Bacterial Genetics - Brewer 4/25/16

Question 1

“clonal”

intro’ing genetic variation into bacterial genome

point of sequencing bact genomes

Answer 1

all bacteria in a pop are genetically identical (clonal) unless they’ve experienced…

1. mutation (increase with use of a drug)
2. gene exchange

bacterial genomes can be sequenced and compared (pathogenic vs nonpathogenic) → ID genes essential for virulence

Question 2

characterisitics of bacterial genomes

Answer 2

typically single circular DNA molecule (bacterial “chromosome”)

• some species have multiple or linear chromosomes
• large cells may have 1+ copy (typically identical)

size reflects “lifestyle”

• fewer genes → simpler org, less self-sufficient, more dependent on host

Question 3

accesory genetic elements

Answer 3

common in bacteria

• plasmids
• viruses [bacteriophages aka “phages”]
• insertion sequences (IS)
• transposons (Tn) : conglomerates of ISs
• pathogenicity islands (PI)

Question 4

replication of bacterial genome and accessory elements

“replicons”

Answer 4

replicons have sites for initiation of DNA synthesis

• many also have sites for partition of replicated DNA into daughter cells
• include : chromosomes, plasmids, viruses

**insertion sequences, transposons, pathogenicity islands are NOT replicons → only replicate when integrated into a replicon

Question 5

plasmids

Answer 5

mostly circular

many types

size and copy number vary

• typically inversely proportional (DNA is energetically expensive → large plasmid, fewer copies made to keep things energy efficient)

easily detectable : epidemiological applications

• if case clusters have a common source, should see same set of plasmids; if case clusters have diverse sources, should see diff sets of plasmids

Question 6

how do you detect plasmid DNA?

Answer 6

lyse cells so that chromosomal DNA stays inside cells, but plasmids escape

• gel electrophoresis: separate plasmids by size
• stain with EtBr

Question 7

bacteriophages

Answer 7

genetic parasites → inject their genomes into bacterial cells, use its machinery for replication

two types:

1. virulent (lytic) bacteriophages : release progeny via cell lysis

2. temperate bacteriophages : insert genomes into bacterial genomes, replicate as part of it

• integrated viral genome = provirus/prophage → can later excise itself from chromosome, replicate, and lyse cell!

Question 8

how can viruses be involved in contributing to genetic exchange among bacteria?

Answer 8

• non-viral genes can become incorporated into a provirus
• expression of provirus genes is typically blocked via provirus-encoded repressor
• however…sometimes genes escape repression → become expressed
  
  • often happens with bacterial virulence genes, esp toxins

relevant in cases of non-viral DNA incorp into provirus + escape from repression

Question 9

insertion sequences

Answer 9

IS

simplest accesory genetic element

contain only the machinery req for their own movement

• gene encoding transposase
• inverted repeats flanking (recognized by transposase) - nt seqs that are the reverse complement of the downstream

Question 10

transposons

3 steps in evolution of Tn

Answer 10

Tn

resemble IS but contain genes unrelated to transposition

• often contain antibiotic-resistance genes

three steps in evolution of Tn:

1. IS inserts near antiobiotic-resistance gene
2. second copy of IS inserts on other side → transposase now capable of moving IS-ARgene-IS
3. damage or loss of internal inverted repeats “locks” structure together

Question 11

pathogenicity islands

Answer 11

PI

very large transposons : contain 50-100 genes

• possible that PI contains ‘complete kit’ of virulence genes → sufficient to turn a non-pathogen into a pathogen
• most pathogenic strains contain multiple PIs

Question 12

transposases

Answer 12

catalyze movement of IS and Tn via recognition of terminal sequences

similar to enzymes that catalyze…

• integration of HIV into human genome
• V/D/J recombo splicing in Ig and TCR assembly

Question 13

modes of transposition

Answer 13

1. cut and paste transposition : Tn or IS removed from donor → transferred to recipient

2. replicative transposition : Tn or IS copied from donor → copy transferred to recipient

• now found in both donor and recipient
• can occur via fusion of circular DNA molecules
  
  • donor/IS + recipient → co-integrate [transposase] → donor/IS + recipient/IS [resolvase]

Question 14

virulence and antibiotic-resistance genes:

location & transport

Answer 14

virulence/AR genes typically found in plasmids and viruses

• these elements have mechs for transfer between bacterial cells!

chromosomes don’t have this ability: genes only get moved by plasmids/viruses by accident

Tn can move chromosomal genes to plasmids and viruses → enable rapid spread within and between bacterial pops

