM by glycosidic bonds |
|
What is the third amino acid in a wall peptide of peptidoglycan? |
A lysine or mDAP (meso-diaminopimelic acid) which has a free amino group on them |
|
What is the purpose of the free amino group of the third amino acid in a wall peptide of a peptidoglycan? |
To form a peptide bond with an amino group attached to a carboxyl group found at the C-terminus of a wall peptide |
|
What happens during a transpeptidation reaction? |
C-terminal D-alanine of one subunit is removed by a transpeptidase enzyme Then links free amino group of neighbouring wall peptide to newly liberated C-terminus of first wall peptide |
|
Can peptidoglycan vary from species to species? |
Yes |
|
What is a difference between typical gram negative and positive peptidoglycans? |
Negative: D-isoGlu mDAP Positive: D-isoGln L-Lys |
|
Why do bacteria peptidoglycans have D amino acids instead of the typical chirality of L? |
Bacteria secrete proteases which recognize L shaped amino acids and degrade them/breaking the peptide-linkage bonds Having D- amino acid instead of L, this allows the protease not to eat its own bacterial cell wall |
|
Do gram-negative bacteria always have cross-link wall peptides? |
Not always but usually |
|
How do gram-negative bacteria do cross-linking for wall peptides? |
They cross link wall peptides by linking amino groups on the side chain of a mDAP (or m-A2pm) to carboxy terminus of the D-Ala on position 4 of adjacent wall peptide |
|
How do gram-positive bacteria do cross-linking for wall peptides? |
Bacteria usually cross link wall peptides by linking amino group on the side chain of a lysine in position 3 to a chain of amino acids The N-terminus of this "crossbridge" connects to the carboxy terminus of the D-Ala in position 4 of the adjacent wall peptide |
|
In a cross link wall peptide, what positions amino acids/molecules are connected? |
For gram negative and positive, its from position 3 to position 4's D-Ala |
|
Does gram negative or positive bacteria have extra peptides at the wall peptide connection? |
Gram positive |
|
Why would gram positive bacteria have extra peptides at their cross-bridge/interbridges? |
Bacteria are constantly fighting to each other and destroying one another where this variation can give one bacteria an advantage over another one Bacteriophages pop bacteria cell wall to get out but with variation of peptidoglycan (such as 5 Gly) can make bacteria resistant to popping Diversity of each bacteria's cell wall help avoid parasites mass extinction/an epidemic |
|
What position of the peptidoglycan wall peptide never (or ~99.999999%) never change? |
Position 4, D-Ala |
|
Describe briefly and broadly how cell wall synthesis (how wall peptides get synthesized attached to peptidoglycan) for S. aureus? |
Made in cytoplasm where NAG (or GlcNAc) will be attached to a nucleotide UTP UDP-GlcNAc (UTP-NAG) enzymatically converted to UDP-NAM (MurNAc) NAM-wall peptide transferred to a lipid which then modified further before being flipped by a "flipase" (MurJ protein) to outside of cell membrane for assembly into cell wall |
|
In cell wall synthesis for peptidoglycan, where are all the energy requiring steps taken place? |
In the cytoplasm |
|
What is the protein MurJ? |
A protein transfer complex (a flipase) Transport peptidoglycan wall peptide brick from inner to outer leaflet of the inner membrane |
|
What does the enzyme transglycosylase do? |
Connects the NAG + NAM with other NAG + NAMs |
|
What does the enzyme transpeptidase do? |
Creates cross-bridges/interbridges between position 3 and 4 of wall peptide amino acids |
|
Where does LPS biosynthesis start in? |
In the cytoplasm with nucleotide linked sugars that get transferred to lipids |
|
Where is the O-antigen of LPS assembled? |
It is assembled on the same type of bactoprenol lipid as what peptidoglycan would bind to during synthesis
They are then transferred to periplasm by specialized membrane proteins (RfbX or MsbA-1 and 2) then ligated together
O-antigen subunits are polymerized onto the LPS core then the completed molecule is transferred to outer membrane |
|
What does LPS transport from inner to outer membrane require? |
It requires a whole multi-protein machinery to transport it It is not spontaneous |
|
Why does LPS require a multi-protein machinery to transport it instead of being spontaneous? |
LPS is a lipid, it would never cross the aqueous periplasm by itself It takes energy to extract LPS from inner membrane and get it to the outer membrane This energy comes from ATP hydrolysis inside the cell which is carried out in the cytoplasm with proteins that carry this energy into exterior membrane |
|
For LPS transport from inner to outer membrane, where does this energy come from? |
ATP hydrolysis inside the cell (cytoplasm) |
|
Staphylococcus aureus has the ability to bind and do what to antibodies (immunoglobulins) of mammals? |
When they bind, this prevent bound antibodies from working properly by binding using "protein A" |
|
What is Protein A? |
Exquisitely high affinity for human antibodies and prevent them from working It is a immunoglobulin-binding factor and displayed on surface of bacterial cell Can be found in gram-positive bacteria It consists of a C-terminal sorting signal that will be in the membrane while its N-terminus protrudes out |
|
What can be found in the C-terminal sorting signal of Protein A? |
Three parts 1) Highly conserved LPXTG sequence (X is any) 2) Stretch of mostly hydrophobic residues for about 20 amino acids long 3) Positively charged C-terminal tag of about 5 amino acids (rich in lysine or arginine amino acids) |
|
What part of Protein A causes it to get temporarily restrained into the membrane? |
The hydrophobic C-terminal domain |
|
What does the enzyme Sortase do? |
It sites on the surface of the cell and recognizes anything with a LPXTG motif (such as Protein A) and cleaves it between T and G residues It will then transport that motif to a penta-Gly crossbridge of a peptidoglycan and attach it by transpeptidation and transglycosylation reaction |
|
What happens to the hydrophobic domain after the C-terminal sorting sequencing has been cleaved by Sortase? |
It is degraded |
|
Where does the enzyme Sortase cut? |
It sites on the surface of the cell and recognizes anything with a LPXTG motif (such as Protein A) and cleaves it between T and G residues |
|
What type of reaction does Sortase use to attach a Protein A LPXT to a peptidoglycan? |
Transpeptidation and transglycoslyation reaction |
|
What are the steps of incorporating Protein A into the peptidoglycan wall? |
1) Protein C go through membrane and hydrophobic domain stuck in membrane 2) Sortase cleaves between LPXTG 3) Transports it to a wall peptide 4) Tranpeptidation and transglycoslyation reaction adds it to the wall peptide |
|
What happens if every wall peptide of peptidoglycan had a protein A attached to it? |
If every brick was attached to a Protein A, it would not be able to create a net/mesh structure |
|
What is Murein (Braun's) lipoprotein? |
Holds outer membrane onto cell and attaches it to peptidoglycan Most abundant protein in gram-negative cells Amino-terminal cysteine residue is modified and carries a glycerol molecule to which two fatty acids are esterified where also linked to a fatty acid (N- to lipid, C- to cell wall) |
|
How are Murein (Braun's) lipoprotein's anchored onto the peptidoglycan? |
Fatty acyl residues are inserted in the outer membrane (inner leaflet) and epsilon amino group of a C-terminal lysine residue from the protein forms peptide bonds with free epsilon-carboxyl group of mDAP in peptidoglycan |
|
What happens if a gram-negative bacteria does not have Murein (Braun's) lipoprotein? |
The outer membrane would peel off |
|
To what position does Murein (Braun's) lipoprotein bind to? |
The side chain of the C-terminal lysine residues are attached to the peptidoglycan via free mDAP carboxyl group |
|
What are porins? |
Most abundant proteins in the outermembrane They make pores in the membrane, spanning the outer membrane to form a narrow, water-filled "pore" or channel through the membrane |
|
What type of property do nutrients that go through porins usually have? |
Hydrophilic |
|
What are the major types of porins? |
Classical porins Specific channels |
|
What are classical porins? |
Form nonspecific pores or channels for rapid passage of small hydrophilic molecules across outer membrane Usually form trimer where each subunit was made by 16-beta strands traversing the membrane to form a beta barrel surrounding a large channel |
|
What is PhoE? |
It is a unique classical porin in that it is produced only under conditions of phosphate starvation by environmental conditions |
|
Classical porins form what type of structure that is made up of what? |
Form trimers that are made up of 16-beta strands traversing the membrane to form a beta barrel surrounding a large channel |
|
What is the diameters of classical porins and what they do allow through them? |
The diameter of classical porin channels are 1.1-1.2nm They allow passage of hydrophilic molecules with molecular weight less than 600-700 Da |
|
Under adverse conditions/poor nutrition, what happens to OmpC? |
It is replaced by OmpF which form pores of larger diameter (1.2m), allow cells to take up larger molecules |
|
Does OmpF and OmpC prefer cations or anions? |
Cations slightly over anions |
|
Does PhoE prefer cations or anions? |
Anions |
|
What is the regulation of OmpF? |
Repressed under conditions of highly external osmolarity |
|
What is the regulation of OmpC? |
Expressed under conditions of high external osmolarity |
|
What is the regulation of OmpD? |
Expression depends on cAMP |
|
What is the regulation of PhoE? |
Repressed by external phosphate |
|
If you have high external osmolarity, which classical porins will be expressed and repressed? |
OmpF repressed OmpC expressed |
|
Would you find OmpD in E. coli? |
No |
|
If a drug inhibits and eliminates cAMP, what happens to OmpD? |
It will not be expressed |
|
What does the structure of OmpF look like? |
Three beta barrels (made from beta sheets) |
|
What makes the eyelet of the beta barrel for OmpF? |
The eyelet is formed by two negative charged residues: 1) Glutamic acid at position E117 2) Aspartic acid at position D113 As well as four residues from the opposing barrel wall: a lysine at position 16 and arginines at position 42, 82 and 132 |
|
What is OprF in Pseudomonas aeruginosa? |
Major porin of P. aeruginosa
Allows much slower diffusion of small solutes than other porins (50-100 times slower than OmpF of E. coli) |
|
What does Pseudomonas aeruginosa cause in humans? |
Cause serious lung infection in immunocompromised individuals and cystic fibrosis patients
Very difficult to treat due to notoriously impermeable outermembrane (because of OprF) where it can live in disinfectants and antibiotics because of this |
|
What are specific channels? |
They mediate the downhill (spontaneous) diffusion of specific classes of nutrients
Not as general and exclude most things because their shape will only let in a certain shape such as maltose or lactose |
|
What is LamB in E. coli? |
Favours passage of maltose and maltodextrins across the outer membrane Porin-like trimeric protein and constructed as beta-barrel from 18 beta-strands It is also a phage lamba receptor for E. coli Does not require energy |
|
What happens if you have a mutant of LamB in E. coli? |
Mutants of this will not be affected by the virus its receptor is for It also could not let in maltose and maltodextrins |
|
Does LamB require energy? |
No, LamB structure does not require energy |
|
What is Maltodextrin? |
A polysaccharide that is used as food additive Produced from starch by partial hydrolysis |
|
Do specific channels require energy? |
No |
|
What is ScrY? |
Specific channel Transporter for surcrose Found in some strains of E. coli |
|
What is Tsx? |
Specific channel Transports nucleosides Receptor for phage T6 Found in some strains of E. coli and Salmonella |
|
How do bacteria capture iron from its environment? |
By secreting Siderophores |
|
What is Siderophores? |
A low molecular weight molecule that can form soluble complex with ferric iron
Too big to just pass through a porin channel |
|
Can vitamin B12 go through a porin? |
No, too big |
|
What are TonB dependent transporters? |
Outermembrane porin transport receptors that require TonB and energy from the cytoplasm
Can transport ferrichrome, vitamin B12 and iron siderophores across them
They are dependent on TonB because there is no energy in the periplasm so this would be energergized from the cytoplasm's proton motive force to energy the transporter |
|
What is ferrichrome? |
Cyclic hexa-peptide that forms a complex with iron
It is a siderophore (binds to Fe3+) composed of three Gly and three modified orinithine residues with hydroxamate groups |
|
What is FepA? |
Ferric enterobactin receptor Beta-barrel structure In the centre they have a plug domain that acts as a binding pocket (receptor) for their specific substrate That substrate could be vitamin B12, heme molecule, a siderophore or something else larger and present at low concentration |
|
Will you find ATP in the periplasm? |
No |
|
What does TonB do to a TonB dependent transporter? |
TonB acts on the "cork" domain of the TBDT that blocks the OM receptor until it is engaged by its substrate (such as Vitamin B12) |
|
What is BtuF? |
A periplasmic binding protein that transports vitamin B12 to the inner membrane ABC transporter protein (BtuCD) or ATP-binding cassett after being added to the perplasm by the TBDT |
|
Which of these are least effective against Gram-negative bacteria? |
Vancomycin because too big |
|
How does Vancomycin inhibit cell synthesis? |
Binds to D-Ala and prevent it from being used as a substrate |
|
What does the antibiotic Chloramphenicol do? |
Inhibit protein synthesis |
|
What does the antibiotic Tetracycline do? |
Inhibit protein synthesis |
|
What does the antibiotic Rifamycin do? |
Inhibit RNA synthesis |
|
What does the antibiotic Penicillin G do? |
Inhibit cell wall synthesis |
|
What does the antibiotic Vancoymcin do? |
Inhibit cell wall synthesis by binding to D-Ala and prevent it from being used as a substrate |
|
What are the four basic mechanisms for antibiotic resistance? |
1) Bacteria can hibernate (go into static growth and not make new cell wall) 2) Bacteria can destroy antibiotics by enzymes 3) Bacteria can mutate target to no longer bind to antibiotic 4) Bacteria can keep antibiotic away from target by decreasing permeability or increasing efflux |
|
How do bacteria keep antibiotics away from the target? |
Decrease permeability by decreasing influx through expression of modified porins, fewer porins or change of membrane structure Increase efflux by pumping the drugs out |
|
What is the AcrA/AcrB/TolC complex? |
Major contributor to antibiotic resistance Strains that lack any of the three components have greatly increased susceptibility to several classes of antibiotics |
|
What does ArcB do in the ArcA/ArcB/TolC complex? |
Uses energy from proton gradient across the inner membrane to drive efflux of compounds at the periplasm-IM interface Passes these compounds to TolC efflux channel for final export outside the cell |
|
What does TolC do in the ArcA/ArcB/TolC complex? |
It is an efflux channel for exporting compounds from ArcB to outside of the cell |
|
What is a lytic transglycosylase? |
Break bonds between NAG and NAM of peptidoglycan |
|
What is a amidases? |
Breaks the bond between NAM and wall peptide |
|
What is a endopeptidase? |
Breaks bonds between any two amino acids in wall peptide and crossbirdge peptides |
|
If a bacteria has D-iGln, is it gram positive or gram negative? |
Gram positive |
|
Where would lytic transglycosylase, amidase and endopeptidase cut between two wall peptide bricks? |
|
|
What is the name for the lytic transglycosylase enzyme that cuts the NAG at its reducing end towards the NAM? |
A glucosaminidase |
|
What is the name of the lytic transglycosylase enzyme that cuts the NAM at its reducing end towards the NAM? |
Muramidase |
|
What is lysozyme? |
A muramidase that is produced by animals to destroy invading bacteria Can be found in human tears as well as human immune cells make certain types of these to destroy bacterial cells First enzyme to have is structure solved |
|
What is Lysostaphin? |
Endopeptidase produced by Staphylococcus simulans to kill Staphylococcus aureus by targeting and cleaving the unique pentaglycine crossbirdge found in its cell wall |
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What are filementation temperature sensitive mutants in E. coli? |
Genetic mutants that have been isolated to form filaments when exposed to higher growth temperatures |
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Why are temperature sensitive mutants significant? |
They enable us to study essential genes Cells can be grown normally at one temperature and then switched to "non-permissive" temperature to study how loss of factor effects the cell |
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What are conditional knockouts? |
Under one condition they work fine but when you change to set condition, it stops working |
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What is the mreC mutant in E. coli? |
Instead of making long filaments, they start producing small circular cells |
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What is side wall formation in bacteria? |
As the cells grow longer, new peptidoglycan is incorporated along the walls of the cell |
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What is septum formation in bacteria? |
At the appropriate time, the rod-shaped cell switches to synthesize the division septum (forms two seperate cells) |
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What does the MreC and MreD do? |
Code proteins for cell elongation If you get rid of these guys, the bacteria cells begin to look like round dots (separate without elongation) |
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What happens if you get rid of MreC and MreD from bacteria? |
Bacteria growth will cause them to look like round dots Without MreC and MreD, the bacteria will separate without elongation |
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What is PBP2 in the elongase complex? |
Penicillin binding protein 2 (when penicillin binds, it stops working) It is a transpeptidase/transglycosylase |
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What is MraY in the elongase complex? |
Makes "Lipid 1" (makes one of the bricks before getting flipped out) |
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What is MurG in the elongase complex? |
Enzyme in synthesis of the "lipid 2" peptidoglycan precursors |
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What is the MreB in the elongase complex? |
A bacterial actin homolog Proposed to make filaments inside the cell |
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What happens when you inhibit the elongase complex? |
Leads to round shaped cells |
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How can inhibition of the elongase complex happen in bacteria? |
Can occur through mutations in any of the elongase proteins by use of certain chemical that interfere with the elongase |
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What does the antibiotic Mercillinam do? |
Inhibit PBP2 thereby causing the formation of small round cells |
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What happens when penicillin binds to PBP2? |
It prevents it from working (do any transpeptidase and/or transglycosylase) Can cause formation of small round cells/inhibition of elongation |
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What is the divisome in bacteria? |
A complex of enzymes and transmembrane proteins that synthesize the bacterial septum |
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What happens if you block any of the Fts proteins in the divisome? |
It blocks septum formation forming long filament formations of the bacteria |
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What happens if you block septum formation/synthesizing? |
The bacteria will grow long like a long filament formation |
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What is the PBP3 and PBP1b of the divisome? |
It does transpeptidase and transglycosylase |
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What is MurG of the divisome? |
Enzyme in synthesis of the lipid 2 peptidoglycan precursor |
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What is the FtsZ of the divisome? |
Bacterial homolog of eukaryotic tubulin Proposed to make filaments inside the cell that form a ring This ring contracts to drive septum formation |
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What happens when you inhibit divisome? |
Leads to filamentous cells |
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How can you inhibit the divisome? |
Inhibition can occur through the use of certain chemicals that interfere with the divisome or through mutations in any of the divisome proteins |
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What does sanguinarine do to B. subtilis cells? |
Inhibits FtsZ of divisome causing cell filamentation |
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Can bacteria be viable while being a filamentous cell? |
They will be viable for a while but will die |
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What is fluorescence microscopy? |
Process where you stimulate a molecule with one wavelength of light and it emits a longer wavelength of light |
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In fluorescence microscopy, why are you only simulating the first wavelength of light in the reaction? |
Reason why is that first wavelength of light that you are stimulating reaction with, is absorbed by some atom or molecule orbital and kicked up to higher energy orbital state then within a few microseconds drop back down and emits a longer wavelength of light |
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Explain the general steps in fluorescence micrscopy? |
1) Energy of UV photon is absorbed by electron 2) Electron is raised to orbital of higher energy 3) Electron loses some energy as heat and drops to slightly lower orbital 4) Fluorescence is emitted at longer wavelength 5) Electron returns to original level |
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What is immunofluorescence micrscopy? |
In this technique, the protein you are looking for is labeled using antibodies (hence "immunofluorescence), typically two antibodies are used |
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How do you label the protein inside of a cell in immunofluorescence microscopy? |
You must kill the cell (by fixation with formaldehyde) and permeabilize the cell (with detergent and lysozome) You cannot use this technique on living cells unless the protein is exposed on the surface |
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Can you use immunofluorescence micrscopy on living cells? |
No unless protein is exposed on the surface of the cell |
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What are the general steps to immunofluorescence microscopy? |
1) Purify protein of interest
2) Round up an animal, inject animal with purified protein of interest
3) Come back a few weeks later, bleed it and get antibodies out of the animal
4) Hope the animal created antibodies
From here there are two options:
I) Put fluorescent tag on that antibody
II) Buy a antibody against that antibody commercially that is already fluorescently labelled |
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What does fixation do during immunofluorescence microscopy? |
Lock all proteins in place using chemicals that crossing the protein so they do not move around while you are killing the cell |
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What is fluorescence micrscopy? |
Using fusions to fluorescent proteins The protein of interest is genetically engineered to be attached to a fluorescent protein like GFP or dsRed |
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What is chimera or chimeric protein? |
When you add an extra domain to a protein |
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Where do fluorescent protein usually attach to on the protein of interest in a microscopy? |
At the N- or C- terminus of the protein of interest |
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Where can a problem arise with during fluorescence microscopy? |
Problems can arise if protein of interest is only made in very small quantities Or if it doesn't work correctly when fluorescent protein is attached Important not to overexpress chimeric protein since this can cause protein to go to locations in the cell it isn't suppose to |
