Lectures 3 & 4 - Microbial Genetics I & II Flashcards
1
Q
What does the bacterial genome size relate to?
A
Gene number
2
Q
List bacteria types from smallest to largest genome. Explain why.
A
- Obligate parasitic bacteria: have evolved to take advantage of their host so do not need a large genome
- Archaea: technically not bacteria and can survive extreme conditions
- Other bacteria
3
Q
What are the 2 types of bacterial genes? Describe each.
A
- Core genes: provide required functions for survival (e.g. amino acid biosynthesis, DNA replication, cell wall biosynthesis, ribosomes, key metabolic pathways, nucleotide turnover, etc.)
- Variable set of accessory genes: provide flexibility to allow growth in a variety of niches, or survival when their expression is necessary and can move horizontally between strains (and sometimes between species)
4
Q
Where are bacterial accessory genes found?
A
Harbored on a variety of mobile elements that take may take up residence on the chromosome (e.g. bacteriophage transposable elements, genomic islands and islets, integrons, transposons, etc.) OR remain extrachromosomal (plasmids)
5
Q
Where are bacterial core genes found?
A
Usually on chromosomes
6
Q
What is a genomic island?
A
Groups of genes that are contiguous on a chromosome and originated from another organism and are associated with a specific function
7
Q
What is a genomic islet?
A
Small genomic island
8
Q
What are (bacterio)phages?
A
Bacterial viruses
9
Q
What are integrons?
A
Gene capture agents comprised of an integrase and an ATT sequence that captures gene cassettes in the environment, which combine the integron (which has a downstream promoter) on a chromosome or plasmid and becomes part of it because it has a homologous ATT sequence
10
Q
What are transposons?
A
Entities that are capable of moving from one genetic location to another
11
Q
Are transposons capable of self-replication in the cytoplasm?
A
NOPE
12
Q
Nature of the genes in the flexible gene pool? 8
A
- Pathogenicity
- Antibiotic resistance
- Secretion
- Symbiosis
- Degradation
- Secondary metabolism
- Restriction/modification
- Transposases/integrases
13
Q
E. coli genome: number of base pairs?
A
Close to 5 M
14
Q
E. coli genome: number of protein coding genes? % of total genes?
A
4,300
88%
15
Q
E. coli genome: average distance between genes?
A
120 base pairs
16
Q
E. coli genome: % of genome coding for stable RNA?
A
1%
17
Q
E. coli genome: % of genome made of repeats?
A
1%
18
Q
E. coli genome: number of core vs variable genes? What is the bottom line?
A
2,000 core genes vs ~2,000-3,000 variable genes (with ~15,000 distinct genes total between all strains)
=> bacterial species maintain a genetic pool that is much larger than the one present in each strain (not true for humans)
19
Q
What is a prophage?
A
Phage integrated into a bacterial genome
20
Q
How can a commensal bacterium become pathological?
A
By acquiring new variable genes
21
Q
What are the 3 mechanisms of bacterial horizontal gene transfer? Can bacteria conduct all 3? Which is the LEAST common?
A
- Transformation***
- Conjugation
- Transduction
Usually can conduct one of these
22
Q
What is genetic transformation?
A
Acquisition of new genetic material by the uptake of exogenous DNA
23
Q
What is genetic transduction?
A
Horizontal transfer of fragments of bacterial DNA from one bacterium to another by a bacteriophage
24
Q
What is genetic conjugation?
A
Transfer of genetic information from one bacterium to another that requires cell-to-cell contact and the presence of an appropriate plasmid
25
What are the 5 steps of genetic transformation?
1. Small polypeptide is synthesized and is secreted into the environment (optional) => on encountering other cells it stimulates new gene expression for gene products that alter cell surface receptors for free DNA recognition and processing = recipient cell is in a state of competence
2. dsDNA encounters cell surface and is recognized by receptors at the cell surface
3. DNA is processed by endonucleases to the appropriate size
4. Exonucleases digest one of the DNA strands and the remaining DNA strand is taken up by the cell, which is bound by single-stranded DNA binding proteins
5. DNA pairs with and is assimilated into the bacterial chromosome by general recombination/homologous recombination, helped by RecA proteins to search for the homologous sequence
26
What is a cellular state of competence?