Question 15

3 mechs for DNA transfer between bacteria

Answer 15

1. transformation : DNA released by lysis of one cell is taken up by another cell
2. conjugation : DNA transfer between cells via direct cell-to-cell contact
   * requires use of conjugative plasmid
3. transduction : bacterial DNA packaged into a virus particle → transferred into another cell upon infection
   * transformation = lysis*
   * conjugation = cell-to-cell thru plasmid*
   * transduction = virus*

Question 16

features of bacterial DNA transfer

Answer 16

one-way (donor → recipient)

common intermediate : merozygote : carries complete copy of recipient chromosome + donor chromosome fragment

• donor fragment is unstable! will be lost unless it combines with the recipient chromosome
• combination requires DNA homology (2 orgs need to be at least near-match in species)

Question 17

transformation

Answer 17

lysis & uptake of released fragments

occurs naturally in some bacteria

can be made to happen in lab setting in almost any cell (bacteria, fungi, plant, mammal)

Question 18

conjugation

Answer 18

DNA tranfer via cell-to-cell contact via conjugative plasmid

plasmid encodes all biochem fx req for DNA transfer

• transfer is efficient; usually only plasmid DNA transferred

best studied: F-factor of E. coli, F-plasmid

1. plasmid transferred from donor to recipient via conjugation bridge
2. conjugation bridge breaks post-transfer; recipient cell contains linear fragment of > unit length
3. transferred DNA (in recipient cell) is recircularized

Question 19

transfer of chromosomal DNA via conjugation

Answer 19

can occur when F plasmid is insterted into chromosome

chromosome/Fplasmid will attempt to transfer like a giant plasmid would

• conj bridge often breaks before transfer is complete

*F plasmid can also transfer non-chromosomal plasmids this way → usually more efficient because plasmids are smaller than chromosome

Question 20

R factors

Answer 20

F-like plasmids that contain multiple antibiotic resistance genes

• resistance genes usually in transposons (within transposons…within transposons…)
• i.e. multiple resistive traits can be conferred by a single plasmid, depending on what it contains → rise of MDR!!!

Question 21

transduction

2 modes

Answer 21

transfer of bacterial DNA via viruses

two modes:

1. virus contains bacterial DNA only

• because there’s homology between the virus-contained DNA fragment and the DNA of the recipient bacteria…within merozygote → replacement of homologous sequence by transduced DNA
  
  • intraspecies recombination!

2. virus genome incorporates 1 or more bacterial genes

• occurs when a piece of bacterial chromosome is picked up in the process of provirus excision → all progeny viruses from that point forward will contain that segment of bacterial chromosome!
• that DNA can then be introduced into any bacterial species that the virus is capable of infecting (no homology required)
  
  • cross-species recombination!

Question 22

benefit of bypassing “homology requirement”

Answer 22

proviruses, IS, Tn all have special mechs for inserting their DNA into bacterial chromosomes that do not require sequence homology

• enables virulence genes to spread to unrelated bacteria (cross-species)

Question 23

restriction endonucleases

Answer 23

barrier to gene exchange

cleave heterologous DNA into fragments

cellular DNA protected from cleavage by DNA methylation at restriction sites :)

*lets bacteria take selective aim at viral DNA invaders

Question 24

antigenic phase variation

Answer 24

continual production of antigenic variants → turns the specificity of the immune response against itself!

• new antigenic variants are not recognized by the specific initial immune response → escape to infect another day
• “cat and mouse game”

created by programmed alterations in DNA

• not random; designed to happen
• DNA determines where changes occur and what alterations happen
• more freq than mutations, reversible, occurs in nearly all pathogens

has prevented devpt of vaccines against malaria, gonorrhea, trypanosomiasis

Question 25

3 mechs of antigenic phase variation

examples

Answer 25

1. inversion

ex. Salmonella flagella (H antigen) variation - H1 or H2

• enzyme hin (H inversion) catalyzes inversion of DNA between two repeats: H1 ⇔ H2
• if promoter for H2 is live → transc/transl of H2 and H1repressor; if not → transc/transl of H1

2. recombination between expressed and silent genes

• Neisseria have expressed (PilE) and silent (PilS) blocks of DNA coding for pili
  
  • pili are key antigens for immune response
• recombo of PilE x PilS → new antigenic types of pili

3. polymerase stuttering during copying of a repeat

• Neisseria outer membrane protein PII can vary number of copies of its CTCTT repeat
  
  • if #nt is an integral multiple of 3…PII produced (reading frame maintained)
  • if #nt is not an integral multiple of 3…PII not produced (reading frame not maintained)
• PII is used for adhesion but is also recognized by immune system → cells that don’t produce PII don’t adhere as well, but also constitute antigenic variant that can escape immune response