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Where was GFP originally isolated from? |
The jellyfish Aequorea victoria
|
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What is the chromophore in GFP? |
It is the fluorescent part of the protein Made up by three amino acids (Glycine, tyrosine and theronine/serine) buried in the core of the molecular Forms spontaneously |
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How does a fluorescent microscope work? |
1) Bacterial cell illuminated with light at wavelength that excites GFP chromophore 2) GFP molecule emits light at longer wavelength (light scatters in all direction and microscope picks up some) 3) Dichroic mirror reflects only wavelength desired of the emitted light 4) Goes through camera to viewer |
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What are the draw back of using standard (wide-field) fluorescent microscopes? |
You lose positional information in the Y-axis (lose visual depth) High degrees of scattering and background fluorescence from molecules both above and below the focal plane bur the image |
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What are the solutions to the draw backs/limitations of using a standard (wide-field) fluorescent microscope? |
1) Use a confocal microscopy 2) Use a TIRF microscopy |
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What is a confocal microscopy? |
AKA Confocal laser scanning microscopy Both excitation light and emitted light are focused together (lower the amount of light at specific spot) Helps visualize cells in 3D by adjusting plane of focus at several intervals, merging the stack of images to build a 3D model Allows observation of live microbes in real time |
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What is TIRF microscopy? |
AKA Total Interal Reflection Fluorescence Micrscopy Relies on phenomenon that occurs when light reflects inside of a prism When angel of light hits prism wall is very small, 100% of light is reflected away However, at point of impact, tiny electromagnetic wave called "evanescent wave" is produced Unlike normal light, this wave decays rapidly (exponentially) This wave can only penetrate into liquid above prism to a depth of 100 nanometers which reduced background fluorescence |
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What is a limitation of TIRF microscopy? |
You can only look at the part of the cell closest to the coverslip |
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TRUE or FALSE: MreB and elongase complex make helical filaments |
FALSE MreB and elongase complex do NOT make helical filaments |
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What is AmiC in the divisome complex? |
A autolysin (self lysin) A cell-wall lytic amidase (cleaves bond between glutamic acid and wall peptide) |
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Why would the divisome include an enzyme that destroys cell walls like AmiC? |
Of the peptidoglycan wall, new bands are being built underneath and being attached to the wall where the amylase would come along and cut a bond and add in a new stand to the old peptidoglycan strand This way, the wall will never be broken |
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Is bacterial adaptive responses reversible? |
Yes, they go away some time after the stress goes away |
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What is a gene? |
A stretch of DNA in a genome that encodes a discrete and individual protein or RNA |
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Can bacterial do splicing? |
There are currently no found examples of "alternative splicing" or "spliceosomes" in bacteria |
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What is an operon? |
A unit of genetic material that functions in a coordinated manner by means of an operator, a promoter and one or more structural genes In an operon, genes can be overlapping, adjacent or have spaces between each gene |
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What does it mean if a gene is monocistronic? |
The mRNA of gene contains a single RNA that codes for a single protein |
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What does it mean if a gene is polycistronic? |
The mRNA of a gene contains a single RNA that codes for multiple proteins (like 3) |
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What is a regulon? |
Operons around chromosome that share regulation Example in image) The "pink" protein regulon consists of 5 genes, two monocistronic and a cluster of three polycystronic |
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What is a promoter? |
Regions of DNA that control the transcription of adjacent genes by binding RNA polymerase to initiate transcription of an adjacent gene |
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In a typical E. coli promoter, where will the Pribnow sequence (TATAAT box) be found? |
-10 |
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In a typical E. coli promoter, where will the UP element be found? |
Rich in AT-base pairs found approximately at -40 to -60 but can vary in position |
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Which base pairs is the UP element rich in? |
AT-base pairs |
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What are the consensus sequences of typical promoters from E. coli? |
-35 and -10 sequences |
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Why is the recA promoter a strong promoter compared to other promoters? |
It largely adheres to the consensus sequence, lacking only a single base and having a slightly sub-optimal spacing between the -35 and -10 sequences |
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How is the araBAD promoter a weak promoter? |
It does not adhere well to either the -10 or -35 consensus motifs and has sub-optimal spacing The promoter has more mismatches than the natural/consensus |
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What is the araBAD promoter controlling? |
The arabinose utilization operon |
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If a researcher increases the space between the -35 and -10 of a bacterial promoter, would this make it a stronger or weaker promoter? |
Weaker |
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If a promoter has many mismatches compared to the consensus promoter of bacteria, would it likely increase or decrease the strength/frequency of the promoter? |
Most likely make promoter weaker |
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Why aren't all bacterial promoters optimal? |
Because you do not want to be transcribing all the time as well as some genes need to be transcribed a lot and some not so much as well as we want some genes to be off/on till some other factor come along |
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What is the alpha subunit of bacterial RNAP (RNA Polymerase)? |
Two identical alpha subunits per holoenzyme with each alpha subunit have two distinct domains: 1) N-terminal domain (NTD) which interacts with RNAP via beta and beta prime subunits 2) C-terminal domain (CTD) which interacts with the up sequence on the promoter of DNA |
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What is the NTD of the alpha subunit from bacterial RNAP? |
N-terminal domain which interacts with RNAP via beta and beta prime subunits |
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What is the CTD of the alpha subunit from bacterial RNAP? |
C-terminal domain that interacts with the upstream sequence of the promoter of DNA |
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What is the beta and beta prime subunits of bacterial RNAP? |
Largest distinct subunits Carry out actual catalytic reaction, reading DNA into an RNA transcription |
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What is the omega subunit of bacterial RNAP? |
Help complex subunits assembly, specifically beta prime Little (if any) role in transcription |
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What is the sigma subunit of the bacterial RNAP? |
Main recognition part for promoter sequences -35 and -10 regions Responsible for correctly targeting RNAP to correct sequences on the chromosome |
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What are the steps in bacterial transcription? |
1) Promoter recognition 2) Isomerization 3) Initiation 4) Promoter escape and elongation |
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What happens during the first step of bacterial transcription: promoter recognition? |
RNAP binds to promoter to generate "closed complex" (driven by affinity of RNAP holoenzyme for its promoter sequence)
Transcription cannot occur from this point because DNA is still in double-stranded conformation |
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What happens during the second step of bacterial transcription, isomerization? |
Promoter is unwound near the -10 sequence to expose a region of ssDNA from approximately -12 to +2 Facilitated by action of sigma factor
Goes from closed complex to an open complex |
|
What happens during the third step of bacterial transcription, initiation? |
Promoter is unwound near -10 sequence to expose a region of ssDNA First few bases are transcribed RNAP still trapped at promoter and "abortive" cycling of transcription can occur where small transcripts of less than 10 base pairs are made and RNAP never leaves the promoter |
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What happens during the fourth step of bacterial transcription, promoter escape and elongation? |
Major conformational change can occur at this point RNAP escapes the promoter and transcribes into adjacent gene RNA leaves sigma factor behind RNAP is now an "elongation" complex where it will transcribe the whole gene until it encounters specific termination signals downstream |
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The rate at which forward progression through the pathway of RNAP transcription depend on what? |
How well the RNAP binds the promoter to form the initial closed complex How easily the RNAP melts the DNA to form the open complex How easily the RNAP can escape from the promoter and form the elongation complex |
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Why would bacterial RNAP have different "alternative" sigma factors? |
Each sigma factor would have an optimal target for a unique promoter consensus sequence This allows detection of different types of promoters |
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What is sigma D/RpoD/sigma 70? |
The "housekeeping" sigma factor or also called as primary sigma factor Transcribes most genes in growing cells |
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What is sigma S/RpoS/sigma 38? |
Starvation/stationary phase sigma factor To save energy |
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What is sigma H/RpoH/sigma 32? |
Heat shock sigma factor Turned on when exposed to heat to protect again the heat |
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What is sigma 28/RpoF? |
The flagellar sigma factor |
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What is sigma 24/RpoE? |
Extracytoplasmic/extreme heat stress sigma factor |
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What is sigma 19/FecI? |
Ferric citrate sigma factor Regulates the fec gene for iron transport |
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What is sigma 54/RpoN? |
The nitrogen-limitation sigma factor Is unrelated in sequence to other sigma factors and requires additional activating proteins to help initiate transcription |
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Sequence specific DNA binding proteins typically target which groove? |
Typically target the major groove where there is a cavity wide enough to accommodate an alpha helix and where more DNA hydrogen bonds are exposed |
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What is Class I activated promoters? |
DNA binding proteins target specific sequences upstream of the promoter and help recruit RNAP to a non-optimal promoter sequence by binding the C-terminal domain of the RNAP alpha subunit (Green circles in image) |
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What is Class II activated proteins? |
Activating protein binds a region just upstream of the -35 box where it can make contact with domain 4 of the sigma subunit |
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How does protein-induced conformation change in DNA help activation of RNAP? |
Some promoters bind RNAP poorly because sequences not oriented or spaced properly Activating proteins enhance transcription by bending DNA to improve ability of RNAP to bind these sub-optimal promoters |
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What is simple repression of RNAP? |
Blocking RNAP from binding to its promoter, causing transcription to be repressed
Repressors bind "operator" sequences that lie within the promoter, blocking access of RNAP |
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How does generating looped DNA cause simple repression of RNAP? |
Some repressors trap promoter DNA into a loop, preventing RNAP binding or traps RNAP into a complex that cannot escape the promoter |
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If you have a depletion of nucleotides and ATP & energy, can you transcribe genes? |
No, those are what are required to do transcription |
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What does the LacI gene do? |
Makes a repressor where if bound to a promoter for LacZYA, it shuts that gene off |
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What does gene LacZ do? |
Produces beta-galactosidase |
|
What does the gene LacY do? |
Produce Lactose Permease |
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How does lactose get into the bacterial cell? |
Lactose gets in by Lactose Permease |
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What causes the repressor for LacZYA (LacI) to unbind to the DNA? |
Once lactose is in the cell, it will bind to LacI, causing conformational change, releasing to from the bound DNA |
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What is the "Two-component" system? |
Most common way in which bacteria transmit signals from outside world to cytoplasm Most abundant multi-step signaling pathways found in nature (over 70,000 two-component systems have been identified from genome sequences)
They have domains that can often be swapped to generate new signaling molecules |
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In a broad, simple two-component system, what is a histidine kinase (HK)? |
Sensor of environmental signal Transfer a phosphate from ATP to response regulator Almost always found as a homo-dimer |
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In a broad, simple two-component system, what is a response regulator (RR)? |
Molecule that controls the response output Often the RR is a DNA binding protein that changes its affinity for DNA once it is phosphorylated on an aspartic acid by the HK |
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Where would you find a histidine kinase sensor residue? |
You would find it on the inner membrane of bacteria |
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What are the steps in environmental signals are transduced through phopshotransfer or phosphorelay? |
1) Signal detected by periplasmic domain of histidine/sensor kinase resulting in conformational change of protein complex 2) Newly activated kinase domain undergoes "autophosphorylation" where it transfer terminal phosphate from ATP to a conserved histidine residue in its catalytic domain (causing it to become activated) 3) Histidine kinase domain transfer phosphate from its conserved histidine residue to an aspartic acid residue in the response regulator, resulting in conformational change of the response regulator, altering its activity 4) Response regulator is now able to bind to DNA and active/repress transcription 5) Reverse. When environment change, some HKs can cat as phosphatase to remove phosphate from response regulator to shut down the system rapidly |
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What are the least chemically stable phosphoryl-mediated signaling? |
Phosphohistidine & Phosphoasparate |
|
What are some very chemically stable phosphoryl-mediated signaling? |
Phosphoserine & Phosphothreonine & Phosphotyrosine |
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What are phosphorelay systems? |
Systems that bounce phosphate between Histidine (H) and Aspartic Acid (D) residues Allows more flexibility, allowing you to get modulated responses compared to two-component systems |
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What are periplasmic sensing histidine kinases? |
Sense stimuli in the periplasm or outside world
|
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What are transmembrane sensing histidine kinase? |
Sense stimuli in the inner membrane |
|
What are cytoplasmic sensing histidine kinases? |
Sense cytoplasmic stimuli |
|
What is the ArcAB system? |
Uses phosphorelay to respond to oxygen levels and regulate gene expression
Repression of enzymes in aerobic metabolism, activation of enzymes in anaerobic metabolism |
|
ArcB protein is specifically sensitive to what? |
Redox-state of the quinone pool |
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If there is a lot of electron flux going on, what will happen to the quinone pool for ArcB? |
Buildup of oxidized quinones |
|
When will the disulfide bonds be oxidized in ArcB protein? |
When there is a build up of oxidized quinones |
|
When will you find active ArcB protein? |
During anaerobic conditions when quinones are maximally reduced (no oxygen to give electrons to) When disulfide bonds are reduced |
|
What is polymyxin B? |
Produced by a gram-positive bacteria Can damage the outer membrane of gram-negative bacteria, killing them Can poke holes in membrane (causing gradient dissolution) or go through it (binding to intercellular molecules) The antimicrobial peptide interact with cell surface and have a preference for negatively charged membranes |
|
What is the basis for cross protection in bacteria? |
Generally cross protection occurs when protection against two apparently unrelated stresses utilize similar or identical defense systems |
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The adaptive response in bacteria to polymyxin B is dependent on what? |
PhoP and PhoQ (still less than a 50% survival rate though) |
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What bacterial treatments increase the survival rate of bacteria against polymyxin B? |
Wild type pretreated cells in low magnesium buffer 30 minutes before adding polymyxin PhoP mutant pretreated cells in low magnesium buffer 30 minutes before adding polymyxin with PhoP added back onto the plasmid |
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How does the PhoP/PhoQ system sense diverse causes of membrane stress? |
PhoQ has a patch of highly negatively charged amino acids (aspartic and glutamic acid) Despite PhoQ should be repelled due to negative charged membrane, the domain is held in place by bridges formed by divalent cations (Mg2+ and Ca2+) Loss of Mg or Ca (or antimicrobial peptides) disrupts the bridge |
|
How does low pH affect the PhoP/PhoQ system? |
Low pH affects aspartic acid and glutamates affecting their charge making them lose their negative charge (becoming neutral), causing conformation change of the PhoP/PhoQ |
|
When is the PhoP/PhoQ system in a repressed state? |
When there is Mg2+ or Ca2+ between them and the membrane |
|
When is PhoP/PhoQ system in an active state? |
Loss of Magnesium/Calcium Antimicrobial peptides Low pH causing amino acids becoming neutral charge |
|
What does the PhoP/PhoQ system do? |
They regulate a variety of genes that toughen up the outer membrane of bacteria |
|
What are Pags? |
PhoP-activated genes Genes being produced when PhoP/PhoQ is active |
|
What are some Pags genes? |
Divalent metal transport LPS modification Outer membrane proteins Superoxide dismutase |
|
What are Prgs? |
PhoP-repressed genes Genes produced when PhoP/PhoQ is repressed |
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What are some Prgs genes? |
Type-3 secretion system (SPI-1) involved in invading cells Flagella |
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How does an activated PhoP/PhoQ make changes to the LPS of the outer membrane? |
Adds an extra sugar to LPS (PmrAB/aminoarabinose modification) Adds extra hydroxyl group (LpxO/addition of 2-hydroxy to myristate) Palmitodylation of 3-hydroxyl group (PagP) |
|
Does turning on PhoP/PhoQ, making changes to the LPs of the outer membrane make it stronger or weaker? |
Stronger, more resistant to insult |
|
What does aminoarabinose/PmrAB do to LPS? |
Adds an extra sugar Lessens negative charge on lipid A |
|
What does PagP mediated palmitolyation do to LPS? |
Palmitoylation of 3-hydroxyl group Increases hydrophobicity |
|
What does hydroxymyristate/LpxO do to LPS? |
Addition of 2-hydroxy to myristate Increases hydrogen bonding between adjacent lipid A units, enhancing the outer membrane's ability to resist penetration by organic ions |
|
Which system is required for Salmonella virulence? |
The PhoP/PhoQ system |
|
What is the SOD enzyme? |
Found in Salmonella, diverged from E. coli (horizontally transferred by Gifsy-2 phage) An enzyme that can deactivate free radicals Expressed in macrophages |
|
Why are phagocytic white blood cells dangerous to bacteria? |
Reactive oxygen species (peroxide, superoxide) Reactive nitrogen (nitric oxide) Antimicrobial proteins/peptides |
|
What is sodCI transcriptionally controlled by? |
The PhoP/PhoQ two-component regulatory systems |
|
What happens to sodCI when you have a PhoP-null mutation? |
Decrease in transcription of sodCI and loss of regulation |
|
What is sodCI horizontally transferred by? |
Gifsy-2 phage
|
|
Are histidine sensor kinases highly modular? |
Yes |
|
Can domains be swapped to change the specificity of signaling? |
Yes, altering target (ex. magnesium sensor with sugar metabolism action) |
|
What is a condition for domain swapping in HK and RR molecules? |
Must have associating charges (negative and positive, hydrophobic and hydrophobic etc...) |
|
What is a bacterial adaptive response to alkylation damage of DNA? |
Agents that alkylate DNA, adds methyl group to various bases and backbones Occurs spontaenously |
|
What is O6-meG? |
A damaged base that will mispair with wrong pair, causing mutations and possibly be lethal |
|
What four genes are required to repair alkylation damage? |
ada - alkB
alkA aidB |
|
What does ada gene repair? |
MPT, O6-meG and O4-meT |
|
What does alkB gene repair? |
1meA and 3meC |
|
What does alkA gene repair? |
3meA, 3meG, O2-meT, O2-meC, 7-meG, 7-meA |
|
What does aidB gene repair? |
Currently unknown |
|
What is the ada gene & protein? |
Ada protein is a DNA repair protein and transcription factor
A sacrificial stoichiometric reagent DNA repair protein which takes a methyl group from a damaged methylated-DNA base or phosphate |
|
Can ada protein turn into a transcription factor? If so, when? |
When it has a methylated N-terminal domain in the presence of damage Binds to a particular sequence at the promoter, allowing RNAP to bind more tightly (fairly weak promoter) |
|
When ada protein is acting as a transcription factor, what is N-Ada and C-Ada doing? |
N-Ada will be methylated, attaching to promoter C-Ada will interact with alpha subunit of RNA polymerase |
|
What does the methylation of the N-terminal domain of Ada do to result in strong DNA-binding? |
Methylation reduces the size of a negatively charged patch in Ada |
|
Is DNA negatively or positively charged? |
Negatively
|
|
What happens to proteins during heat shock? |
Hydrogen bonds and noncovalent bonds will be broken Denaturation/unfolding causes exposure of hydrophobic regions |
|
What are chaperones? |
Proteins in E. coli used to combat against changes in temperature/heat shock Have ability to bind to unfolded or aggregated proteins and use energy and allow them to fold properly |
|
What is the GroEL and GroES proteins? |
They form a chamber for folding proteins
They are able to enclose unfolded protein, use energy (by ATP-hydrolyses) to refold the protein using misfolds and then spit them out
They use hydrophobic surfaces, pulls apart the aggregated structure and allow them to fold back to original form |
|
Heat shock response is controlled by what alternative RNAP sigma subunit? |
Heat shock sigma factor AKA sigma H or sigma 32 |
|
When there is a number in the sigma subunit for RNAP, what does that number mean? |
The kilodolton size/measurement/mass/molecular weight |
|
What factor increases the rate of sigma 32 translation? |
Increased upon heat treatment |
|
What does FtsH in bacteria do? |