Transient state that develops during bacterial growth (usually end of log phase) although some species of bacteria may be competent all the time (constitutive) (e.g. Neisseria gonorrhae), during which certain genes are expressed (e.g. cell surface receptors) to allow DNA to enter the cell
27
What does it mean for transformation to be a non-additive process?
Donor DNA replaces a comparable segment of DNA in the recipient chromosome through homologous recombination
28
Is cellular competence harder to achieve in gram (+) or (-) bacteria?
Gram (-) because of more complex cell wall
29
What does transformation require?
Extensive sequence homology (or homology created by forming a loop) between donor and recipient DNA
30
2 types of cell surface receptors on bacteria? Can a bacteria have both?
1. General receptors: more common in gram (+) bacteria and recognize DNA
2. Specific receptors: recognizes a specific DNA sequence
NOPE
31
How can restriction enzymes impact genetic recombination?
Restriction modification system uses restriction endonucleases to cut DNA where it is unmethylated, and methylation may not occur fast enough after recombination so the recombined DNA may be lost
32
How is genetic transformation used in the lab? Example?
1. Molecular biology techniques make cells that are not normally transformable, transformable by inducing them to take up exogenous DNA by expressing cell surface receptors
2. Electroporation: applying high voltage to appropriately treated cells in the presence of exogenous DNA to drive DNA into the cell by creating transient pores
=> Such artificial competence/transformation has proven extremely important in the biotech industry to introduce foreign DNA into desired host bacteria
33
3 properties of bacteriophages?
1. General morphology: usually filamentous, icosahedral, or complex, consisting of a head and a tail with or without tail fibers and baseplate
2. Nucleic acid can be dsDNA, ssDNA, ssRNA, or dsRNA (listed in order of decreasing frequency)
3. Phages usually contain only nucleic acid and protein (lipid components are found only rarely), with the protein coat called a capsid, which surrounds the nucleic acid referred to as a nucleocapsid
34
Most structurally complicated group of bacteriophages?
T4 (E. coli) with dsDNA with ~100 genes that inserts DNA into a cell
35
Host range specificity of bacteriphages?
Very specific
36
2 types of bacteriophages? Describe each.
1. Virulent bacteriophages: always enter the lytic cycle of phage propagation and successful infection results in death of the infected cell and release of new phage particles
2. Temperate bacteriophages: can enter lytic cycle but can also establish a quiescent relationship with the infected cell where the phage DNA integrates into bacterial chromosome and is maintained and replicated as part of the bacterial chromosome for an indefinite time
37
What do we call a bacterial cell infected with a prophage?
Lysogenic cell
38
Can a lysogenic cell revert back from the lysogenic stage to enter the lytic cycle?
YUP
39
Describe the lytic cycle of phage infection of bacteria.
1. Phage attached to host cell and injects DNA
2. Phage DNA circularizes
3. New phage DNA and proteins are synthesized and assembled into phages spontaneously (arms and legs with DNA packaged into the head)
4. Cell lyses (lysin protein synthesized, which chews up the peptidoglycan), releasing phages
40
Describe the lysogenic cycle of phage infection of bacteria. What to note?
1. Phage DNA integrates into the bacterial chromosome via site-specific recombination (small homologous ATT sites) with an integrase (and other host and viral proteins but not recA) => prophage
2. Bacterium reproduces normally, copying the prophage and transmitting it to daughter cells
3. Many cell divisions produce a colony of bacteria infected with prophage
NOTE: occasionally, a prophage exits the bacterial chromosome initiating a lytic cycle
41
Is the site-specific recombination in the lysogenic cycle of a phage additive?
YUP
42
How are phages clinically relevant? What is an issue though?
Lytic phages can be used to treat antibiotic resistant infections (esp. to treat wounds, middle ear and gut infections, listeria, etc.)
