Bacterial Genetics

Question 1

How large is the typical gene in prokaryotes, and how large is the genome? Does it have introns?

Answer 1

Typical gene is about 1000 bases (333 amino acids).

Genome is 4000kb, meaning about 4k genes.

Haploid chromosomes with no introns or histones

Question 2

What is a plasmid vs an episome? What is the significance of these?

Answer 2

Plasmid - autonomously replicating, circular DNA
Episome - plasmid that can integrate into chromosome, some of which can encode for conjugation “transfer genes”

Many virulence factors are plasmid-encoded

Question 3

What is an R factor and their two subunits?

Answer 3

R(esistance) factor is a conjugative episome which encodes for Abx resistance

1. RTF - resistance transfer factor, coding autonomous replication and conjugal transfer
2. Resistance determinant - composed of 1+ transposons, which carries the antibiotic resistance gene

Question 4

What is a complex transposon?

Answer 4

More than one insertion sequence (including transposase gene) flanking another gene, often including antibiotic resistance (in the case of R factor).

This is able to hop into chromosome and overcome the host restriction barrier

Question 5

What is a lytic vs temperate phage?

Answer 5

Lytic - infects and reproduces via bacterial lysis

Temperate = lysogenic, can integrate into chromosome and only become lytic during stress. Includes many toxin genes

Question 6

What is phage therapy?

Answer 6

Infection of bacterial pathogens which may be effective for those who have antibiotic resistance

Question 7

What is the bacterial innate immune system?

Answer 7

Restriction / Modification - breaks probable viral (unmethylated at certain spots) DNA via enzymes. Methylation is species-specific, and sterically blocks digestion via restriction enzymes

Question 8

What are the three bacterial gene transfer methods?

Answer 8

1. Transformation
2. Transduction
3. Conjugation

Question 9

What is “competence” in transformation?

Answer 9

Ability of bacteria to accept DNA, including naked DNA.

Not all external DNA is accepted via bacteria, and many species have specific sequence requirements to accept foreign DNA. It will also be subject to restriction enzyme degradation

Question 10

What enzyme mediates integration of external DNA into host chromosome during transformation?

Answer 10

RecA, which requires some sequence homology for DNA to be integrated. Otherwise, the DNA will be destroyed.

Question 11

What is generalized transduction?

Answer 11

As phage is packaging during bacterial infection, it “accidentally” packages host DNA into pseudovirion particle.

DNA integrates into recipient chromosome via RecA following infection via pseudophage

“head full” mechanism

Question 12

Does generalized transduction happen via lytic or lysogenic phages? Specialized?

Answer 12

Generalized: Can happen via both types
Specialized: Only lysogenic phages (prophage)

Question 13

What is specialized transduction?

Answer 13

Transfer of specific chromosomal sequences via prophage (i.e. lambda phage) exiting out of lysogenic state and carrying some of the host chromosome with it. This reintegrates into new recipient genome upon infection of the phage

Question 14

What is lysogenic conversion, and what are some examples?

Answer 14

Change in bacterial phenotype after phage infection (bacterial virulence factors carried by specialized transduction)

Examples:
Diphtheria toxin, botulinum toxin, cholera toxin, exotoxin A (S. pyogenes), Shigatoxin

Question 15

What is conjugation and what type of bacteria does it occur in?

Answer 15

DNA transfer via cell-cell contact (sex)

Occurs in gram positive or gram negative

Question 16

What is required for conjugation and what is the E. coli example?

Answer 16

Specialized episome, including DNA encoding for sex pilus

E coli - F plasmid - F+ always transfers to F- via rolling replication. F+ is resistant to conjugation via another F+.

Question 17

What is an operon?

Answer 17

Functional transcriptional unit, includes promoter and associated genes (can be mono or poly-cistronic).

All genes are coordinated regulated in case of polycistronic

Question 18

What is a cis vs trans-acting regulatory sequence?

Answer 18

Trans (away) - encode regulatory proteins which diffuse to site
Cis - DNA binding sites which bind the regulatory proteins

Question 19

What are three types of cis-acting regulatory regions?

Answer 19

1. Promoter - DNA sequence recognized via RNA polymerase
2. Operator - near promotor, binds repressor protein and reduces transcription when bound (i.e. lac repressor)
3. Activator - near promotor, binds activator protein to increase transcription (i.e. CRP protein)

Question 20

What is an attenuator?

Answer 20

mRNA secondary structure which modulates transcription (i.e. hairpin loop)

Question 21

What are the 5 genes associated with the lac operon?

Answer 21

Structural

1. LacZ - betagalactosidase
2. LacY - galactoside permease
3. LacA - galactoside acetylase

Regulatory

4. LacI - codes repressor
5. LacO - operator site which binds the lac repressor

Question 22

What is the inducer of the lac operon?

Answer 22

Allolactose (indicates lactose presence), binds lac repressor to inactivate it, increasing transcription

Question 23

What is CRP’s function in lac operon?

Answer 23

CRP binds cAMP and binds the activator sequence to increase transcription when glucose is low

Question 24

When will you have low vs medium vs high levels of transcription

Answer 24

Low - glucose low but no lactose present (repressor bound, activator bound)
Medium - glucose high but lactose is present (repressor not bound, activator not bound)
High - glucose low and lactose present (repressor not bound, activator bound)

Question 25

What are the levels of transcriptional regulation?

Answer 25

1. Positive only - use of activator
2. Negative only - use of repressor
3. Combination - i.e. lac operon, uses both activator and repressor

Question 26

What are some methods of translation regulation?

Answer 26

Usually negative control, prevent bindong of ribosome to mRNA (mRNA looping or silencing RNA), can also make RNA unstable via digestion via RNases

Question 27

How can post-translational regulation be done?

Answer 27

Protein lysis or reduction of protein activity via small molecules / proteins

Question 28

Why is virulence factor regulation highly regulated?

Answer 28

Virulence factors are costly and often only advantageous in specific host environments

Question 29

How is flagella type expression controlled in Salmonella enteritidis (pathogen of raw eggs)?

Answer 29

Antigen variation via inversion of promoter direction, changing phase 1 to phase 2 flagella.

Process is dictated via Hin protein (H invertase) which does site-specific inversion / recombination

Question 30

What is the general principle underlying virulence factor production in Vibrio cholerae?

Answer 30

Transmembrane sensor responds to environmental conditions, and signal is transduced via protein kinase

Cytoplasmic sensory is a DNA binding protein which can modify transcription of a gene

These genes use multiple sensors to respond to environmental cues (quorum sensing, presence of bile salts indicating entrance into GI tract, sensing of bicarbonate in intestinal lumen) to produce virulence factors

Question 31

What is the mechanism of Pertussis toxin production activation in Bordetella pertussis?

Answer 31

BvgS - transmembrane sensory kinase, recognizes a change in temperature to 37 degrees Celsius (indicates entrance into human body)

BvgA - virulence Activator - amount phosphorylated by BvgS determines activity. Relative concentration determines if Pertussis toxin is synthesized

Question 32

What is a pathogenicity island? Why are they clinically important?

Answer 32

Stretch of chromosome which encodes for virulence attributes, especially a number of operons with a common function helping bacterial survival in a host.

often code for adhesins and toxins

These sections can be removed from chromosome to eliminate virulence, so strains without them can be used in potential vaccines

Question 33

What is a regulon?

Answer 33

Series of operons under control via one transcription factor

Question 34

What DNA patterns do pathogenicity islands often contain?

Answer 34

Repetitive terminal sequences - indicating transposition
Also have higher AT concentration than normal, indicating it is foreign DNA (some bacteria can regulate these abnormal genes and prevent transcription)