It is a protease in the membrane of E. coli which eats/degrades sigma 32 |
|
What do the chaperons DnaK and DnaJ do? |
Makes sigma 32 a better substrate for FtsH |
|
How can a whole field become infected with Anthrax? |
Doesn't spread across humans but rather through livestock like cows
Animals that are infected with Anthrax will be filled with bacteria that eat their nutrients and make spores causing the animals to die and decompose in the grass, releasing more spores |
|
Who discovered Anthrax? |
Robert Koch |
|
TRUE or FALSE: Bacterial spore formation is a stress response |
TRUE |
|
Sporulation (endospore formation) occurs primarily in gram positive or negative bacteria? |
Typically in gram-positive Rare in gram-negative |
|
In a laboratory, how can you induce sporulation? |
Sporulation can be induced by starvation for a carbon, nitrogen and/or phosphorus source Sporulation begins immediately following exponential growth |
|
Is sporulation a reproductive process? |
No When environmental conditions return to normal, one spore will transform into one vegetative cell which then undergoes cell division |
|
Are spores metabolically dormant? |
Yes |
|
What are spores highly resistant to? |
Variety of environmental insulds
UV and gamma radiation
Free radicals
High (120) and low temperature
Acid and alkali conditions
Hydrolytic enzymes
Organic solvents |
|
What allows spores to be seen with a light microscope? |
They can be seen with a light microscope due to high protein levels causing a high refractivity |
|
What is the cortex of a spore? |
Surrounds spore core and composed predominantly of peptidoglycan with different structure from that of vegetative cell wall
~50% of NAMare muramic acid lactam (MAL) ~25% of NAM carry single L-Ala therefore ~25% as many DAP residues available to participate cross-link formation in spore cortex Important for maintenance of spore core dehydration which increases spore wet-heat resistance |
|
Do all spores have an exosporium? |
No, it is an optional feature/structure |
|
What is the spore coat? |
Outmost structure common to spores of all species Consists of a species of one or more morphologically distinct layers that protect spore from variety of toxic molecules and from mechnical damage |
|
What is the core wall of a spore? |
Immediately underlying the cortex is a second layer of peptidoglycn with different structure, the germ cell wall The core wall becomes the cell wall of newly growing vegetative cells |
|
Will you find teichoic acids in spores? |
No |
|
What is a spore core? |
Contains the cytoplasmic membrane, cytoplasm and nucleoid |
|
What is the core cytoplasm of the spore core? |
Highly dehydrated Contains high concentration of dipicolinic acid (DPA) and calcium which forms calcium dipicolinic acid (this associated with low water content and heat resistance) pH ~1 unit lower then growing cells |
|
Why is the core cytoplasm of spore cores highly dehydrated? |
Allows the cytoplasm to last for a long time Less chance for reactions to occur |
|
What is the spore nucleoid like in the spore core? |
Highly condensed and bound by SASPs (small acid-soluble spore proteins) which protect DNA from potential damage from UV radiation, desiccation and dry heat |
|
Why are spore nucleoid high condensed and bound by SASPs? |
To protect DNA from potential damage from UV radiation, desiccation and dry heat |
|
What is the process of sporulation like? |
1) Vegetative cell goes through vegetative cycle for growth of medial division 2) Polar division for asymmetric cell division 3) Engulment of prespore 4) Cortex development 5) Spore coat development 6 & 7) Maturation and cell lysis (lyse mother cell to release spore) 8) Germination 9) Go to step 1 |
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What is Spo0A in sporulation? |
Master regulator that turns on genes for sporulation Activates genes required for later events in sporulation Represses genes involved in stationary-phase growth A phosphorelay pathway produces Spo0A~P which initiates sporulation |
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What initiates sporulation? |
Spo0A~P |
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The switch from vegetative growth to sporulation involves a switch of what? |
Switch from sigma A to vegetative sigma factor H and F in forespore compartment of RNAP |
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Bacteria motility is driven by what? |
Surface appendages |
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What are the four type of bacteria movement/motility? |
Swim Swarm Twitch Glide |
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What is the flagella? |
Most common form of mobility in bacteria Needed for both swimming and swarming Movement in liquid media or moist surfaces Helical, threat-like appendage |
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What does the structure of flagella like? |
|
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What does the L Ring and P Ring do for the flagella? |
Anchor the flagella tail/filament to the membrane |
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Which parts of the flagella help the flagella filament/tail spin? |
The ROTOR and STRATOR |
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What does MotA/B do in flagella? |
Form proton channel |
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Where does the energy for flagella movement come from? |
Proton movement or Proton motive force
|
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If flagella is rotating clockwise, is it going forwards or backwards? |
Backwards |
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If flagella is rotating counter-clockwise, is it going forwards or backwards? |
Forwards |
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How does flagella turn direction when it has a single flagella? |
Stops moving/rotating and reorients itself |
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How does flagella turn direction when it has a bundled flagella? |
Tumbles by having flagella push apart |
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What is bacterial swarming? |
Widespread flagella-based motility Movement on solid surface with some fluid/moisture Often form spearheaded raft along advancing front Bacteria appear to be in a different state |
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How do bacteria appear to be in a different state during swarming? |
Bacteria longer in length Have more flagella (hyperflagellated) to overcome friction Bacteria move as a group (swimming is independent) Thought to be important in pathogenesis |
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What is bacterial gliding? |
Movement on dry, solid surfaces without flagella or pili Slower than flagella-based movement ~40 proteins spanning cytoplasm, inner membrane and periplasm (no clear mechanism) Engerzied by proton motive force |
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What is another name for bacterial gliding? |
Adventuours motility |
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What is bacterial twitching? |
Movement on solid surfaces Type 4 Pili acts as 'grappling hook' (extend, attach and retract) Needed for biofilm formation and signal transduction on host cell surface Tend to be rafts of cell moving outward |
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What is Type 4 Pilus? |
Mostly found in gram positive bacteria Main structure is made of a protein called Pili Movement is through sequential pulling and attaching Need ATPase to polyermize the pilli and another to retract |
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What does PiliA of Type 4 Pilus do? |
Bind inanimate objects (plastic) and host cells glycolipids |
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What does PilB of Type 4 Pilus do? |
Polymerizes PilA into growing pilus |
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What does PilTU of Type 4 Pilus do? |
Retracts pilus
|
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What does PilD of Type 4 Pilus do? |
Prepillin peptidase processes PilA by removing leader sequence |
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What is taxis? |
Bacteria direct movement based on environment |
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What is chemotaxis? |
Directed motility Towards/aways from chemicals Receptors detect external attractant concentrations Signals relayed to activate CheY which binds to motor switch |
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What is phototaxis? |
Directed motility Towards/away from light Many photoreceptors but all seem to bind chromophore co-factors which absorb visible light Electrochemical gradient and redox sensors seem involved but mechanisms unclear Bacteria have dedicated photoreceptors |
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What is aerotaxis? |
Directed motility Towards/away from oxygen Aer and Tsr receptors interact with electron transport chain Sense redox changes Similar to chemotaxis where activates CheY to control flagella motor switch |
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What is magnetotaxis? |
Directed motility Orientation in magnetic fields Some bacteria can align themselves with Earth's magnetic field Have organelles called magnetosomes containing magnetic crystals Live in transition zones between high and low oxygen |
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What is biased random walk of bacteria? |
Randomly transitions between 'runs' (CCW) and 'tumbles' to help find things to eat When there is a gradient present, their runs become longer towards that attract |
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How does a two-component system control chemotaxis? |
1) Membrane sensor for the chemical (this case repellent) 2) Protein CheW interacts with CheA 3) CheA is a kinase that will phosphoryalte CheY and CheB 4) CheY causes CW motion (default is CCW) 5) CheB demethylates sensor (CheR methylates it which increases activity) |
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What does methylation of the two-component chemotaxis system do? |
Allows memory of the system which tells if it is moving towards or away from an attractant |
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What is a magnetosome? |
Bacteria organelle for magnetotaxis Contains magnetic crystal |
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Do bacteria display ultraism and when? |
Yes during Myxococcal fruiting body where bacteria in the stems will not propagate so those on top would (they become stock cells) |
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What is a biofilm? |
Bacteria that adhere to a surface (living, dead or abiotic) through various self-generated adhesions Occur on most surfaces in nature and are the preferred mode of growth for majority of bacterial species (instead of the commonly laboratory planktonic growth) |
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What is planktonic? |
"Without a plank" Refers to bacteria free in solution, not attach to a surface Opposite of a biofilm Common laboratory bacterial growth method |
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What happens to bacteria genes when they are in a biofilm? |
They begin to switch/change gene expression, changing their phsiological state |
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Do bacteria have a choice whether they want to be biofilms or not? |
Yes, it is a choice Kinda like a social behevaiour where they coordinate their behaviour in response to a stimulus |
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Why are biofilms important? |
They are incredible hard to get rid of as they are resistant to antibiotics and disinfectants They can invade pipes, industrial/marine systems, and medical devices |
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How can we use biofilms for 'good'? |
Wastewater treatment and pollutant degradation (waste water gets passed over imbedded rocks and over microbes where it absorbs nutrients Plant and animal growth promotion Protection against pathogens by "normal flora" or "probiotic" biofilms |
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What is normal flora? |
AKA Normal microbial flora
Relatively stable with specific general populating of various body regions during particular periods in individual's life
May aid or harm host or exist as commensals |
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How would a normal flora aid a host? |
Competing for microenvironments more effectively than pathogens like Salmonella Producing nutrients host can use Block binding sites for pathogens Production of anti-microbial compounds Priming of immune response |
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How may a normal flora harm the host? |
Causing dental caries, abscesses or other infectious disease |
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What is Rhizosphere? |
Narrow region of soil that is directly influenced by root secretions and associated with soil microorganism |
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Why would a plant want a biofilm on them such as red clover root hairs? |
Bacteria feed off dead cells, proteins and sugars released by roots Bacteria fix nitrogen and make other nutrients for plants |
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How do biofilms on humans help prevent infection? |
Block binding sites for pathogens by releasing soluble factors and competition for same receptors Competition for nutrients Production of anti-microbial compounds Priming immune response |
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Why is implant contamination a huge issue? |
Once a patient gets infected from a hip infection, the biofilm willconstantly grow and are very problematic Even if cleared once, it can keep growing back |
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What are the advantages for bacteria/biofilms sticking to a surface? |
Safety in numbers Buffet benefits Commerce |
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In biofilms, how are there safety in numbers? |
Herd effect (predators might graze on your neighbour instead of you) Protection from immune cells and molecules Group detoxification of harmful substances |
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In biofilms, what are buffet benefits? |
(An advantage for sticking to surfaces) Food comes to you instead of going to food (reduce energy expenditure) Neighbour may produce food for you from more complex substrates you cannot use |
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In biofilms, what is commerce? |
(An advantage for sticking to surfaces)
Swapping of DNA fragments and replicons (plasmids, transposons, bacteriophages)
Stimulate further biofilm formation |
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What are the disadvantages for bacteria to form a biofilm? |
Small town syndrome Famine Bad neighbourhoods |
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For bacteria in biofilms, what is Small town syndrome? |
Progeny need to leave home and make their way in the world to acquire nutrients elsewhere Disadvantage for bacteria to form biofilms |
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For bacteria in biofilms, how can famine happen? |
Food stop going by certain bacteria which can cause the bacteria to eat its neighbours Disadvantage for bacteria to form biofilms |
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For bacteria in biofilms, how can there be bad neighbourhoods? |
Some bacteria might attract undesirable elements (such as immune cells) into a territory Disadvantage for bacteria to form biofilms |
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Are biofilms reproducible? |
Yes under a given set of conditions but it all depends on both genetic and environmental factors |
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Are mushrooms and channels the only arrangements biofilms can form? |
No, other arrangements are possible
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Is it possible for a biofilms to contain more than one bacterial species? |
They often do
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Do biofilms have circulatory systems? |
Many biofilm structures contain water channels that permit convective flow of liquids through the biofilms This shows there is an active exchange of nutrients from external environment |
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TRUE or FALSE: Biofilms are elastic |
TRUE Clusters are quite elastic with intentional contraction and extension |
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What is the substratum of biofilms? |
Underlying layer or substance beneath the surface
|
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How does the surface and the substratum of biofilms differ? |
Decreased oxygen and pH near substratum Growth rate different in each area Gene expression changes |
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How does oxygen levels change in biofilms? |
It decreases near the substratum |
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How does the pH change in biofilms? |
It decreased near the substratum
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How are growth rate different in each area of the biofilms? |
Often lower average growth rate or none at all in some areas |
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What has a higher overall growth yield, planktonic or biofilm bacterial cultures? |
Biofilms Reach a higher cell density/unit volume than planktonic |
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What is going on in this biofilm? |
This image shows how gene expression changes in biofilms and is not uniform Specifically what is going on is a two component regulatory system that recognizes phosphate where the active cell turns on alkaline phosphatase which can chop off phosphate from other molecules in the environment and use it for themselves (yellow ones expressing the substrate) |
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What are some methods to study biofilms? |
Robbins device
Chemostats Flow cells Static systems |
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What is the biofilm study technique, Robbins Device? |
Microbial culture flow through a tubular section made of glass, metal or clear plastic containing removable studs that permits study of various materials in same system |
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What is the biofilm study technique, Flow cells? |
Spying on bacteria growing on a slide affixed to a chamber where fluid (sterial media) flow through |
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How is Static Biofilm Assay done? |
1) Overnight broth culture 2) Remove broth and was unattached cells 3) Stain cells with crystal violet 4) Solubilize crystal violet (by alcohol or organic solvent) and read optical density |
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Why would you remove broth and wash unattached cells in a Static Biofilm Assay? |
When washed out, the platonic bacteria gets washed out but the biofilm is stuck
|
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Here are the results of a static biofilm assay, what does these results show? |
The wild type as well as M3 and M4 can produce a biofilm |
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How are biofilms commonly formed? |
1) Initial attachment 2) Firm attachment/microcolony 3) Initial encasement of microcolony by production of expolysaccharide (EPS) production 4) Maturation 5) Detachment and disperal under right conditions |
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How bacteria attach to a surface depends on what? |
Properties of surface (made of, charge, hydrphobicity, etc...) and adhesions (flagella, pili, cell surface proteins, EPS) |
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What are bacterial "conditioning film"? |
Surfaces typically have absorbed environmental components that change their properties |
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What are the two steps of bacterial attachment? |
Dock (reversible) and Lock (irreversible)
|
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What happens if the bacteria of a biofilm have a pili mutant? |
Bacteria will accumulate on surface but will not organize themselves |
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What happens if the bacteria of a biofilm have a flagellar mutant? |
The bacteria will barely stick to surface at all |
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What happens if the bacteria of a biofilm can no longer do chemotaxis (or any taxis)? |
They will be confused Make biofilm but cannot make chemotaxis (ability to swim towardsnutrient or away from repellent) so without this, you swim in a disorientatedway |
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What happens if the bacteria of a biofilm no longer have motor function? |
They will be paralyzed as they cannot swim |
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What aspect of the flagellum is necessary for biofilm formation? |
Presence of a functional flagellum |
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What is bacterial FlhDC? |
Regulatory proteins that control flagella expression These decrease in concentration as biofilm forms |
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What is bacterial OmpR/EnvZ? |
Two-component regulatory proteins that control adhesin expression They peak in concentration as biofilm forms irreversible (lock) attachment |
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What is bacterial RcsCDB? |
Regulatory protein that control capsule production Increases and peaks at irreversible attachment and decrease after biofilm formation |
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What is the function of the exopolysaccharide? |
Cement/adherance to substratum Structural/scaffold Protection for desiccation by trapping water in Protection from predation Protection from biocides, antibiotics, antibodies and noxious substances Nutrient storage and edible scaffold |
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What are the three matrix proteins required in Vibrio cholerae biofilm formation? |
RbmA (produced first), RbmC (produced last) and Bap1 (produced second) |
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What happens to Vibrio cholerae biofilm formation if the bacteria has an inability to produce VPS? (aka VPS- bacteria) |
VPS- prevents retention of daughter cells and stops accumulation of RbmA and RbmC that blocks biofilm formation |
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What does the biofilm development of V. cholerae look like? |
1) Initial attachment occurs and RbmA accumulates on several sites on cell surface 2) Founder cell divides and RbmA continues to accumulate while Bap1 appears at cell surface interface at initial division site ensuring that daughter cell adhere to surface (lock) 3) Cells further divide and a cell cluster gradually forms, encased in a flexible envelope containing RbmC, Bap1 and VPS with the production and accumulation of RbmA 4) Mature biofilm forms as individual cell clusters expand and contact each other 5) Dissociation and dispersal |
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What appears to be the responsibility of V. cholerae Bap1? |
Ensure that daughter cells of biofilm adhere to the surface |
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TRUE or FALSE: Biofilms can survive exposure to high levels of antimicrobials that kill planktonic cells of the same strain |
TRUE |
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What does it mean that biofilms have antibiotic tolerance by Lack of Penetration/Diffusion? |
Of the antibiotic, the concentration takes a long time to hit anything relevant (they have a diffusion limitation) Their protection is enhanced if the bacteria also have antibiotic resistance genes (ex. breakdown of antibiotic) |
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What does it mean by biofilms have a diffusion limitation? |
Cells at the surface would be killed by high concentrations of antimicrobial compound while cells at bottom would be exposed to lower concentrations and survive |
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What does it mean that biofilms have antibiotic tolerance by Slow Growth effects? |
The ones on the bottom are hibernating which means you are notmaking new cell wall, new proteins, or no transcription which means drugs thattarget cell wall, proteins and transcription will not work |
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Why do the bacteria on the bottom of a biofilm have a slower growth rate than those higher up? |
They have reduced growth rate due to limiting oxygen and nutrients |
