Bacterial Genetics - Brewer 4/25/16 Flashcards
“clonal”
intro’ing genetic variation into bacterial genome
point of sequencing bact genomes
all bacteria in a pop are genetically identical (clonal) unless they’ve experienced…
- mutation (increase with use of a drug)
- gene exchange
bacterial genomes can be sequenced and compared (pathogenic vs nonpathogenic) → ID genes essential for virulence
characterisitics of bacterial genomes
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
accesory genetic elements
common in bacteria
- plasmids
- viruses [bacteriophages aka “phages”]
- insertion sequences (IS)
- transposons (Tn) : conglomerates of ISs
- pathogenicity islands (PI)
replication of bacterial genome and accessory elements
“replicons”
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
plasmids
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
how do you detect plasmid DNA?
lyse cells so that chromosomal DNA stays inside cells, but plasmids escape
- gel electrophoresis: separate plasmids by size
- stain with EtBr
bacteriophages
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!
how can viruses be involved in contributing to genetic exchange among bacteria?
- 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
insertion sequences
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
transposons
3 steps in evolution of Tn
Tn
resemble IS but contain genes unrelated to transposition
- often contain antibiotic-resistance genes
three steps in evolution of Tn:
- IS inserts near antiobiotic-resistance gene
- second copy of IS inserts on other side → transposase now capable of moving IS-ARgene-IS
- damage or loss of internal inverted repeats “locks” structure together
pathogenicity islands
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
transposases
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
modes of transposition
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]
virulence and antibiotic-resistance genes:
location & transport
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
3 mechs for DNA transfer between bacteria
- transformation : DNA released by lysis of one cell is taken up by another cell
-
conjugation : DNA transfer between cells via direct cell-to-cell contact
* requires use of conjugative plasmid -
transduction : bacterial DNA packaged into a virus particle → transferred into another cell upon infection
* transformation = lysis*
* conjugation = cell-to-cell thru plasmid*
* transduction = virus*
features of bacterial DNA transfer
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)
transformation
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)
conjugation
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
- plasmid transferred from donor to recipient via conjugation bridge
- conjugation bridge breaks post-transfer; recipient cell contains linear fragment of > unit length
- transferred DNA (in recipient cell) is recircularized
transfer of chromosomal DNA via conjugation
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
R factors
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!!!
transduction
2 modes
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!
- 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!
benefit of bypassing “homology requirement”
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)
restriction endonucleases
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
antigenic phase variation
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
3 mechs of antigenic phase variation
examples
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