Advantages: specific bacterial target, resistance less of an issue than for antibiotics
Issue: need to know the specific type of bacteria to attach as phages have high host specificity + risk of shock
43
What is lysin therapy?
Using the protein lysin normally synthesized by phages to cause bacterial cell lysis to treat bacterial infections
44
What is lysogenic conversion?
Lysogenic phages can be used as the presence of prophage can confer or alter a phenotypic trait of the host bacterium => important clinically in that many virulence factors are specified by the prophage including toxins, e.g., C. diptheriae, C. botulinum, V. cholorae toxins and other virulence factors important to adhesion, antigenicity, intracellular survival etc.
45
What are the 3 types of transduction? Describe each.
1. Generalized: “any” gene of the host bacterium can be transferred to recipient and doesn’t require lysogeny
2. Specialized: only specific genes near the attachment sites of a lysogenic phage in the host chromosome can be transferred
3. Abortive: do not need to know
46
Describe the mechanism of generalized transduction.
Transduction phage particles are generated during the course of a lytic infection. Normal phage replication occurs, but during packaging of phage DNA into the capsid an infrequent mistake is made such that a random fragment of the bacterial chromosome is encapsulated instead of viral DNA. The particle is normal in all other ways. Should the particle encounter and infect another bacterium only the bacterial chromosome fragment is introduced. The fragment can pair and recombine with the resident chromosome via general recombination
47
Describe the mechanism of specialized transduction.
Only discrete/specific portions of the bacterial chromosome are packaged in specialized transducing particles
Existence as a prophage (i.e., lysogeny) is a prerequisite
Specialized transducing phages result from errors in excision of prophage DNA during induction to lytic growth. Bacterial sequences that flank the prophage integration site end up as part of the “viral chromosome” because of the imprecise excision. Thus every phage resulting from propagation in the cell in which the imprecise excision occurred will carry the same bacterial sequences.
48
What are 3 examples of virulence factors that are prophages into Streptoccocus pyogenes strains?
1. DNAse: degrades DNA trailing from immune cells that could remove bacterial cells
2. Hyaluronidase: allows the bacteria to invade tissues more easily
3. SpeH/A: toxins
49
How was conjugation discovered?
The first conjugal system was discovered and studied in E. coli and is mediated by the F plasmid
50
What is the F plasmid? Is it essential to the bacterial cell?
Covalently closed, circular double-stranded DNA molecule of 60 Mdal obligately intracellular and containing genes for conjugal transfer and autonomous replication
Non-essential to the cell
51
What are E. coli cells containing the F plasmid called? What about those that do not have it?
E. coli cells that contain the F plasmid extrachromosomally are referred to as F+ cells; those that do not contain F are referred to as F-
52
Describe the mechanism of conjugation using E. coli and the F plasmid as an example.
Unidirectional mating of F+ cells with F- cells using the donor's pilus (gram -) or chemotaxis (gram +):
1. Cells come in contact and conjugation process is initiated
2. Single-stranded nick at OriT and binding of protein at 5' end => rolling circle replication initiated
3. Single-stranded DNA is transferred to F- and the complementary strand is synthesized + F+ cell also replicates leftover strand to maintain double-stranded plasmid
4. Cells separate at the end of the transfer => both donor and recipient harbor the plasmid at end of mating
53
2 theories of pilus function for bacterial conjugation?
1. Old theory: pilus connects both cells and plasmid is transferred through the pilus
2. New theory: pilus connects both cells and retracts, pulling both cells so they are close
enough to form a second bridge to transfer genetic material
54
How does chemotaxis work in bacterial conjugation?
Pheromone type compound produced by F- cells to attract F+ cells
55
Role of OriT plasmid gene in conjugation?
Origin of replication used for DNA transfer
56
Role of OriV plasmid gene in conjugation?
Origin of replication used for vegetative extrachromosomal replication
57
Role of insertion sequences (IS) plasmid genes in conjugation?