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What does it mean that biofilms have antibiotic tolerance for Phenotypic tolerance? |
Changes in gene expression induced by growth in a biofilm that result in a tolerant subpopulation (not permanent mutations) Includes many stress response genes |
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How was the Pseudomonas aeruginosa PA14 gene 45E7 found? |
A experiment to find biofilm antibiotic resistant genes used a random library of 4000 strains with different mutations caused by a transposon randomly interrupt different genes Then screen for mutants for loss of antibiotic resistant in biofilms where they found 45E7 which was more sensitive to several antibiotics while in biofilm It had an insertion in the NdvB gene |
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What is the Pseudomonas aeruginosa & B. japonicum NdvB gene? |
It encodes glucosyltransferase (links sugars to one another)
Makes cyclic-beta-(1,3)-glucans which is a circular polymer of glucose The NdvB protein sits on the inner membrane |
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What happens to a bacteria with a NdvB knockout? |
When gene is knocked out and cannot make NdvB protein, the bacteria becomes more susceptible to antibiotics Without the NdvB gene, it does not make cyclic glucans |
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What is the Tobramycin killing assay? |
Fractions eluted from columns preloaded with glucan/no glucan Tobramycin will be trapped and released by 25% acetonitrile only when glucan is present |
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Will bacteria that have the NdvB gene express that gene when in planktonic growth? |
No, only in biofilm growth
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How do the glucans produced by the NdvB gene produced antibiotic resistant to Tobramycin (Tb)? |
The diffusion of Tb into the cytoplasm may be slowed by the glucans produced by NdvB The decreased diffusion of Tb may allow the biofilm cells additional time to adapt to and resist the action of the antibiotic |
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What does it mean that biofilms have antibiotic tolerance by their Persister phenotype? |
The bacteria would go into long term deep hibernation They undergo a deep unmetabolic state that makes them appear like spores and are highly resistant to different types of stress |
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What happened to the city of Caffa in 1346 that caused the spread of the Black Death? |
Caffa was a trading post between Europe and Asia At this time, there was a fight between the Italians and the Mongolians which caused the city to be put under siege The Mongolians caught the Black Death and shot their corpses over the walls of Caffa, infecting the people inside who then traveled by sea to the rest of Europe |
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What is the symptoms of the Black Death, AKA the Bubonic Plague? |
Severe malsia Headache Shaking chills Fever Pain and swelling Adenopathy Buboes (affected regional lymph nodes) Gangrene |
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What bacteria caused the Bubonic Plague? |
Yersinia pestis, a gram-negative rod closely related to Salmonella and E. coli |
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How is Bubonic plague acquired? |
By flea bites through skin |
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How is Pneumonic plague acquired? |
Caught through inhalation of air |
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What is deadlier, bubonic plague or pneumonic plague? |
Pneumonic plague |
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What is malaise? |
Feeling of general discomfort or uneasiness |
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Where does Yersinia pestis live in fleas? |
In the flea's proventriculus, the valve-like organ between the esophagus and midgut |
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How does the Yersinia pestis spread using its flea host? |
When bacteria gets into flea, builds up in esophagus and begins to starve so its bite more and more to feed itself When they suck something up, it only goes half way and spits it back out, great for transmission |
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Is the blockage of the flea's proventriculus necessary for bacterial infection? |
No, but it increases the rate of the flea's biting |
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What is quorum sensing? |
Bacterial cell-to-cell communication using small freely disffusible small molecules called "autoinducers" where they would activate transcription Also a way of telling how many bacteria and what type are present in the immediate environment |
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What does quorum sensing do to gene expression? |
How the bacteria to switch between two distinct gene expression programs:
One that is favored at low-cell-density for individual, asocial behaviour Another that is favored at high-cell-density for social, group behaviour and start producing goods called common groups |
|
What is luciferase? |
Produce bacterial luminescence Involves oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (FMNH2) with the liberation of excess free energy in the form of a blue-green light at 490nm |
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Why does the Juvenile bobtail squid do bioluminescence? |
They are colonized in their light organ by Vibrio fisherii and specifically only this species and uses the light to blend in with the starry night sky (not expressed during day) to protect against predatory fish swimming underneath |
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How does the Juvenile bobtail squid do bioluminescence? |
When squid under sun, bacteria turn off the gene that turns on light as it would be wasteful and in morning, the bacteria at its lowest count Throughout the day, the bacteria are dividing in the light organ, fed by amino acids provided by the squid At high concentrations of bacteria, enough autoinducers have accumulated in the light organ to trigger bacterial synthesis of luciferase/luciferin and this would produce a blue-green light out of the light organ shinning downwards In the morning, approximately 95% of the bacteria culture will be expelled from the light organ and process begins again |
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Which biological autoinducer has a Boron in it? |
An AI-2 autoinducer such as S-THMF-bacteria |
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What is quorum sensing like in gram-negative bacteria that involve LuxI-like and LuxR-like enzymes? |
LuxI would secret an AI-1 molecule, a large hydrophobic and small molecule that is able to get across porins and inner membrane and can get into the cytoplasm Once AI-1 in cytoplasm, it will bind to LuxR which is a DNA binding protein The LuxR-AI-1 complex would activate or repress target gene expression The LuxR-AI-1 complex would also lead to synthesis of more LuxI for more inducers |
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In the LuxI and LuxR quorum sensing system, what is the purpose of the rapid amplification of autoinducers? |
To produce a quick change from very low to very high concentrations of autoinducers to ensure all cells nearby trigger at nearly the same time |
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What is LuxI? |
An quorum sensing enzyme that produce AI-1 |
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What is LuxM? |
A quorum sensing enzyme that produce AI-1 |
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What is LuxS? |
A quorum sensing enzyme that produce AI-2 |
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What is CqsA? |
A quorum sensing enzyme that produce CAI-1 |
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How does the quorum sensing in V. harveyi work? |
They are complex, involving three different autoinducers (AI-1, AI-2 and CAI-1) |
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What is the quorum sensing mechanism that is required in V. harveyi to inhibit LuxR? |
If there are no autoinducers being synthesized, LuxN, LuxP & Lux Q and CscQ with have phosphorylation, using ATP, its component regulatory system Which will phosphorylate LuxU Which will phosphorylate LuxO Which will produce small RNAs Which will inhibit LuxR Which allows the secretion of Type 3 secretion |
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What is the quorum sensing mechanism that is required in V. harveyi to produce Lux ABCDE genes? |
If there is AI-1 produced by LuxM, LuxN will not phosphorylate its complement system If there is AI-2 produced by LuxS, LuxP & LuxQ will not phosphorylate its complement system If there is CAI-1 produced by CqsA, CqsS will not phosphorylate its complement system With LuxN, LuxP & LuxQ and CqsS systems inactivated, LuxU and LuxO will not get phosphorylated (inactivated) This allows LuxR to inhibit Type 3 secretion and produce Lux ABCDE genes |
|
What is AHL? |
N-acyl homoserine lactone structures, an autoinducer-1 (AI-1)
Membrane permeant Specific to each pathogen Can be made by gram negative bacteria |
|
What is Furanosyl borate diester? |
An autoinducer-2 (AI-2) Membrane impermeant Conserved structure (except in Salmonella) Only biological structure that has Boron origin in its structure Made by gram negative and positive bacteria Often made by LuxS enzyme and recognized by LuzQ surface receptor |
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What molecule is needed for the formation of AHL and AI-2? |
SAM |
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In Pseudomonas aeruginosa, what happens if you have a LasI mutant? |
It forms flat, undifferentiated biofilms that unlike wild-type biofilms, are sensitive to biocide sodium dodecyl sulfate
|
|
What is the enzyme LasI? |
A quorum sensing enzyme that is a LuxI homlogue and produces AI-1 |
|
What is this image showing? |
If you have a mutant is LasI, you will not get mushroom (or upward development) formation of bacteria but if you add a purified AI-1 (what LasI makes), the mushroom formation will somewhat return |
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What is quorum interception in Salmonella? |
Salmonella lack LuxI gene and cannot make AHL but can detect multiple AHL's from other species using LuxR homologue, SdiA They can also recognize modified AI-2 via a LuxP homologue, LsrB |
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What is SdiA? |
A LuxR homlogue in Salmonella that detect other species AHL and actives several genes, one being rck (resistance to complement killing) Becomes active during the transit of Salmonella enterica through gastrointestinal tract of turtles (other species too?) |
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What is LsrB? |
A LuxP homologue in Salmonella that detect modified AI-2 Actives expression of AI-2 transporters and clear AI-2 from the medium |
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How does a "typical" gram-positive bacteria do quorum sensing? |
Mature autoinducer peptide will be transcribed and translated It will export by a membrane transporter and get detected by a histamine kinase on the inner membrane Using ATP, it will active a response receptor by phosphorylation This complex will activate or repress target genes |
|
What are some anti-QS strategies observed in bacteria? |
Metabolism of autoinducers by V. paradoxus
Degrade AHL using aiiA gene by B. subtilis Autoinducing peptides active own virulence genes but inhibit those of other S. aureus groups in S. aureus |
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How does the host cell (ex. human cells) respond to bacterial QS molecules? |
IL-8 production by lung epithelial cells IFN(gamma) production by T cells Cox-2 expression by fibroblasts Acceleration of apoptosis by macrophages and neutrophils |
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How does the upper airway epithelium do anti-QS? |
High degradation activity for C6 molecules and C4-HSL with very little diffusion |
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Are bacteria QS degradation heat sensitive? How can this be shown? |
Yes It can be shown by boiling cell lysate which inactivated QS degrading activity |
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What is a glycan? |
Compound consisting of a large number of monosaccharides linked glycosidcally |
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What enzyme is required to break down a glycan? |
Different enzymes are required to break down each bond |
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Can humans create the proteins to break down the bonds of glycan? |
Our own genome only encodes enzymes with the capacity to break down only a few glycans including lactose, maltose, and sucrose as well as monosaccharides
We rely heavily on microbes to break down complex glycans |
|
What are Bacteroides thetaiotaomicron? |
Very successful glycophile and an abundant member of our microbiota and has the capacity for digesting (human) indigestible dietary polysaccharides Can be found in the human gut and is anaerobic Has 163 homologs of two TonB-dependent outer membrane proteins (SusC and SusD) that bind and import starch |
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What are the purposes of TonB-dependent transporters and surface/perpiplasmic enzymes? |
These work to break large olgiosaccharides into simple sugars
TonB-dependent transporter SusC work in concert with starch binding lipoproteins, SusD, SusE, SusF and SusG which break down the olgiosaccharides into mono-/di-saccharides which then cross inner membrane through specific membrane transporters |
|
What is SusG? |
It is a surface enzyme that breaks large starch into smaller olgiosaccharides |
|
What are the steps of Bacteroides breakdown of olgiosacchardies into simple sugars? |
Starch binding initiated by SusD, SusE and SusF
Initial degradation carried out by SusG of large starch into smaller olgiosaccharides
Olgiosaccharides transporter into periplasm via SucC in concert with inner-membrane protein TonB
In periplasm, malto-oligosaccharides further degraded to glucose by SusA (AKA neopullulanse) and SusB (a alpha-glucosidase)
Simple sugars then imported across inner membrane by permease |
|
What is Arabinoxylan? |
A heteropolymer complex carbohydrate with multiple monosaccharides and glycosidic linkages Each alpha/beta linkages must be broken by different enzymes |
|
Where is most of the glycan breakdown taken place in the human body? |
In the large intestine |
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What has faster transit of materials; thin mucous layer or thick? Why? |
Thin Thick has slower transit as it provides the bacteria time to digest the tough material |
|
How is the human gut like a fermenter? |
Our intestines are devoid of oxygen and the microbes that live in our gut are all capable of living anaerobically Many of the gut bacterial species are strict anaerobes and will die in the presence of oxygen Because we lack oxygen, the microbes lack the capacity to break down short fatty acids into anything smaller where the bacteria would then secret the byproducts of fermentation (which our tissues utilize) |
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What is the small intestine like for nutrient digestion? |
Small intestine is where simple and easy to digest nutrients get absorbed into blood stream |
|
Where would you find colonocytes? |
The cells that line the intestine |
|
What is the primary energy source for colonocytes? |
Butyrate
|
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What happens if we allow oxygen into out gut? |
Some bacteria will die Other bacteria will convert sugars, using the oxygen, into carbon dioxide which means they will use all the energy from fermentation for themselves and leave none for us |
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What happens when you starve the bacteria in our gut and how would you starve them? |
Prolong periods of simple sugars and fat diets can cause the gut microbes to starve When they starve, this can result in loss of diversity in the gut microbiota |
|
How is our microbiota established? |
Before birth, we are stile, protected by the amniotic sac At birth, we first encounter bacteria as we pass through the vaginal canal which habors many types of commensal bacteria As we eat complex food and grow, we develop a microbiota and stabilize our microbiota after the age of 3 |
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What does breast milk apparently do for a baby's microbiota? |
Break milk contain several polysaccharides that our bodies cannot break down This suggests that breast milk is designed to help specific bacteria colonize the infant gut Breast-fed have more total proteobacteria and bacteriodetes than formula fed which only have more firmicutes and no bacteriodetes |
|
Why are bacteriodetes important for the gut microbiota? |
They break down glycan |
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What is the purpose of germ-free Gnotobiotic mice in relation to research into the microbiota? |
Take gut microbiota from people with different conditions, transfer them into mice and observe physiological effects the microbes produce |
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Is there a link between obesity and the microbiome? |
Yes, an obesity-associated gut microbiome have increased capcity for energy harvest |
|
What happens if you put the microbiome of a lean mice into a cohouse obese mice? |
The cohouse mice will begin to lose weight (this suggests transferable property and changeable phenotype) |
|
TRUE or FALSE The microbiota have no association with pathology |
FALSE Changes in the microbiota can be associated with pathology |
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What is Clostridium difficile? |
A gram-positive spore-forming anaerobe Highly virulent toxin-producing strains that have emerged, causing debilitating and sometimes lethal intestinal infections NOTE: There are other Clostridium in the human gut that are safe for us |
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What is the number one risk-factor for C. difficile disease is what? |
Antibiotic use |
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How is antibiotic use the number one risk factor for C. difficile disease? |
Antibiotics are wiping out protective microbes in our gut, enabling C. difficile to invade and establish a niche in our gut Normally, the gut microbiota will be able to outcompete the C. difficle but due to the antibiotics, the C. difficile population can rapidly expand and produce a lot of toxins |
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What appears to be wrong with this colon? |
It appears to have a C. difficile infection |
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What is one method for treating C. difficile infections? |
Fecal transplantation |
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What is fecal transplantation? |
Taking poop from a healthy individual and delivering it by mouth or anus route to the individual's infected with C. difficile |
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How does the liver fight off C. difficile growth? |
The liver produces cholic acid which gets convereted into deoxycholic acid by Clostridium scindens C. difficile are very sensitive and grows very poorly in the presence of deoxycholic acid which can end up stopping C. diff vegetative growth |
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What are some common methods that bacteria acquire new genes naturally? |
Transformation/uptake of free DNA Conjugation/mobile plasmids Transduction/phages |
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In bacterial conjugation, which bacteria is considered the "male" bacteria? |
The one that sends out the pilus to conjugate |
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In the genetic exchange in prokaryotes, how does transformation occur? |
Donor bacterium will go through cell lysis Competent recipient bacterium takes up DNA fragment Recombination occurs |
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In the gene exchange in prokaryotes, how does transduction occur? |
Donor bacterium will get infected by bacteriophage New phages contain phage genetic bacterial and bacterial DNA Bacterial DNA fragment injected into receipient bacterium by abnormal phage Recombination occurs |
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In the gene exchange in prokaryotes, how does conjugation occur? |
Donor bacterium + recipient bacterium will have a direct transfer of DNA by pilus Seperating of the mating bacteria will then occur Recombination |
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What was Griffith's 1928 experiment? |
Inserted live S strain (capsuled bacteria) into mice, it died Inserted Live R (no capsule) strain into mice, it lived Inserted heat killed S strain into mice, it died Inserted heat killed S strain with live R strain into mice, it lived Showed the transformation principle |
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Give a summary of Avery's transforming principle experiment? |
DNA alone changed R to S cells and this effect was lost when extract was treated with deoxyribonuclease DNA carried the genetic information required for R to S conversion or transformation |
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Can all bacteria take up DNA? |
No, only competent bacteria can (which can be natural or chemically modified to allow it) |
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What is genetic transformation? |
A process by which free DNA is incorporated into a recipient cell and brings about genetic change |
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What is a competent cell? |
A cell that is able to take up a molecule of DNA and be transformed
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What is natural competence? |
Genetically programmed physiological state permitting the efficient uptake of macromolecular DNA
Can be gram positive or negative |
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What is artifically-induced competence? |
State of competence achived by chemical or physical treatment of the cell
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Are gram positive bacteria's competence regulated by something? |
Gram positive bacteria are regulated by growth phase to enter state of competence They become competent later in their life cycle Quorum sensing can help them be competent |
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Why does quorum sensing have an impact on gram positive state of competence? |
The cells are growing well, they believe it would be good time to exchange DNA
A way to exchange DNA with their own species, because picking up DNA from different species can be dangerous whereas picking DNA from your own kind is safer
Can be wasteful if they exchange DNA in an environment where there's not enough bacteria around |
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What is quorum sensing-based control of competence like in Streptococcus pneumoniae? |
Pheromone precursor (pre-CSP) is encoded by ComC and processed and secreted by action of ComAB proteins This pheromone detected by histidine kinase ComD, leading to phosphorylation of ComE Phosphorylated ComE drives transcription of ComeCDE and ComAB operons as well as ComX ComX is minor sigma factor that appears to active transcription of late competence proteins which includes proteins involved in taking up DNA, RecA and negative regulatory protein that eventually turns off expression of Com operons |
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In the quorum sensing-based control of competence in Streptococcus pneumoniae, what is the function of ComAB? |
Cleave the pre- off of pre-CSP and secrete CSP |
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In the quorum sensing-based control of competence in Streptococcus pneumoniae, what is the function of ComD? |
A histidine kinase pheromone (CSP) detector that phosphorylates ComE when comes in contact with its pheromone |
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In the quorum sensing-based control of competence in Streptococcus pneumoniae, what is the function of ComE? |
When phosphorylated by ComD, it drives transcription of ComCDE, ComAB and ComX operons |
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In the quorum sensing-based control of competence in Streptococcus pneumoniae, what is the function of ComX? |
ComX is a minor sigma factor that appears to active the transcription of late competence proteins including proteins involved in taking up DNA, RecA and negative regulatory protein that eventually turns off expression of Com operons |
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What is the gram-positive competence system composed of? |
A pseudopilus encoded by ComG operon that is related but not identical to Type-IV pilus (Type-2 secretion system) A NucA nuclease A ComEA DNA binding protein A ComFA ATPase helicase/motor protein A ComEC transmembrane pore |
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How does DNA get into gram positive bacteria? |
DNA gains access to ComEA protein by pseudopilus NucA nuclease introduces double stranded breaks, allowing free end to enter the cell Enters cell through ComEC channel ComFA protein is an ATPase that may act as a helicase to unwind DNA so DNA enters cell single stranded (entering by 3' end) where ssDNA bind to SSBP 5' end of incoming DNA chopped into nucleotides RecA works a recombination protein, binding to ssDNA |
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What is required on gram-negative bacteria for efficient transformation to occur? |
The incoming DNA to have a sequence called DUS (DNA-Uptake Sequences) |
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Do bacteria have biases when picking up DNA? |
Gram positive no Gram negative yes |
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How does DNA get uptake into gram-negative bacteria? |
DNA containing DUS sequence is bound to known receptor on cell surface called DR
DNA passed through the PilQ-secretion complex ComE, a periplasmic protein delivers DNA to ComA DNA degraded to ssDNA before being passed through ComA channel ssDNA binds to SSBP then RecA for recombination |
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Does DNA uptake/import require energy? |
ATP yes as it generates significant force (Bacteria like B. subtilis has an uptake rate of 80 +- 10 bp/s) |
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What are two commons methods of artificially-induced competence? |
Calcium-induced competence Electroporation |
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What is calcium-induced competence? |
E. coli treated with high concentrations of calcium ions and then stored in the cold so become transformable at low efficiency
No specific DNA sequences required (although DNA methylation pattern will affect efficiency)
Simple reagents, no special equipment
Less than 10% of cells typically become competent |
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What is electroporation? |
An artificially-induced competence method that uses pulsed electric fields to produce small, transient pores in lipid membranes
When DNA present outside the cell during electric pulse, they can enter through pores
Require sophisticated power supply which allows pulse current, duration and electrical resistance to be carefully controlled
Works with most species with ss- or ds-DNA
Approximately 95% of cells become competent but high death rate |
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TRUE or FALSE and explain DNA can be used as a nutrient by competence |
TRUE Experiment done by Finkel and Kolter showed that when bacteria (wt that can do competence, and mutant that cannot) are put in a environment where DNA is their own food source, the wildtype will be able survive longer Putting same bacteria in environment with no DNA, they both do not survive well |
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What are these experimental results showing? |
Young biofilms are held together with DNA and will disperse if treated with DNase Older biofilms are DNase resistant |
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Why does ComGB mutant produce less/no biofilms? |
This is because it lost its competency due to the mutation of the pseudopilus involved in competence |
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The fact that DNase treatment does not lower biofilms of ComGB mutants any further than their already lower levels, what does this suggest? |
DNA is not involved in the small amount of biofilm made by ComGB mutants
In other words, if you do not have a pseudopilus, it does not matter if DNase is there or not |
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What does ComGB do? |
ComGB is a late competence protein required for DNA binding and uptake |
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What are plasmids? |
Small, circular (usually), supercoiled DNA molecules
Present is more species but not all strains May have ~1 to 1000 copies per cell Uses replication machinery of the host Have own replication origin and are autonomously replicated and stably inherited Dispensable for growth under many conditions NOTE: There are exceptions to all of the above |
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What are some common strategies plasmids do to stably maintain themselves in the cell? |
Autonomous replication and copy number control (control their replication number where some plasmids can only be present in one copy in a cell)
Providing useful function for the cell such as drug resistance
Partition systems that actively segregate the plasmid to daughter cells
Addiction systems |
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What is the function of the Col plasmids (ColE1, ColE2, ColE3)? |
They make toxins that kill other E. coli cells as well as an immunity protein so the toxin does not kill its own cell |
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What is the function of the F plasmid? |
F-pilus and conjugation |
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What is plasmid "Theta" replication? |
Using an origin of replication, having either uni- or bi-directional replication to form catenates and then individual plasmids |
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What are plasmid catemers and how are the seperated? |
Catemers are products of plasmid replication where the leading and lagging strand form two rings looped into one another
Usually, these structures are resolved through action of Type 1 and 2 topoisomerase which transiently break the DNA backbone and one plasmid pass through the strands of the other plasmid |
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Does plasmid replication always require an RNA primer to start replication? |
No, sometimes it can start with a nick in the DNA so that the replication can start in one direction from that nick (5' to 3') |
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What is Rolling-circle replication? |
A plasmid replication method that goes in one direction where the branch goes in a circle The displaced strand either goes into a single strand of the plasmid or replicate into a double stranded genome |
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What is a stringent plasmid? |
Low copy number |
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What is a relaxed plasmid? |
High copy number |
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What is the ColE1-derived plasmid? |
Maintained at 10 to 20 copies per cell Consists of three different components: RNA 1, RNA 2 and Rom/Rop RNA 1 transcription is upstream of RNA 2 (and terminates at start of RNA 2) where it is ~100x higher in concentration than RNA 2 even though it is very unstable and degraded by RNase E RNA 2 needs to fold into particular secondary structure in order to be processed by RNase H which allows it to prime DNA polymerase 1 (which the secondary structure can be inhibited by RNA 1) |
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Is RNA 1 of ColE1 stable? What is it degraded by? |
It is very unstable and degraded by RNase E
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What can inhibit RNA 2 of ColE1? |
RNA 1 |
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If there is high concentration of RNA 2, will replication of ColE1 occur? |
Yes |
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If there is high concentration of RNA 1, will replication of ColE1 occur? |
No |
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What cleaves RNA 2 of ColE1? |
RNase H |
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What is the function of Rom/Rop of ColE1? |
Stabilizes the RNA1 and RNA2 complex, turning off RNA 2 |
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When will there be an accumulation of RNA 1 and Rop? |
When there is high concentration of ColE1 plasmid |
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What is the plasmid partitioning like for plasmids? |
ParR binds to ParC and pairs plasmid which form a nucleoprotein complex or partition complex
The partition complex serves as nucleation point for polymerization of ParM where ParM-ATP complex inserts at this point and push plasmids apart
The insertion leads to hydrolysis of ParM-ATP complex, leading to depolymerization of filament
At cell division, plasmids copies are at each cell extremity and will end up in future daughter cells |
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What happens when ParM is bound to ATP? |
When bound, it contacts the partitioning complex Starts hydrolyzing ATP to form filaments at the replication origin Pushing plasmid apart so they appear on the two sides of the cell and division occurs |
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Can two plasmids that use a common mechnaism for controlling plasmid replication, can they co-exist? |
No, they are incompatible |
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Give an example of a plasmid that is incompatible with pBluescript (ColE1 origin) |
pUC (pMB1-derived origin) |
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What are compatible plasmids? |
Two plasmids have different ways by which they control their copy number they do not interfere with each other |
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What are incompatible plasmids? |
If two plasmids control their copy number using the same mechanism The plasmids cannot be stably maintained in the cell with one another |
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What is plasmid addiction? |
Some plasmids encode genes that will kill the host if the plasmid is lost after cell division Typically they involve a stable toxin and an unstable anti-toxin that must continually be synthesized in order to suppress the activity of the toxin |
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What happens is a bacteria gets rid of a plasmid they are "addicted" too? |
Without the plasmid, there will be no more anti-toxin being produced and the remaining anti-toxin are stable enough to stick around, the remaining toxin will begin to kill the cell |
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What is the F plasmid "addiction"? |
Bacteria with the F plasmid will produce CcdA (inhibits CcdB and has short half-life) and CcdB (inhibits gyrase and long half-life) The CcdB is a toxin that inhibits Gyrase, an enzyme necessary to maintain proper chromosome supercoiling When F plasmid present, there is sufficient transcription of CcdA to keep CcdB from inhibiting Gyrase When F plasmid is lost, the CcdA protein is longer being synthesized and begins to degrade allow the CcdB protein to become active and inhibit gyrase, killing the cell |
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What was Lederberg and Tatum's 1946 experiment on bacteria? |
Their first experiment, they mixed two strains who couldn't grow on minimal media because couldn't make methionine or biotin, other threonine, leucine or thiamine and mixed them together and the mixed was able to grow which showed bacteria can exchange DNA Their second experiment, they put the same bacteria on different ends of a tube with a fine filter that only let small molecules through and found no colonies grew, which meant that bacteria must touch each other to exchange DNA |
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What does the F ("fertility") plasmid do? |
It is a large plasmid that causes plasmid addiction and has two origins of replication and an origin of transfer Encodes all genes necessary for transfer and to repel transfer from similar plasmids (plasmid exclusion) Encodes a transposon and some insertion sequences |
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What is the F pilus? |
Encoded by the F-plasmid They are receptor for some bacteriophages Encodes a kind of Type 4 secretion system which transfers DNA instead of proteins |
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What are the steps to conjugation? |
Contact between recipient cell and F+ cell (that has F- plasmid) is communicated to the plasmid to the origin of transfer by protein TraM OriT is nicked on one strand by "relaxase"/helicase protein TraI Cells brought into proximity by retraction of the pilus Cells make tight contact with each other by a process called mating-pair stabilization (which is not essential for transfer but does occur in liquid environments) Replication then occurs as a mechanism that is a modification of the rolling-circle replication At same time, TraI-DNA complex is shot into the recipient cell by the T4SS (5' end first) Synthesis of second strand begins in the recipient cell where the priming of the reaction may be catalyzed by TraI or by a host protein Cell separate and conjugation is done |
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What is plasmid surface exclusion? |
When a cell with one plasmid cannot make with a cell with the same plasmid |
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What is the protein TraI? |
It is a helicase (or "relaxase") that is a monomer that is covalently attached to newly liberated 5' end of DNA |
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Is the mating-pair stabilzation mandatory/essential for conjugation? |
It is not essential for transfer but does allow conjugation to occur in liquid environments It is needed if the liquid/medium is moving |
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What happens if you vortex a bacteria during conjugation? |
You will disrupt the stabialization process |
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What is the TraT protein? |
Major outer membrane component of F+ bacteria Inhibits formation of mating aggregates where the models include either competing with OmpA for binding to pilus tip or masking a region of OmpA |
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What is the TraS protein? |
Inner membrane protein that prevents trigger of donor conjugal DNA metabolism Thought to block transmission of mating signals |
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What does the TraS and TraT proteins do? |
They are surface exclusion proteins by F+ bacteria, preventing mating of F+ bacterias |
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What is the conjugation core complex structure? |
A conjugation system that spans both the outer and inner membrane that serve various functions such as including to act as a receptor for TraI-DNA complex
Includes the TraN, TraO and TraF in its core complex |
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What is TraF? |
A subunit of the conjugation core complex structure that makes contact with lipids of the outer membrane with the lipids of the outer membrane
It is not a beta-barrel structure
Spans both the inner and outer membranes simultaneously |
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What is Agrobacterium tumefaciens? |
A plant pathogen that is the cause of "crown gall disease" If bacteria encounters a wound in a plant, it colonizes it |
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What causes Crown Gall disease? |
When Agrobacterium tumefaciens enter a wound in a plant
The bacteria uses a conjugation apparatus encoded on a large conjugative plasmid (Ti) to deliver a segment of "T-DNA" into plant cells
This T-DNA is able to integrate into plant cell genome and from there, it directs the plant cell to divide (by producing hormones) to produce opines (a amino acid food for bacteria) |
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How can the Ti plasmid be used for biotechnology? |
The Ti plasmid can be modified to deliver non-toxic genes to plant cells to make transgenic crops |
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How can Ti plasmids be used to make transgenic crops? |
Gene of interest is inserted into DNA segment containing the delivery signals (oriT) of T-DNA Non-virulent strain of Agrobacterium can transfer this DNA into a plant cell, enabling manipulation of what the plant produces |
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TRUE or FALSE Phages genomes can integrate itself into bacterial genome |
TRUE |
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What are responsible for the abatement of 80% of prokaryotic heterotrophic production? |
Viral infections
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How do phages impact seasonal epidemics of cholera? |
Phages are inversely correlated with the prevenelance of environmental cholera phages When phage levels are high, cholera bacteria levels are low |
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What are three common lifestyles of phages? |
Virulent Temperate Filamentous |
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What is the phage lifestyle virulent? |
Lytic
Viruses lyse or kill their host after infection |
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What is the phage lifestyle temperate? |
Lysogenic Viruses can achieve a state where their genome replicates along with the host genome without killing their host until an opportune time In many cases these phage integrate into the host genome |
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What is the phage lifestyle filamentous? |
Phage can replicate and direct the host to shed new phage particles without causing cell death Population can keep growing after infection as it will not kill the host These look like filaments |
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What is the bacteriophage lytic cycle generally like? |
Bacteriophage attaches to the cell surface Absorption will have, attaching the phage to the bacteria Injection of the DNA from the head of the phage to the bacteria where no exposure of the DNA to extracellular environment Transcription of the phage DNA will occur at multiple stages Phage proteins are made of the phage DNA and conversion of bacterium to phage factor The bacterium would work like a factory to produce phage structures DNA get packed into new phages, assembling new phage particles Proteins will be created like holin (disrupts cytoplasmic membrane) and lysozyme (degrades peptidoglycan) to lyse the cell, releasing the phage particles |
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Can a bacteriophage release its phage particles without lysis of the cell? |
Yes, in the case of filamentous phages |
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What does the protein Holin do? |
Disrupts the cytoplasmic membrane of bacteria |
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What does the protein Lysozyme do? |
Degrade peptidoglycan of bacteria
|
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How do bacteriophages inject their genome into a bacteria? |
The bacteriophage would recognize the surface of a bacteria by using fibers that are on the tip of the tail
Begin attachment of phage to cell surface
Have tail contraction
Penetration and unplug
Inject the DNA |
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TRUE or FALSE
Phages are very specific |
TRUE
Phages that infect E. coli will unlikely infect other species regardless of how related they are
Sometimes, they can even be strain specific |
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Why are phages very specific? |
The phages recognize specific molecules on the surface like sugar, LPS, transporters or other proteins |
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What type of virus is this? |
T4 virulent bacteriophage |
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What is the T4 virulent bacteriophage? |
It is a mode for lytic phage growth Its genome is a linear dsDNA |
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How does the T4 phage inject their DNA (do infection)? |
There is a tube (needle) in the middle of the T4 that is going through the outermembrane of the bacteria While injecting the DNA, it brings the outer and inner membrane together while making a hole in the peptidoglycan by lysomes The DNA would leave the head and somehow go through the tube through the membranes |
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How do you quantify plaque-forming units (PFU) by plaque assay? |
Dilute phage solution and mix with molten top agar and bacterial suspension Pour mixture onto nutrient agar place Where ever a phage landed, it will infect a few hundred other bacteria which will cause holes in the bacterial suspension Can quantify suspension (holes/plaques) |
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In a plaque assay (with viruses), why are you sandwiching the viruses between top agar and nutrient agar? |
Prevents the viruses from moving around
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Are the gram positive tail tips of bacteriophages the same for gram negative? |
No, they are different
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How is phage lamba head assembled? |
Begins with 12 gpB
Other proteins add on such as gpNu3, gpC, and 2 groE which will begin to form the head
A terminase with DNA and gpF1 gets added on to add the DNA into the empty head
A gpD protein will then stabalize the head
A gpW protein will plug the whole of the head
Then gpF2 which is required for attached the trail that is being assembled at the same time |
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How is phage lambda tail assembled? |
The tail tip is formed using proteins gpJ, gpI, gpL, gpK, gpH, gpG and gpM
gbH is the tube/tape measuring protein The tail tube formed with protein gpV which makes the rings around gbH
Then gpU (final ring) and gpZ will be added that will bind to gpF2 of the head complex, forming the phage particle |
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In phage lambda, how many types of proteins make up the head of the phage particle? |
Only one type, gpE |
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How is the empty phage head of lambda phage made? |
After gpB-containing-primer ring is formed, a conformational change will turn on proteases (gpC) that will cut off all scaffolds in head, letting it leave the head, making it empty
The gpNu3 protein is the scaffold which will bind to the capsule (gpE) protein and help assembly of the head shape |
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How do most phages replicate? |
They replicate using the rolling circle replication, forming concatemers
|
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How are phage DNA packaged? |
The phage concatemers are cleaved into unit size by enzymes called terminases (green circles) that recognize specific packaging sequences These are loaded into phage head by portal proteins (pink box) in an energy-dependent (ATP) process Once head is full, the DNA will be cleaved by terminase protein |
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What type of lifestyle does bacteriophage lambda life? |
A temperate phage (and a model for lysogeny) |
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What is the usualy life cycle like for the temperate lambda phage? |
Begins lytic, infecting the bacteria by absoprtion and penetration Phage DNA will then cyclize and then continue lytic, or become lysogenic In lysogenic, it will integrate into DNA to form prophage and be replicated with the host (still in lysogenic), the prophage can be induced by UV live to reform cyclic phage DNA and then continue lysogenic or become lytic In lytic, it will replicate, assemble particles and lyse the cell |
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What is lysogen? |
Bacterium containing a complete set of phage genes |
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What is a prophage? |
Phage DNA in lysogens, whether integrated or not |
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What are the general properties of lysogen? |
Lysogens are resistant to re-infection by a phage of the type that first lysogenized the cell (this resistance to superinfection is called immunity) Even after many cell generations, a lysogen can initiate a lytic cycle (this is called induction where phage is excised as single DNA segment and lytic cycle begins) |
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How does a bacteriophage decide between lysis and lysogeny? |
Lysis and lysogeny are controlled by the proteins encoded by cro and cI (AKA lambda repressors) genes Lambda phage will remain in lysogenic state if cl proteins predominate, but will be transformed into the lytic cycle if cro proteins predominate |