Transposable elements that influence the state of F plasmids in the cell => provide a region of homology on the bacterial chromosome and F plasmid for integration => Hfr cell formation
58
Role of incompatibility plasmid genes in conjugation?
They place plasmids in incompatibility groups, meaning if both plasmids are integrated into a cell they will not be able to both be maintained over time as the cell divides (due to how plasmids are segregated during replication) => daughter cells will contain one or the other
59
Where does the F plasmid integrate into E. coli cells?
The F plasmid can integrate into the E. coli chromosome at a variety of locations
60
What does Hfr stand for?
High frequency recombination
61
Are Hfr systems widespread?
NOPE
62
What does the mating of Hfr cells with F- cells result in? Describe the mechanism.
Results in the transfer of variable amounts of the bacterial chromosome from donor to recipient
1. F+ cell: F plasmid integrates into chromosome by recombination => Hfr cell
2. Hfr cell joins F- cell via conjugation pilus
3. Portion of F plasmid partially moves into recipient cell trailing a strand of donor's DNA
4. Conjugation ends with pieces of F plasmid and donor DNA in F- cell, which synthesizes complementary DNA strands
5. Donor DNA and recipient DNA recombine making a recombinant F- cell => F- never received a full plasmid and the portion it received is hidden in the chromosomal DNA + remaining Hfr cell
63
What is the F' plasmid?
F plasmid can excise from the chromosome of Hfr cells => F' cell
64
2 types of excision to form F+ cell from Hfr cell?
1. Type 1 excision (precise): only plasmid DNA is excised
2. Type 2 excision (imprecise): a small fragment of the bacterial chromosome may be physically associated (covalently) with F => F' behaves as F would in all matings with F- cells, i.e., the small chromosomal fragment linked to the plasmid is also transferred in every mating => recipient cell (F' as well) thus will become diploid for the sequences present on the fragment
65
2 major types of resistance to antibiotics? Describe each and provide an example.
1. Intrinsic resistance: resistance that is inherently built into the organism (e.g. organism that has no cell wall, making it intrinsically resistant to antibiotics that target cell
walls)
2. Acquired resistance: resistance that results from previous exposure to an antibiotic
66
2 mechanisms by which bacteria can acquire antibiotic resistance? Describe each.
1. Mutation vertical transfer: simple chromosomal mutation that alters the target of
antibiotics, making the antibiotic ineffective - passed on through generations of cell
division
2. Horizontal transfer: mutation or resistant gene in plasmids can be transferred between
organisms in response to selective pressures - this can rapidly change the resistance of a population of cells
67
Example of intrinsically resistant bacteria?
Pseudomonas aeruginosa, a common cause of nocosomial infections
68
Example of acquired resistant bacteria?
Tuberculosis mycobacterium RNA polymerase
69
How can plasmid specify antibiotic resistance? 4 ways.
1. Modifying enzymes: modify antibiotics or targets (through adenylation, acetylation, or methylation) so that the antibiotics cannot bind to the target (e.g. erythromycin binds to rRNA, so a base can be changed on rRNA so that it can no longer bind)
2. Degrading enzymes: destroy antibiotics (e.g. Penicillin)
3. Bypass enzymes: (e.g. Tetracycline targets an elongation factor during translation and some plasmids can express an alternate elongation factor)
4. Efflux pumps: enable the cell to pump out antibiotic as fast as it flows in, making it ineffective (e.g. Tetracycline resistance)
70
Describe chromosomal mediated resistance.
Resistance is produced by chromosomal mutations that result in target alterations, making the antibiotic ineffective
71
What does it mean for hemolysins to be oxygen labile and that there is a need to go deeper in agar plate where the O2 tension is lower?
It means that exposure to oxygen inactivates the hemolysin
72
What does it mean to be a virulence factor?
For a component to be considered a true virulence factor antibodies against it must be able to neutralize the capacity of the organism to cause disease, or when the gene that encodes the component is inactivated the organism is rendered non-virulent
73
What are plasmids that encode enzymes that degrade oil slicks?
They are enzymes that can degrade hyrdocarbons