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How does a bacteriophage re-enter the lytic mode? |
When the host DNA is damaged (like UV irradiation), cl protein may be cleaved by protease activated by RecA protein
Cleaved cl protein cannot bind to operators
Thus, Cro proteins can be produced to transform the lambda phage into lytic cycle |
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How does the lambda phage chromosome insert itself into the bacterial chromosome? |
Integrase is used to make staggered cuts and strand transfer which will insert the lambda into the bacterial chromosome |
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What protein is required for lambda phage chromosome excision from the bacterial chromosome? |
Xis |
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What is the Lysis pathway for producing a cro repressor? |
Synthesis of cro repressor from rightward transcription (PR) shuts off the cI promoter for repressor maintenance (PRM) but allows more PL and PR transcription |
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What is the purpose of lambda phage's protein, cIII? |
It stabilizes cII from being degraded
|
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How is the lysogeny pathway activates PRE? |
At high multiplicity of infection, cII protein is made from rightward transcription
cIII is also made from leftward transcription
cIII prevents degradation of cII by FtsH proteases
cII accumulates and induces synthesis of cI repressor from PRE |
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How is the lysogeny pathway activates PRM? |
cI binds
Activates promoter for cI maintenance and repressing transcription from PL and PR |
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What happens when a host bacteria, infected with phage lambda, has DNA damage? |
It induces lysogen of the phage |
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How does DNA damage induce phage lambda lysogen? |
When DNA is damaged, it turns on SOS response when ssDNA is exposed When RecA becomes activated, the phage stimulates cI repressors to cleave itself causing N-term domain to bind to DNA and C-term domain to be a dimerization domain When the cI interacts with RecA, it is cleaves and comes off the DNA, no longer having a repressor on the phage DNA Phage is now in lysogen |
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Of the lambda phage, besides working as a repressor required for lytic growth, what other functions does cI have? |
Makes cell immune to infection against other lambda phages |
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What does rexA and rexB in lambda prophage do? |
Confer resistance to other phages |
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What does the lambda prophage gene bor do? |
Increases survival of the E. coli in animal serum |
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TRUE or FALSE Prophages can help assist a bacteria to become (more) pathogenic |
TRUE |
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What genes make the E. coli O157 lethal? |
A toxin gene called Shiga Toxin (these are also found in prophages) |
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In Carynebacterium diphtheriae, what is a commonly well known prophage-encoded toxin? |
Diphtheria toxin |
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What are the evolutionary advantages to keep a huge percentage of phages in the bacterial genomes? (For the bacteria) |
These phage genomes can help make their host more adaptive and fit to the environment which will increase the survival of the host |
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What are some mechanisms that phages can use for genetic mobilization? |
Phage can randomly package wrong DNA at low frequency (generalized transduction) Phage accidentally carry genes nearby their integration site on the host chromosome (specialized transduction) Phage carry genes in their genomes that alter host physiology (ex. superoxide dismutase in prophage in Salmonella) Phage can mobilize parasitic pathogenicity islands |
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What is transduction and how does it occur in nature? |
Virus-mediated transfer of host DNA (chromosomal or plasmid) from one host cell (donor) to another (recipient) In nature, this occurs (infrequently) due to error made during phage life cycle |
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What is generalized transduction occur by bacteriophages? |
Occurs during lytic cycle of virulent or temperate phage and can transfer any part of bacterial genome Random fragments of partially degraded bacterial DNA will be packaged by mistake Generalized transducing particle contain no phage genes |
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How is an abnormal phage created for specialized transduction? |
When the phage DNA excises from the bacterial genome, once in a while (rare), it will screw up and not use the proper recombination sites to excise Instead it would excise some bacterial genome, forming a abnormal outlooping This creates low frequency of phage particles |
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What is a SaPI? |
S. aureus Pathogenicity Island They contain genes for toxins such as the "super-Antigen" that causes Toxic Shock Syndrome They encode some phage-like proteins but possess a few genes that are required to construc a phage particle |
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How do SaPI get from one cell to another? |
The SaPI1 contain sequences that enable it to be packaged by phage 80alpha while excluding the 80alpha genome during packaging at high frequency |
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What happens when a 80alpha phage infects a Staphylococcus aureus with SaPI1? |
The SaPI1 island directs its own packaging into the phage head while excluding the 80alpha genome during DNA packaging at high-frequency Many phage particles would burst out of cell filled with SaPI1 DNA instead of 80alpha, spreading the islands to new strain |
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What toxin does SaPI encode for? |
Toxis Shock Syndrome Toxin (TSST) |
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How does SaPI parasitize helper phages for their own life cycle? |
SaPI is usually reperssed by Stl When helper prophage gets induced and exists themselves from the genome to start expression genes, they will produce an enzyme called dUTPase that will inhibit Stl This allows SaPI to become activated, excising itself from the host genome and begin to replicate and create proteins that allow it to hijack phage particles This results in the particle having a normal tail but a small head |
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What is the point of the SaPI particle (or pathogenist island particle) having a small head? |
The small particle head is not big enough to hold the original phage genome but now big enough to hold the SaPI genome One way of hijacking the phage particle |
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What is TerS? |
A enzyme produced by SaPI used to hijack the head of the phage by changing the terminase DNA binding specificity It works with TerL |
|
What causes cholera? |
A gram negative bacteria called Vibrio cholerae that colonizes the small intestine
Cholera is caused by two virulence factors that was given to the bacteria by two phages: TCP and CTX |
|
What is the virulence factor TCP? |
Toxin co-regulated pilus operon
Encodes a self-binding Type-IV pilus that holds the Vibrio cells together
Also enodes a TcpF which is a secrete protein required for colonization |
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What is the virulence factor CTX? |
Cholera toxin (this causes the symptoms of cholera) A two-subunit toxin secreted from the bacteria that disrupts ion flow in epithelial cell of the intestine Encoded by prophage CTX-phi |
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What is CTX-phi? |
A filamentous phage that uses TCP as its receptor to enter the bacterial cell Its prophage encodes CTX (Cholera toxin) |
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What are some common bacterial defense systems against phages? |
Restriction-modification systems Alterations to cell surface Suicide systems Xenogeneic silencing Codon biases CRISPR Prophage encoded system |
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Briefly, how does altering the cell surface of a bacteria work as a defense against phages? |
Phages are generally very specific to species and strains that they infect Species specific diversity in LPS, peptidoglycan, etc... can change so it does not let the phage bind |
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Brielf, how does having a suicide system in bacteria work as a defense against phages? |
Form of altruism where the bacteria kills itself to prevent the phage from replicating |
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Briefly, how is xenogeneic silencing in bacteria a defense against phages? |
H-NS-like proteins silence foreign DNA |
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Briefly, how is codon biases in bacteria a defense against phages? |
Bacteria use specific codons that are different from other bacteria which prevent translation of foreign DNA |
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Briefly, how is prophage encoded systems in bacteria a defense against other phages? |
Protect against challenges with the same type of phage but sometimes phage encode secondary protection systems against other phages |
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How are restriction-modification systems a method of bacterial immunity against phages? |
Restriction-modification systems cut DNA that come from a foreign source by recognizing specific sequences They serve as powerful means of protection against phage and limit gene transfer between species |
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What is a Type 1 restriction enzyme? |
Multisubunit enzyme that recognize a given sequence but cut far away from the recognition site |
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What is a Type 3 restriction enzyme? |
Multisubunit enzyme that require two recognition sequences in opposite orientation on the same DNA molecule and cut outside the recognition sequence |
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What is a Type 2 restriction enzyme? |
Enzyme that cut within or immediately adjacent to their cognate recognition site Typically recognize a palindromic sequence |
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What is BamHI? |
A type 2 restriction enzyme from Bacillus amyloliquefaciens strain H Exists as dimer Binds to GGATCC and cuts between first and second G residues on each side of palindrome |
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How does a bacterial protect its own DNA from the restriction-modification system? |
By encoding second enzyme that selectively modifies the target sequence of restriction enzyme by methylating one of the bases in the recognition sequence This modification enzyme is called methyltransferase These enzyme genes usually adjacent to the restriction enzyme gene |
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What is CRISPR? |
Clustered Regularly Interspaced Short Palindromic Repeats It is a type of DNA repeat found in archaea prokaryotic genomes The CRISPR regions that are commonly found in bacteria have spacer regions that match sequences of phage genomes Bacteria adapt to phage infections by adding/modifying the spacer sequences similar to phage genome to prevent infection (phage resistance) |
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How is CRISPR used in bacteria for adaptation and resisting phage or foreign DNA? |
When DNA comes into the cell that the bacteria has never seen before, it will chop up pieces of the phage DNA and incorporate them into the beginning locus/spacer sequences so it has memory of that phage infection and ready to fight against it again |
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Do phages have a defense/counter against the CRISPR system? |
Yes, they have a mechanism that can inactive CRISPR |
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What is the Cas9 of the CRISPR system? |
Cas9 CRISPR System can be used to edit any genome It is a type 2 restriction enzyme and can be used to preciously edit a genome of an organism and cut at a specific 20 to 30 sequence It is highly specific and can be a method to make mutations or repress genes |
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How does Cas9 function as a nickase? (nCas9) |
Cas9 can function as a nickase (nCas9) when engineered to contain an inactivating mutation in either the HNH domain or RuvC domain active sites When nCas9 is used with two sgRNAs that recognize offset target sites in DNA, a staggered double-stranded break is created |
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How does Cas9 function as an RNA-guided DNA binding protein? |
Cas9 functions as a RNA-guided DNA binding protein when engineered to contain inactivating mutations in both of its active sites This catalytically inactive or dead Cas9 (dCas9) can mediate transcriptional down-regulation or activation, particularly when fused to activator or repressor domains |
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Is there a way to visualize Cas9? |
dCas9 can be fused to fluorescent domains such as GFP for live-cell imaging of chromosomal loci |
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What did Erwin Chargaff do? |
He experimentally determined that the AT ratios and GC ratios are roughly the same in DNA |
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Briefly, what are some problems posed by horizontal gene transfer? |
Disruption of essential genes Regulatory disruption Metabolic disruption Disruption of chromosomal packaging |
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Briefly, what does it mean that horizontal gene transfer can cause regulatory disruption? |
Results in fitness problems where genes that are supposed to be turn off are turn on (or vice versa) |
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Briefly, what does it mean that horizontal gene transfer can cause metabolic disruption? |
Even if gene is useful to help metabolize, that gene might be turned on or off at the wrong time |
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What is the H-NS nucleoid-binding protein? |
Acts almost exclusively to repress transcription Binds DNA with relatively little sequence specificity preferring AT-rich DNA The H-NS binding site is not highly sequence specific but does have a strong bias for AT rich DNA to generate extend footprints on DNA template H-NS binds weakly to DNA |
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What do the blue lines/spikes mean? |
When that gene is repressed by H-NS, the line would be longer and more negative When that gene is not repressed, the line is shorter and more positive When H-NS is removed, genes increase in expression |
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How can H-NS be used by the bacteria's horizontal gene transfer? |
H-NS can regulate large number of genes obtained by the horizontal gene transfer
Specifically the can bind and regulate newly introduced AT-rich genes |
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What does the grey, blue and orange box mean? |
Grey box is foreign pieces of DNA picked up horizontal gene transfer The blue box known as pathogenicity islands Orange box is prophages or lysogenic phages integrated into the chromosome |
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How do you do chromatin immunoprecipitation on microarray method? |
Step 1) Crosslink protein to DNA with formaldehyde (which also kills the the cell)
Step 2) Sonicate (pop cell open with sonic waves) to sheer chromosomal DNA (protein of interest remains stuck onto piece of DNA due to treatment with formaldehyde)
Step 3) Incubate protein-DNA complex with antibodies against factor
Step 4) Precipitate with protein G beads and wash extensively (these beads will bind to antibodies and hold them down so you can wash away unwanted DNA)
Step 5) Incubate at 55 degrees for 5+ hours to break crosslink
Step 6) Clean, amplify and label co-precipiated fragments
Step 7) Hybridize to array to identify the bound fragments |
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If hp0226 is a newly inserted gene into a AT/GC-neutral sequence on Salmonella from H. pylori, what is this result showing? |
Even if a gene is brand new and never seen before, the H-NS binding protein can still bind to newly introduced gene if that gene is AT-rich |
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Briefly, how can a DNA binding protein (like H-NS) tell AT-rich DNA from GC-rich or GC-neutral DNA? |
New DNA comes in, if it is AT rich compared to the host genome that is 50% GC, they know it is not host DNA because it is too AT rich, thus H-NS will shut down the expression of the foreign DNA |
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What groove does the H-NS binding protein bind to? |
The minor groove |
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What is the A-tract of DNA? |
Were it has a lot of consecutive A or T residues |
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What base pairs can dramatically expand the minor groove? |
TA steps (or adjacent pairs) can dramatically expand the minor groove and make it more flexible |
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What are GC-base pairs like in the minor groove? |
They have an extra amino group pocking out that causes steric hindrance |
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What is the H-NS DNA binding protein's domain "QGR" or "RGR"? |
A conserved peptide sequence motif This is an AT-hook motif that nestles deeply into the minor groove of the AT-rich DNA, usually sections that have a TA step or two-flanked narrow regions H-NS would be excluded from GC-rich DNA due to presence of extra amino group |
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Why is H-NS binding protein excluded from GC-rich DNA? |
Due to the presence of an extra amino group in GC pairs in the minor groove |
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How does H-NS selectively target DNA sequences derived from a foreign source? How does it not target self DNA? |
Weak binding of each individual H-NS subunit makes it like a velcro Are whole series of weak interactions (strung together like a velcro), can make a strong overall interactions A series of very weak interactions, when coupled together, it can make a stronger interaction In other words, H-NS can discriminate on the basis of AT-content and length |
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How does the H-NS binding protein discriminate on the basis of AT-content and length? |
Weak interaction by H-NS allows it to avoid interacting with DNA at accidental small patches of DNA that is AT rich Instead, it focuses on long patch of AT-rich DNA within big blocks of foreign DNA |
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What is xenogenic silencing? |
The silencing of foreign DNA in bacterial cells Characteristic GC-content of the bacterial genome can be exploited to recognize the silence alien sequences Explains how bacterial cells newly acquired genes are regulated by the bacterial cell without compromising its genomic and regulatory integrity Has allowed bacteria to simultaneously acquire and protect themselves from foreign DNA Requires minor-groove binding proteins that target structural features unique to AT-rich DNA |
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What is a mechanism that bacteria can use to counter or regulate H-NS binding proteins? |
Certain transcription factors can displace H-NS off specific genes in appropriate context, time and place These transcription factors are sequence specific to specific promoters This can be activated by an external signal or membrane stress |
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The PhoP/PhoQ system is a response to what? |
To membrane stressors that turn on a regulatory systems |
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What genes get activated by PhoP? |
Divalent metal transport LPS modifications (ugtL) Outer membrane proteins Superoxide dismutase SlyA |
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What genes does PhoP repress? |
SPI-1
Flagella |
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What is the outcome of the PhoP activation? |
The outer membrane getting toughened up |
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What does the gene sodC do and what does it get activated by? |
It fights superoxides Gets activated by PhoP |
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What does the gene ugtL do and what does it get activated by? |
Modifies outer membrane and protects against AMPs Gets activated by PhoP |
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What happens if you cause mutants in the PhoP and SlyA genes? |
When there are mutant genes in both of these components, it makes the bacteria more susceptible to killing by Polymyxin B |
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If you originally had mutants of PhoP and SlyA genes, what would happen to the susceptibility of bacteria by killing from Polymyxin B if you put back the SlyA gene? |
You restore activity of the bacteria |
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What is needed for activation and transcription of ugtL? |
Both PhoP and/or SlyA to be active SlyA cannot increase ugtL transcription itself but required H-NS to be around to help PhoP can stimulate transcription of ugtL unless H-NS is around but is low |
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How does transcription of ugtL happen? |
Usually, there will be a H-NS that blocks PhoP binding for ugtL SlyA is needed to bind to the DNA and bend it to release H-NS from ugtL Then PhoP can bind and activate transcription of ugtL |
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The is Type 4 secretion system sec-dependent or sec-independent? |
Sec-independent |
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What common features do all secretion systems have? |
Each secretion system secretes their own cargo They require energy High structural homology to other structures like flagella and pili Other require use of chaperones to keep the protein in the cargo unfolded and delivered to translocation machinery |
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When a sec secretion system is sec-dependent or sec-independent, what does the sec mean? |
Sec is a transmembrane complex in the inner membrane
Found in both gram positive and negative bacteria |
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What is the Sec transmembrane complex like? |
Conserved across all domains of life
Secretion is post-translation
Exports proteins across cytoplasmic membrane (this requires SecA and SecB)
Can insert proteins into cytoplasmic membrane (requires signal recognition particle (SRP)) |
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What is Sec61? |
Sec transmembrane complex in eukaryotes that is located on rough endoplasmic reticulum (ER) Cotranslationally pumps proteins destined for secretion to ER lumen |
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How does Sec61 function? |
It is found on the ER Protein enters the ER where it is packaged in membrane vesicles Transferred to Golgi Vesicle goes to cytoplasmic membrane Membrane fuses with cytoplasmic membrane and cargo is deposited outside |
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What does the cargo protein of a sec system's gene contain? |
Signal peptide that consists of positively charged N-terminal domain, hydrophobic domain of ~20 AA and a polar C-terminal cleavage site The its mature protein sequence |
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What is required to be conserved in the gene of proteins destined for Sec-secretion system? |
A conserved N-terminal domain called a signal sequence or "signal/leader peptide"
|
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How does the sec-secretion SRP-independent system work? |
1) Signal/leader peptide is created by the ribosome and binds to the SecB chaperone 2) SecA will then bind to the SecB-signal peptide (the SecB chpaerone prevents premature/incorrect folding 3) The SecAB complex will bring the partially folded protein to secretion apparatus SecYEG channel 4) SecB will dissociate 5) This release triggers translocation 6) SecA initiates translocation by acting like a pump, hydrolyzing ATP and pushing 2.5kDa of protein into pore 7) Hydrophobic domain and the C terminal end will start going through the SecYEG channel, pumping the protein out through the pore while hydrpohobic domain remains at membrane 8) Signal peptidase cleaves the protein and leader peptide gets degraded 9) Protein is not outside gram positive (or in periplasm of gram negative) |
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How does sec-secretion SRP-dependent system work? |
In this case, the leader peptide is typically more hydrophobic 1) Ribosome begins translating protein 2) A signal recognition particle (SRP) will bind, telling the ribosome it wants to dock 3) SRP docks onto FstY 4) GTP converted to GDP to release SRP from untranscribed protein and ribosome brings protein to translocation apparatus, SecYEG 5) Synthesize starts happening again of protein into the SecYEG channel 6) Hydrophobic domain slips into the membrane |
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What is one enzyme that cuts sec secreted proteins at the C-terminal end of the leader sequence? |
Sortease at the G of LPXTG |
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For gram negative bacteria, there is a problem for transporting sec secretion proteins and that is there is no ATP in the periplasm. How do they overcome this problem? |
They use autotransporters (Type 5 secretion)
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How do type 5 secretion systems (autotransporters) work? |
On the sec secretion protein at the C-terminal end, there would be a beta barrel domain, typically consisting of 14 beta strands This would undergo spontaneous insertion into the outer membrane to create a channel through which central passenger domain gets secreted across |
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Where does the energy for autotransproters come from? |
Comes from folding the protein from being unfolded |
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Is type 5 secretion sec dependent or independent? How come? |
Sec dependent as it requires SecYEG first to get sec secretion protein from inner membrane to periplasm
|
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What is the IgA protease autotransporter? |
Degrades IgA on mucosal surfaces
Gets made and autotransported out of bacteria cells where its passenger domain encodes for proteases that are highly specific for IgA |
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Are type 5 secretion found on gram positive or negative bacteria? |
Gram negative |
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What is chaperone/usher secretion? |
Specific chaperons (like PapD) would bind to C-terminal motifs of the sec secretion protein An 'Usher' protein (like PapC) would translocate proteins across membrane to make certain type of pilus with various subunits being brought to this translocation apparatus Assembles bottom up |
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Is the chaperone/usher secretion system sec independent or dependent? How so? |
Dependent as it uses SecYEG to get sec secretion protein to periplasm |
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Does the chaperone/usher secretion system require external energy? |
No |
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What are Uropathogenic E. coli (UPEC)? |
Causes urinary tract infections (UTI) that can progress to kidneys and are 10-20% recurrent infections in women They have two types of pilus, Type 1 and Type P |
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What is UPEC Type 1 pili? |
Infects bladder Binds to mannosylated glycoproteins: uroplakin la and lb Attachments to uroepithelial cells Causes normal E. coli to get peed out |
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What is UPEC Type P pili? |
Aka Pap Pili Attaches to kidney cells Binds glycophingolipids carrying Gal-alpha(1-4)Gal Cause urinary tract infections |
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What is type 2 secretion (General Secretory Pathway) |
Contains many different components to get cargo across the periplasm intermediate 1) Sec secretion protein gets into periplasm by SecYEG 2) PilD (stable 12-14 subunit pore, inserted on outermembrane by PulS) will process pseudopilin (PulGH) and recruit ATPase PulE on inner membrane 3) Minor pseudopilins (Pull-K) will self-assemble 4) Through using ATP (and PulC transmitting energy to outer membrane), molecules will make pseudopilin larger, pushing sec secretion protein through PilD |
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What type of secretion system is cholera toxin secreted from Vibrio cholerae using? |
Type 2 secretion system |
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What is cholera toxin? |
Secreted by Vibrio cholerae
Secreted by type 2 secretion system
Binds GM1 gangliosides on host surface and is endocytosed
Then secreted by ER to cytoplasm by Sec61
This causes efflux of chloride ions into intestinal lumen, trigger rapid fluid loss
You will die from dehydration from so much diarrhea |
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What are ATP binding cassettes? |
Largest family of bacterial transporters as they import and export Conserved across all life (example is Cystic fibrosis transmembrane receptors) Transport variety of substrates Secretion signal is structural Secretion signal is not cleaved Their ATPase has a unique ABC signature motif (LSGGQ) |
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How does Type 1 secretion (ABC transporters) export haemolysin? |
The ABC transporter will consists of HlyB, HlyD and outer membrane porin Tol C
Haemolysin grabbed by HlyB (has ATPase function) and shuttles across inner membrane to HlyD (no periplasmic intermediate, contain in HlyD)
Transports across HlyD to TolC porin which goes through to outside of cell |
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How does Type 1 secretion (ABC transporters) import of D-Ala? |
Dal-S binds to D-Ala (as a periplasmic protein) and brought to periplasmic transporter where D-Ala is imported through specific OM channels/porins ABC transporter, using ATP, brings in the D-Ala, dissociating Dal-S |
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What is a haemolysin? |
Generic term for a protein that when added to RBC, it can pop them |
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Is Type 1 (ABC transporter) sec dependent or independent? |
Sec-independent |
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What is Type 3 secretion system? |
Does direct delivery of protein (effectors) into host cell They are contact dependent (channel stays closed until makes contact with host cell) Energy-dependent secretion (ATP) Large machine wiht 23 components Structurally conserved |
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What is the basel body of a type 3 secretion like? |
Has an InvG that is an outer membrane ring that spans periplasm, contacting the inner membrane ring
Has an inner rod (like PrgJ) that may control the needle length
Two inner membrane rings (PrgH outside, PrkG inside) |
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What is the "weapon" of the type 3 secretion? |
3 proteins (2 hydrophobic and 1 hydrophilic) at the tip (SipB, SipC, and SipD) that forms a pore in other cell's plasma membrane A needle of repeating PrgL stacks with central channel |
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Do type 3 secreted effectors have an N-terminal leader peptide? |
Typically they do not but there will be a secretion signal at the N-terminal end
|
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What bacteria causes Typhoid fever? |
Salmonella enterica
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Salmonella enterica has two type what secretion systems for causing Typhoid fever and what are they for? Do they have type "this" secretion system? |
Two type 3 secretion systems needed for: Invasion into host cell AND intracellular survival They also do have more though |
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How is one of Salmonella enterica's type 3 secretion system used for invasion? |
Encoded by SPI-1 Secretes across host place membrane Causes formation of invasive ruffle where effectors cause actin rearrangements |
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How is one of Salmonella enterica's type 3 secretion system used for intracellular survival? |
Encoded by SPI-2 Secretes across host vacuolar membrane Causes formation of Salmonella induced filaments (Sifs) where the effectors manipulate endosome trafficking and fusion with SCV to form Sifs |
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Are all type 3 secretion systems bad (pathogenic)? |
No, some can be used for endosymbiotic relationships |
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What are type 4 secretion systems? |
They have many different functions (like conjugation, DNA uptake/release, effector translocation)
Effectors have C-terminal, non-cleavable secretion signals |
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What are type 4 secretion systems like? |
They have outer membrane pore formed by secretin-like protein Their periplasmic channel may require peptidoglycan hydrolase to be formed (to allow channel to go through) Could house intact translocon chamber ATPase energizes secretion Could be staging area for pilus assembly |
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How does Helicobacter pylori make you sick and what does it cause? |
Causes peptic ulcers and can lead to gastric cancer Secretes CagA to alter host signal transduction, morphology and proliferation |
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What is Type 6 secretion system? |
Present in ~25% of sequenced gram negative bacteria and common in free-living bacteria in "crowded" environments It is a contractile injfection system similar to tailed bacteriophages (that can kill other bacteria) |
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How do Type 6 secretion systems work? |
The tail tip is believed to be an effector (VgrG trimer) that is bound to Hcp which forms to hexameric rings The TssM ATPase is needed to recruit Hcp and may trigger polymierization It will recruit VipA/B The dynamic polymierzation, contraction and disassembly will occur (essentially reloaded the secretion system) |
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What are the main roles of type 6 secretion system effectors? |
Evolved VgrG proteins (effector domain fused to VgrG)
Classical toxins (secreted up Hcp channel) |
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Generalize: What are the purposes of type 6 secretion system effectors? |
Antipathogenesis (preventing triggering diseases)
Pathogenesis Intra- and inter-bacterial interactions |
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How do gram positive bacteria secrete effectors into host cells? |
(By using cytolysins) Typically it will bind very closely to host cell The Sec secretion system is scattered all over the membrane, not found in one place It will interact closely with the host cell and make a hymolysin to poke hole on the plasma membrane of the host cell and delivers the protein via Sec secretion system They don't have OM to worry about This mostly work to secrete some effectors into host cell |
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Is Sec sec-dependent? |
Yes |
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Is Type 2 secretion system sec-dependent? |
Yes |
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Is Chaperone/usher sec-dependent? |
Yes |
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Is Type 5 secretion system sec-dependent? |
Yes |
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Is Type 1 secretion system sec-dependent? |
No |
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Which secretion systems are found in gram negative bacteria? |
Type 5 Chaperone/Usher Type 2 Type 1 Type 3 Type 4 Type 6 |
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Is Type 4 secretion system sec-dependent? |
No |
|
Is Type 3 secretion system sec-dependent? |
No |
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Is Type 6 secretion system sec-dependent? |
No |
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Which secretion systems use ATP hydrolysis as an energy source? |
Sec Type 2 Type 1 Type 3 Type 4 Type 6 |
|
What are primary pathogens? |
Cause disease in healthy hosts |
|
What are opportunistic pathogens? |
Cause disease only in immunocompromised patients |
|
What is LD50? |
Define as the amount of bacteria neccessary to kill half of the infected test animals Can be affected by several independent factors including bacterial strain differences, differences between animals (mice vs hamster), conditions under which bacteria are grown and how they are administered It is NOT a fixed numner and must be reported along with how experiment was performed |
|
Is LD50 a fixed number? |
No When given in a report, experiment of how was performed will be found along with it |
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What is an infection cycle? |
The route an organism takes from one individual to another |
|
What is horizontal transmission? |
From one member of a species to another (human to human) Can occur through fomites (inanimate objects), vectors (mosquitoes), direct contact or air |
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What is vertical transmission? |
From parent to child (usually mother to offspring) |
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What is accidental transmission? |
A host who is not part of the normal infectious cycle accidentally encounters the pathogen and contracts disease |
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In a host-pathogen interaction, what is a reservoir? |
Animal, bird or insect that normally harbors the pathogen |
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What is immunopathogenesis? |
Occurs when the immune system's response to an infection damages host cells or tissues
|
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What is a virulence factor? |
A protein/gene that allows the pathogen to cause disease (can be toxic)
|
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What are exotoxins? |
Secreted protein toxins that subvert host function |
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How do exotoxins damage cellular membranes/matrices? |
These are pore forming toxins where they insert themselves into membrane of cell and form pores
Cause ions to leak out, leading to depolarization Most are not soluble because embedded into membrane with hydrophobic domains (are soluble in water though) |
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How do exotoxins inhibit protein synthesis? |
One part of the toxin goes into membrane and other goes into cell to stop protein synthesis Tends to kill the cell Usually the intention is to make the environment more suitable for the bacteria, not to kill |
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How do exotoxins activate second messenger pathways? |
They have proteins that affect second messenger pathways and signaling pathways They can also interrupt these signaling pathways and stop immune system from mounting by disrupting the signal |
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What is alpha hemolysin? |
AKA Alpha toxin
The hemolytic alpha toxin is produced by Staphylococcus aureus
Forms a transmembrane, seven-member pore in target cell membrane |
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What are AB toxins? |
Duel component (A and B) toxins that bind to host cell and have toxic activity |
|
What is the B subunit of the AB toxin? |
Binds to host cell
Dlievers A subunit to cytoplasm Often there are B subunits to form pore for A subunit entry |
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What is the A subunit of the AB toxin? |
Toxic activity Can be an ADP-ribosyltransferase: diptheria toxin or cholera toxin |
|
What is diptheria toxin? |
An ADP-ribosylating toxin
Made by gram positive bacteria, Corynebacterium diphtheriae
Encoded on a bacteriophage
Blocks ribosome function, causing cell death
Forms pseudomembrane over trachea (lethal) |
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What is cholera toxin? |
An ADP-ribosylating toxin Made by vibrio cholerae Ribosylates adenylate cyclase causing hyperactivation (producing a lot of cAMP) cAMP activates ion transport causing water to follow leading to uncontrollable diarrhea |
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What is the mechanism of cholera toxin? |
1) AB toxin complex binds to ganglioside GM1 on host membrane lipid rafts
2) Toxin endocytosed
3) Phagosome containing cholera toxin taken to endoplasmic reticulum
4) A1 subunit removed from B sununits and exported into cytoplasm
5) A1 peptide attaches an ADP ribose to amino acid within host G protein that regulates adenylate cyclase
6) cAMP levels rise and active ion transporter causing electroylte imbalance which will cause water from ceclls to follow the ions, causing diarrhea |
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What is botulism toxin? |
A clostridial toxin Produced by Clostridium botulinum On a per weight basis, most potent biological neurotoxin known A protease that inhibits the release of acetylcholine from excitory neurons to muscle cells Cause "flaccid paralysis" where muscles fail to work (no muscle tone) |
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What is tetanus toxin? |
AKA Tetanospasmin Produced by Clostridium tetani Second most potent biological neurotoxin known Binds inhibitory interneurons in spinal cord and prevents release of inhibitory neurotransmitters necessary for muscle relaxation Causes "spastic paralysis" (muscles cannot relax) |
|
What is flaccid paralysis? |
Loss of muscle contraction due to botulism |
|
What is spastic paralysis? |
Excessive muscle contraction due to tetanus |
|
How can botulinum toxin be used for medical purposes? |
It is called Botox (using botulinum toxin type A) Can be used to treat strabismus (cross eyes) Treat muscle spasms (back pain, overactive bladder, prostate issues) Can even treat excessive sweating Though the effects of the toxin last 3 to 6 months |
|
What is Anthrax toxin? |
Made by Bacillus anthracis Has two active toxins in it: 1) Edema factor (raises cAMP levels, causes fluid secretion and tissue swelling) 2) Lethal factor (cleaves protein kinases, blocks immune system from attacking) |
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How does Anthrax toxin enter the cell? |
1) Protective antigen (PA) subunit made as single peptide 2) PA binds to host cell surface where a human protease cleaves off part of Anthrax toxin receptor 3) Seven PA fragments autoassemble in the membrane to form a pore 4) Other two components of anthrax toxin (EF and LF), bind to ring and carried into cell by endocytosis 5) EF and LF are expelled through PA pore into cytoplasm |
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What was the 2001 Anthrax Attack? |
After 9/11, seven letters were sent to membranes of the American congress and selected news outlet including Tom Brokaw at NBC television The letters contained highly purified spores from Bacillus anthracis bacterium This resulted in complete halt of governmental mail survice, close of US Capitol building Total, 22 people contracted disease and five pepople died due to inhalation of anthrax spores |
|
What are endotoxins? |
AKA LPS
Made only by gram-negative bacteria
Present in LPS of outer membrane such as Lipid A (released as bacteria die, can cause sepsis) |
|
What are intracellular pathogens? |
Spend all or some of their time inside host cell during infection (can be within membrane-bound compartments or free within cytosol)
Can enter cells actively (bacterial driven) or passively (by host receptor-mediated uptake)
Speculated that most bacterial pathogens (except those that cause disease through toxin production) spend at least some portion of their infectious cycle within host cell niche |
|
What are vacuole-adapted intracellular pathogens? |
Can be within membrane-bound compartments |
|
What are cytosol-adapted intracellular pathogens? |
Can be free within cytosol |
|
What are the advantages to being an intracellular pathogen? |
Evasion of humoral immunity (antibodies, complement) Evasion of specific innate immune cells (neutrophils) Access to nutrients in host cell Can manipulate host cell signaling, and therefore the host |
|
What are the disadvantages to being an intracellular pathogen? |
Many cellular defenses to contend with Human cells have ways to detect many aspects of pathogen attack and initiate inflammatory response Need a complex strategy to get into host cells, avoid being killed, acquire nutrients and then escape (failure at any step is lethal for bacterial invader) |
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TRUE or FALSE All of the intracellular entric pathogens rely on a Type-II secretion system for the ability to reside within a host cell |
FALSE
All of the intracellular enteric pathogens rely on a TYPE-III (3) secretion system for the ability to reside within a host cell |
|
TRUE or FALSE Some non-enteric intracellular pathogens rely on Type-IV secretion for ability to reside within a host cell |
TRUE |
|
How do phagosome going through maturation by phagocytosis on a bacterium? |
NOTE: Each step, the pH inside endosome/phagosome decreases 1) Cell detects bacteria at surface and engulfs bacteria into early endosome/vacuole by endocytosis Now in Early Phagosome 2) Bunch of proteins and enzymes would bind to early endosome and a multivesicular body comes and fuses to put its internal components into the early endosome Now in Intermediate Phagosome 3) Bunch of materials interacting with bacterium as late endosome vesicle fuses and adds its components like cathepsins Now in Late Phagosome 4) Now a lysosome will fuse with the late endosome, adding in hydrolytic enzymes that can kill the bacteria and keeping it contained in the vacuole Now is a Phagolysosome |
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What are some phagosomal defenses against bacteria? Give examples of each |
Acidification (V-ATPase) ROS production (Cytochrome b558 & NADPH oxidase) Antimicrobial peptides (Defensins) Reactive nitrogen intermediate production (iNOS producing NO) Nutrient limitation by pumping out or sequestering metal ions (Lactoferrin for iron) Digestion of microbe (Proteases |
|
How is acidification a phagosomal defense against bacteria? |
Uses ATP to make protons to fill vacuole with, making it more acidic, lowing pH, inducing signaling pathways |
|
How is ROS production a phagosomal defense against bacteria? |
Produces NADPH oxidase Gets electron from NADPH to generate oxygen radicals to react with water to make hydrogen peroxide or other radicals Radicals are very reactive that can even split DNA |
|
How are antimicrobial peptides a phagosomal defense against bacteria? |
Punch holes in membrane
|
|
Why are phosphate free detergents better for lakes and streams? |
Phosphate is a limited bioavailable nutrient for bacteria If no phosphate in detergent, the algae will have a difficult time to grow |
|
Why does iron not float freely in our body? |
Iron is a rate-limiting nutrient/resource for microbial growth
A lot of bacteria are trying to uptake or steal iron form the host By withholding iron, we can limit growth of bacteria in our tissues |
|
What is Fenton chemistry? |
Using iron to generate highly toxic hydroxyl radicals and peroxides |
|
In the human body, where is Fe3+ stored? |
In intracellularly complexes with:
Ferritin bound by serum transferrin
OR
Lactoferrin at mucosal surfaces |
|
In the human body, where is Fe2+ stored? |
Fe2+ is complexed with haem, which is bound by haemoglobin within red blood cells |
|
What are three general/common iron acquisition systems do bacteria tend to use to acquire iron? |
Haem/hemoglobin uptake systems Siderophores Free iron acqusition systems |
|
What is bacterial hemolysis? |
Using hemolysins to lyse red blood cells and release the enclosed hemoglobin |
|
What is the role of hemoglobin? |
Deliver oxygen to deep tissues |
|
How do gram negative bacteria do hemoglobin uptake after hemolysis? |
Hemoglobin receptors (either Surface hemoglobin receptor or Hemophore receptors) will bind to liberated hemoglobin and takes it heme Heme will be imported into the cell through ABC transporter Heme will be then broken down by cytosolic oxygenase to liberate free iron for bacteria to use (sometimes bacteria will keep the whole heme) |
|
When bacterial hemolysis occurs, why does it sometimes look yellow? |
The beta-globin in it has only the heme product in it (same case for pee) |
|
What is enterobactin? |
AKA Enterochelin Secreted by bacteria to grab iron (or more specifically ferric iron) against its concentration gradient Extremely high affinity for ferric iron (Ka=10^52 M^-1) This high affinity allows this molecule to scavenge iron from host proteins and from environments where it is present in very low concentrations Outer membrane receptors (porin like) on bacteria will recognize this and import it into bacteria |
|
What is FepA? |
Outer membrane receptor that is similar to a porin but contains additional domain in center that plugs up hole These plug domains have high affinity for their substrate (like iron bound to enterobactin) |
|
How does TonB system/complex work? |
TonB acts as a "cork" domain that blocks an outer membrane receptor until it is engaged by its substrate When happens, TonB with ExbD and ExbB uses the proton gradient across the inner membrane as energy to drive release of the bound substrate from the outer membrane receptor The substrate will be passed onto a Periplasmic Binding Protein (PBP) which shuttles to inner membrane to allow substrate to go through ABC-transporter to cytoplasm using ATP |
|
When red blood cells are being lysed, how does the host respond? |
Haptoglobin (HP) will bind to hemoglobin and the free haem is scavanaged by haemopexin (HPX) Also, haem-binding protein, lipocalin 2 (neutrophil gelatinase-associated lipocalin or NGAL) binds and sequesters bacterial sideophores that chelate Fe3+ |
|
How does the human host counter bacterial siderophores that chelate Fe3+? |
Haem-binding protein, lipocalin 2 (neutrophil gelatinase-associated lipocalin or NGAL) binds and sequesters bacterial sideophores that chelate Fe3+ Essentially preventing siderophore that is bound to iron from being used by bacteria |
|
How does enterobactin chelate iron? |
Using its three arms to chelate iron by coordinating it between six oxygen atoms (labeled in red)
|
|
What is salmochelin? |
A specialized siderophore made by Salmonella There are different variations of this that have high affinifty for feric iron |
|
What is Salmochelin S4? |
Produced by Salmonella as specialized specific siderophore and is closely related to enterbactin
Extremely high affinifty for ferric iron Contains additional glucose molecules of two of the three arms that bind iron to prevent being bound by lipocalin-2 |
|
Why can lipocalin-2 not bind to Salmochelin S4? |
Salmochelin S4 has two glucose molecules on two of its three arms that bind iron, preventing lipocalin-2 to bind to it |
|
Why would Salmonella want to cause inflammation in the intestine upon infection? |
A consequence of causing inflammation is the host will produce large amounts of lipocalin-2 Lipocalin-2 will prevent commensal E. coli from obtaining iron but has no effect on the ability of Salmonella Therefore Salmonella can use inflammation to get iron it needs and simultaneously kill off its competitors |
|
What protein does macrophages and neutrophils use to limit iron availability in phagosomes? |
Natural resistance-associated macrophage protein 1 (NRAMP1) |
|
What is NRAMP1? |
Natural resistance-associated macrophage protein 1
Localizes to phagosomal membrane where it pumps Fe2+ and Mn2+ out of the phagosomal compartment, further reducing access to these metals
This starves bacteria of iron and mangenanese |
|
What does Borellia burgdorferi cause and how? |
It is a causative agent of Lyme disease Substitutes Mn2+ in place of Fe2+ in some of its critical metabolic enzymes For this reason, Borrelia does not require Fe2+ to infest its hosts |
|
What is nutritional immunity? |
The ability of the host to sequester critical nutrients from pathogens These nutrients can include copper, zinc, megnesium or manganese |
|
Why did the WHO gave iron supplementation to young children in third world countries and why did they have to stop? |
Over there, there was a lack of dietary iron which was linked to poor health so WHO thought iron supplementation would improve health in children in low income regions in the world Studies stopped prematurely when discovered that children receiving high doses of iron in form of supplements were at greatly increased risk of infectious diseases |
|
What happened to Malcolm Casadaban? |
Doing research on Yersinia pestis that was attenuated to have reduced/lack of iron uptake Unfortunately, Malcolm Casadaban had suffered from hereditary hemochromatosis (iron overload in blood), when he was infected with the attenuated Yersinia pestis, the bacteria was able to use the excessive blood and he died |
|
What happens when you infect a intestine with virulent (wild-type) and avirulent (mutant) Salmonella at the same time? |
The avirulent will do just as well as the virulent The virulent will create the perfect environment for the avirulent to live in Virulent will induce inflammation which enhances colonization of avirulent |
|
What is the Salmonella's metabolic paradox? |
Under aerobic conditions, Salmonella typhimurium uses ethanolamine as sole source of carbon, nitrogen and energy (one of the few bacteria that do this) This growth depends on exogenous cobalamin, a required cofactor that Salmonella cannot synthesize in presence of oxygen Under anaerobic conditions, vitamin B12 is made but Salmonella cannot use thanolamine as carbon or energy source even with alternative electron acceptors nitrate or fumarate |
|
How was the Salmonella's metabolic paradox solved? |
An anaerobic electron aceptor tetrathionate allows Salmonella to use endogenously synthesized vitamin B12 to support anaerobic degradation of ethanolamine as a sole source of nitrogen, carbon and energy The gene that encodes this tetrathionate respiration (ttr operon) is found on SPI-1 |
|
When does tetrathionate become available in the gut? |
During inflammation as the white blood cells are dumping oxygen species into intestinal lumen which is usually anaerobic |
|
What is an antibiotic? |
Substance produced by a microorganism or a similar product produced wholly (synthetic) or partially (semi-synthetic) by chemical synthesis and in low concentrations inhibits the growth of or kills microorganisms
|
|
What antibiotic is this? |
Salvarsan (Notice central Arsenic group) |
|
What is Salvarsan? |
An old treatment for syphilis Unfortunately consists of highly toxic arsenic which gave a lot of side effects |
|
What is Prontosil Red (or sulphonamido-chyrsoidin)? |
A dye for staining leather Contains sulphanilamide in it |
|
What antibiotic is this? |
Sulfanilamide (or 4-aminobenzensulfonamide) |
|
What is the antibiotic Sulfanilamide? |
Inhibits bacterial growth by interferring with synthesis of folic acid |
|
How is folic acid produced using dihydropteroate synthetase? |
|
|
Why must most microorganisms must synthesize folate de novo? |
They lack the active transport system of higher vertebrate cells that allow these organisms to use dietary folates |
|
How does Sulfanilamide interfere with folic acid? |
Sulfanilamide is a competitive inhibitor of dihydropteroate synthease Its similar structure to PABA can block active site of enzymes such as dihydropteroate synthetase |
|
Why does sulfanilamide not harm humans? |
Humans can still obtain sufficient folic acid from their diet
|
|
Even though penicillium was discovered in 1928, why was it not widely used till the early 1940s? |
Alexander Fleming could not isolate the penicillium form the fungus mold |
|
Who were able to isolate penicillium from the fungus mold? |
Florey and Chain in the 1940's |
|
What core aspect makes a antibiotic good? |
Selective toxicity (against target pathogen but not against host) LD50 to be high in MIC and low in MBC |
|
In clinical terms, what is MIC? |
Minimal inhibitory concentration
Measure of concentration of antibiotic (or drug) necessary to inhibit growth of target bacterial cell In other words, lowest concentration that prevents growth |
|
In clinical terms, what does MLC stand for? |
Minimal lethal concentration |
|
In clinical terms, what is MLC? |
Measure of the concentration of antibiotic necessary to kill target pathogen |
|
How do you calculate therapeutic index? |
|
|
Why is alcohol not a good antibiotic? |
Mostlikely, the amount needed to kill bacteria, will kill a human too |
|
What is Disk Diffusion Assay? |
Also called Kirby-Bauer Assay Measures the sensitivity of bacteria to a given antibiotic Contains multiple disks with different antibiotics where sizes of cleared zone reflects relative sensitivity |
|
How is Disk Diffusion Assay gone? Explain each step |
Paper disc soaked in antibiotic and is placed on a petri dish coated with an overlay of bacteria (called a bacterial lawn) As antibiotic diffuses away form the disk, it becomes increasingly dluted, providing a concnetration gradient that radiates from the center of the disk More resistant bacteria can grow closer to the disk, where antibiotic is more concentrated Susceptible bacteria will have a large zone around the disk where they will not grow |
|
What is favourable pharmacokinetics? |
Survive in high concentration and reach the target site (site of infection) |
|
What are pharmacokinetics? |
Actions of drugs in the body over a period of time including: Absorption, Distribution, Localization in tissues, Biotransformation, Excretion |
|
What are the general characteristics of antimicrobial drugs on effects on bacterial growth? |
Bactericidal Bacteriostatic Bacteriolytic |
|
What is bactericidal? |
Kills microbes and bacteria which will not re-grow after removal of drugs |
|
What is bacteriolytic? |
Bacterial cell lysis (Can cause immune reactions in humans) |
|
What is bacteriostatic? |
Stops growth of microorganisms without killing them |
|
What is a broad-spectrum antibiotic? |
These be indicated against polymicrobial infection Targets multiple bacterial species, even commensal bacteria (not good) Should not be overused as can contribute to antibiotic resistance |
|
What are narrow-spectrum antibiotic? |
Ideal for an infection caused by a single pathogen
Should only be used when pathogen has been identified to prevent selection of resistance |
|
Why are natural forming antibiotic compounds usuall better than synthetic ones? |
They are shaped by evolution and have been tailored for a specific purpose |
|
Why is the Streptomyces life cycle important for antibiotic research and development? |
During their late stages of life, they produce different kind of compounds called secondary metabolism which some have the purpose of killing other specific bacteria to increase its chances of survival |
|
Why is the beta-lactam ring of penicillin important? |
This is an active bond R groups can be substituted near it with any number of substituents that make each pencillin different |
|
How does resistance against penicillum rise? |
Resistance arises by having a bacterial acquire a plasmid that carry a penicillinase (beta-lactamase) enzyme |
|
What is natural penicillium most active against? |
Gram-positive bacteria
|
|
What happens during the first steps of transpeptidation during peptidoglycan synthesis? |
Involves a transient (temporary) acyl-enzyme itnermediate
This enzyme specifically hydrolyzes the amide bond formed between the two D-alaine amino acids During this process, peptidoglycan subunit is attached to a serine residue on the transpeptidase enzyme Acyl-enzyme intermediate is resolved when amino group of neighboring peptidolycan subunit attacks the intermediate and releases the free enzyme |
|
How does the antibiotic penicillin work? |
The beta-lactam ring has an amide bond that mimics the D-ala-D-ala bond in the wall peptide which is recognized by transpeptidase during cell wall synthesis This new crossbridge between pencillin and transpeptidase inactives the enzyme |
|
When you modify the position 6 of the beta-lactam ring of penicillin, what properties do you mainly change? |
Change in stability in water Change availability in tissues |
|
When someone is allergic to penicillin, what alternatives can be used? |
Cephalosporins and its derivatives |
|
What is Cephalosporin? |
Produced by fungus Cephalosporium Has low toxicity Frequently given to patients that are allergic to penicillin Active against Staphylococci, P. aeruginosa, E. cloacae and gram-negative bacteria |
|
What antibiotic is this? |
Vancomycin (Notice large, cup-like structure) |
|
What is the antibiotic vancomycin? |
It is a large complex glycopeptide produced by Amycolatopsis orientalis Blocks transpeptidation reaction by binding to terminal D-ala-D-ala of wall peptide and preventing it from serving as a substrate for penicillin binding proteins It would essentially cup an enzyme that goes into the cell wall towards the D-ala-D-ala and prevent it from going in Synthesized by non-ribosomal protein synthease Because this can only inhibit one substrate per enzyme, it needs to be in high concentration to be efficient and is used as a last resort, usually given in IV drips Also, poor solubility |
|
Why is vanomycin a last resort antibiotic? |
It only inhibits one substrate per enzyme, it needs to be in high concentration to be efficient Because of this, and its poor solubility and poor ability to get through cell-wall membrane, it is used as a last resort |
|
Why is vanomycin used almost exclusively on gram-positive bacteria? |
Too big to get through a pore |
|
When a drug gets bigger, is it more or less effective against gram-negative bacteria? |
Less because it gets more difficult to get through peptidoglycan layer or fit through outer (or inner) membrane pores/channels |
|
What antibiotic is this? |
Bacitracin (Notice exceedingly complex structure and with three layers and weird bend) |
|
What is the antibiotic bacitracin? |
Exceedingly complex peptide produced by Bacillus strains Binds undecaprenyl phosphate (bactoprenol), a lipid caarrier, and prevents it from serving as a substrate for the attachment of NAG |
|
What antibiotic is this? |
Cycloserine (Notice its just a simple pentagon with double O and NH2) |
|
What is the antibiotic cycloserine? |
Dy-cyclosern is a simple antimicrobial produced by Streptomyces garyphalus Inhibits enzymes (racemases) that convert L-ala to D-ala and the enzyme that ligate the two D-ala residues to make the cell wall peptide precursor Without the D-ala-D-ala residues cell wall synthesis cannot occur |
|
Which antibiotics bind to 30S ribosomal subunit? |
Streptomycin
Gentimicin
Tetracycline |
|
What antibiotics bind to 50S ribosomal subunit? |
Erythromycin
Chloramphenicol
Clindamycin |
|
What antibiotics bind to to elongation factor G (EF-G)? |
Fusidic acid |
|
What antibiotics inhibit tRNA-synthetase? |
Mupirocin |
|
Why is targeting bacterial ribosome a great method for antibiotic targeting? |
Bacterial and eukaryotic ribosomes are substantially different |
|
What antibiotic is this? |
Clindamycin (Notice only one chair structure and one penta-ring) |
|
What is aminoglycosides? |
Class of antibiotics containing amino-modified sugars They usually interfere with protein synthesis, either with tRNA translocation from A site to the P site or with proofreading during protein synthesis such that incorrect amino acids are incorporated at high frequency They also appear to disrupt bacterial membrane Not well absorbed by small intestine and have to be delivered to patient by injection or intravenously |
|
Why do aminoglycosides have to be delievered to a patient by injection or intravenously? |
Why are not well absorbed by the small intestines |
|
What antibiotic is this? |
Tetracycline (Notice 4 cyclic rings) |
|
What is the antibiotic tetracycline? |
Isoalted from Streptomyces Consists of four rings that can be derivatized to generate variant molecules They bind to 30S subunit of ribosomes and inhibit docking of charged tRNAs, preventing protein synthesis Readily absorbed into growing bone and tissue (therefore rarely used on children and pregnant mothers) Cause yellow staining on growing teeth |
|
Why is tetracycline rarely used with children or pregnant mothers? |
Readily absorbed into growing bone and tissue Also causes yellow stain on growing teeth |
|
What antibiotic is this? |
Erythromycin (class macrolides) (Notice marcolide rings) |
|
What is the class of antibiotics macrolides? |
They have 14 to 16 marcolide rings that can be substitued at certain positions with various modifications
Inhibit protein synthesis by binding to 23S rRNA in the 50S ribosomal subunit |
|
What is the antibiotic erythromycin? |
Belongs to class of antibiotics, marcolides Binds to ribosomes so they cannot shift (translocate) tRNAs from A-site to P-site and fail to transfer amino acids from tRNAs into growing peptide chains |
|
What antibiotic is this and what does it do? And what class on antibiotics does it belong to? |
Ciprofloxacin (class fluoroquinolones) Inhibits DNA gyrase (Notice triangle) |
|
What antibiotic is this? |
Rifampin (Notice how bulky it looks in 2D) |
|
What antibiotic is this? |
A basic fluoroquinolone (Notice the fluorine) |
|
What are the class of antibiotics fluoroquinolones? |
They inhibit gyrases which are lethal for bacterial cells |
|
What is rifampin? |
Bacteriocidal antibiotic that binds to beta-subunit of RNA polymerase Blocks channel that newly synthesized mRNA goes through and stops RNA elongation when transcript is a few nucleotides long Only active on RNA polymerase if it binds before transcription starts |
|
What antibiotic is this? |
Polymyxin
(Notice giant circular ring) |
|
What is the basis of antimicrobial peptide selectivity? |
Antimicrobial peptides have positively charged residues and ydrpohobic residues that are arranged in a fashion that can interact first with negative charges on bacterial surface then hydrophobic lipids in bilayers Animal cell lipid bilayers are generally low in negative charges in their outer leaflet but rather usually loaded with cholesterol and zwitterionic lipids |
|
How come Vanomycin took a long time to develop antibiotic resistance? |
We used it as a last resort Its evolutionary and mechanically changing to the change the D-ala-D-Ala it was targeting |
|
What is the superbug NDM-1? |
New Delhi Metallo-beta-lactamase 1 Cleaves almost anything with beta-lactam bond in it with broad specificity Often found in strains that were already resistant to fluoroquinolones and amino-glycosides (making them superbugs) |
|
How is the Netherlands fighting against antibiotic resistance? |
They have strict laws on how they use antibiotics and where they can be sold
|
|
What is inherent resistance or natural resistance against antimicrobial drugs? |
Lack of target (lacking targets like peptidoglycan) Membrane permeability barrier (outermembrane of gram-negative bacteria) Efflux (pumps like the ArcAB/TolC) Metabolic shutdown (dormant persister state) |
|
What is acquired resistance against antimicrobial drugs? |
Alteration of target (genetic mutations leads to loss of affinity for drugs) Efflux (production of proteins that pump out specific drugs) Alteration or inacivation of drugs (production of enzymes to destroy the drug itself) |
|
How has Streptomycin resistance develop? |
Streptomycin binds to the 30S subunit of bacterial ribosome via contact with 16S RNA and ribosome subunit S12 Mutations arose spontaneously in S12 protein that decreased affinity for streptomycin, making the bacteria antibiotic resistant |
|
What is MRSA? |
Methicillin resistant Staphylococcus aureus Group of Staphylococci that have high levels of resistance against the majority of beta-lactam antibiotics MRSA strains harbor an alternative transpeptidase called MecA that has low affinity for beta-lactams but can still function to cross-link cell wall subunits MecA gene is encoded on a genomic island (acquired by horizontal gene transfer) and appears to have been derived from a transpeptidase from closely related but non-pathogenic bacterial strain Staphylococcus scuiri |
|
What is MecA? |
MRSA strains harbor an alternative transpeptidase called MecA that has low affinity for beta-lactams but can still function to cross-link cell wall subunits MecA gene is encoded on a genomic island (acquired by horizontal gene transfer) and appears to have been derived from a transpeptidase from closely related but non-pathogenic bacterial strain Staphylococcus scuiri |
|
What is resistance to tetracycline due? |
Due to efflux from specific transporter called TetA This transporter is under negative regulation of repressor called TetR When TetR binds tetraclycline, it falls off DNA and allows transcription of TetA antiporter The TetRA genes are usually acquired by horizontal gene transfer |
|
What is the beta-lactamase enzyme? |
It cleaves beta-lactam rings in penicillins and cephalosporins
Use serine to attack beta-lactam ring and allows water to enter that can hydrolyzed penicillin from the active site, deactivating it |
|
What antibiotic is this? |
Carbapenems |
|
What are carbapenems? |
Class of beta-lactam derived antibiotics from naturally occuring beta-lactam produced by Streptomyces cattleya
These antibiotics are structurally distinct from penicillin and cephalosporin deriviates and highly resistant to inactivation by most beta-lactamases
Typically last resort in clinics |
|
What is Aspergillomarasime A (AMA)? |
A inhibitor of metallo-beta-lactamase |
|
What modification can be done to small molecules to make them resistant to aminoglycosides? |
1) Acetylation by acetylase enzymes using acetyl-CoA as donor
2) Phosphorylation by phosphotransferase enzymes (ATP as donor) 3) Adenlyation by adenyl-transferase (AMP attached to aminoglycoside) |
|
Can Aspergillomarasime A (AMA) kill bacteria on its own? |
No, it needs to work with meropenem |
|
What is combinatorial chemistry? |
Make libraries of compounds that are related or have different functional groups or atoms around the molecule which you would test its activity on certain bacteria or enzymes
Find different versions of drugs that are better or less effective |
|
What is rational drug design? |
Decide on particular target that you want drug against Try and find some particular enzyme/protein that is only found in that bacteria and not human so if you inhibit that E/pep, you will kill that bacteria but not the humans In silico drug screening (bioinformatics) |
|
What are anti-infective drugs? |
They inhibit virulence proteins
Drugs that inactivate the toxin and once done, can remove harmful effects of bacteria without needing to kill bacteria itself
If you do this, you are less likely to resistance to drug as you are not killing the bacteria, allowing the surviving ones to reproduce, here you kill none, just the toxin |
|
What is Phage Therapy? |
Make concoctions of phages and give it to patients so the phages can kill/disable the bacteria |
|
What is the fear/problem with phage therapy? |
There is no inherent danger to having phages in our body as they cannot infect us The fear is that there is immune reactions to them or they can pick up virulence genes and spread them So you could end up treating one infection while spreading genes to other bacteria, creating different infection |
|
What are the advantages to phage therapy? |
Bacterial host range and killing ability of a phage is generally restricted to one species of bacteria which helps maintain balance of natural flora (killing only target) Limited host range decreases risk of developing resistant in strains not specifically targeting Resistance to phage cocktails are unlikely Pathogens that forms biofilms are innately resistant to antibiotics and host immune defenses but phages are able to degrade biofilms using exopolysaccharide-degrading enzymes that exist as spike attached to tails (digging through biofilms) |
|
What are phage tail-like bacteriocins (also known as tailocins)? |
Bacteriocins are bactericidal compounds made by bacteria which are generally effective only against same or closely related species Pseudomonas aeruginosa makes bacteriocins called Pycins where R-pyocins resemble myophage tails (contractile) and F-pyocins that resemble siphophage tails (non-contractile) |
|
What do R-pyocins resemble? |
Myophage tails (contractile) |
|
What do F-pyocins resemble? |
Siphophage tails (non-contractile) |
|
TRUE or FALSE R- and F-Pyocins are related to phage tails |
TRUE |
|
How are expression of Pyocins controlled? |
In a manner similar to prophages When they are induced and released, they kill the cell they were released from A repressor, PrtR, would be cleaved by RecA, allowing transcription of PrtN which creates pyocin proteins Altraistic mechanism |
|
How does R-Pyocin kill bacteria? |
By making holes in their cell membrane and letting intracellular molecules leak out and ATP levels drop Usually, when viruses poke into bacteria, the hole will remain plugged so cell does depolraize and die but not with R-pyocin |
|
How can R-pyocins be engineered to kill specific bacterial species? |
You can modify the fibers at the end of the tail through gene manipulation that bind to specific proteins on specific bacteria |
|
What is Lysin CF-301? |
Can be used in combination therapy with antibiotics, it can treat MRSA Together they will kill the bacteria more effectively than antibiotic alone |