4/5 bacterial genome and gene transfer Flashcards
chromosomes
- one or few
- essential genes
- .5-10Mbp (500-10,000kb pairs)
- 1 gene/kb
plasmid
- do NOT encode essential functions
- autonomously replicating nuecleic acid molecules
- sizes range
conformations of plasmids and chromosomes
most are circular and supercoiled, some are linear
plasmid sizes
mobilizable are smallest, and non transmissable
conjugative are biggest
copy number of plasmids
- if small, usually high & no partition mech. encoded
- if large plasmid, usually low copy number and encode PARTITION MECHANISM
partition mechanisms
for low copy number plasmids, ensure plasmid is passed on, simialr to centromeres in euks, VERTICAL TRANSMISSION stability
poison/antidote = addiction system
- another way for vertical transmission stability of low copy number plasmid
- plasmid makes stable poison and unstable antidote
- if no plasmid in daughter, teh antidote will degrade before the poison
- so poison kills daughter cell
what can ensure vertical transmission in plasmids with low copy number
- partition mech
2. addiction system (poison/antidote)
transmissibility of plasmid
- horizontal transmission
- from themselves to another cell
- self-transmissable (conjugative) plasmids
“selfish DNA” of plasmids
- selection at DNA Level, not cell level
- ex: poison/antidote (benefits plasmid, but not cell (at least in short term))
population level plasmid selection
- often fitness cost to carrying plasmids, takes more energy/time to replicate
- not every cell needs them as long as some of them do
- when sleective pressures arise, the ones that have it will survive
accessory traits
-nnot needed for survival (at least in lab conditions)
- R plasmids = conjugation
- abx-res
- heavy metal res
- bacteriocin production
- substrate catabolism (chakrabartys multi-plasmid HC-degrading pseudomonas for oil spills)
- virulence factors
bacteriophage
- phage = virus that infects bacteria
- reproduce by lysing bacteria (lytic) OR by integrating themselves stably into bacterial chromosome as prophage (lysogenic)
lysogenic pathway, bacteriophage infections
- phage insterts its genome into bacteria
- bacterial chromosome takes it up
- stably integrates into bacterial DNA and confer new properties on host
transposable genetic elements
move between sites on on DNA via NON-HOMOLOGOUS RECOMBINATION (between sequences that lack similarity)
structure of transposable element structure
- occurs within larger structure of DNA, usually chromosome or plasmid
- boundaries defined by inverted-repeat sequences of DNA at each end (read same from each end, 15-1700 ntides in length)
- transposase enzyme in there, this allows it to move
2 mechanisms of tranposing a transposable element
- non replicative: cut and paste
2. replicative: copy and paste, original stays adn a copt moves (often end up with many copies)
parts of transposon
- insertion sequences = inverted repeats, boundaries (transposase cuts here)
- transposase gene
- additional gene (like abx-res)
Southern blots of replicative transposition
each element produces one or two bands, so lots of bands means lots of copies of that element
how does a transposon replicate?
relies on host (cannot do autonomous replication)
where can transposons move
anywhere. from chromosome to plasmid, plasmid to chromosome, p to p, to a different site on the same P or C
2 mechanisms of transposons acquiring another fxnal gene
- 2 related transposons insert near each other and transposase only cuts outer inverted repeats on them, so the 2 are cut out as one
- one transposon inserts into another
consequence of transposition
- facilitates rapid spread of resistance to many abx
- effect is greater when transposons integrate into plasmids
how do scientists use plasmids
- recombinant DNA/molecular cloning
- isolate/overproduce DNA for recombinant proteins
- insulin
- DNA vaccines
- gene therapy
- sequence analysis
microbiome vs metagenome
microbiome = entire colelction of microbes present in particular habitat
metagenome = entire collection of microbial genes/genomes in particular habitat
16s rRNA sequencing
- essential gene to all bacteria
- has very conserved parts and very variable parts, and it does not accumulate mutations quickly
- ID bacteria easier bc you dont have to cultivate it, just get sample and amplify DNA with PCR and sequence it
- use databases
16s rRNA outcome
determine bacteria present in clinical setting w/o having to grow them
16s rRNA limitations
- sequences often so short that they dont contain enough info to distinguish bacteria at species level, can only get to genus or broader
- not all DNA can be isolated/amplified with equal efficiency
bacterial conjugation
- cell to cell contact DNA transfer between all bacteria
- conjugative genes in donor, usually on plasmid (or plasmid integrated in chrom.)
E Coli F plasmid model of conjugation
F = fertility factor; F plasmid is conjugative
donor = F+ and donates to F- recipient
donor has pili, nicking activity, DNA transfer machinery
process of conjugation, 6 steps
- cells drawn togetehr by pilus retraction
- mating aggregates form
- cytoplasmic bridge formed
- F-encoded endonuclease makes SINGLE strand cut at oriT on F
- SS DNA passes to F-cell
- both cells make complementary strand & cells separate and are now both F+ donors
experimental results of mixing F+ and F- cells togetehr
get F+ recipients
Hfr formation (high frequency recombination)
- F integrates into chromosome
- single cross over event occurs between HOMOLOGOUS sequences on F and chromosome
- this happens within a single cell
Hfr = high freq recombination
what happens in Hfr transfer?
- requires prior Hfr formation via homologous sequence x-over
- genes then transferred from Hfr donor (which is in chromosome) to an F-recipient at any time
- genes transferred in regular order, so tra operon enters last
- this means that very few recipients become donors because this requires transfer of whole chromosome – transfer interupted
Hfr transfer experimental results, mix Hfr and F-
get F- recipients (they got new genes, but they did not get F = fertility genes of tra operon, needed to become a donor)
F’ formation (happens within a single cell)
-excise out an integrate F either exactly the same (clean excision, returns as F) or with adjacent chrom.genes (transferred like F, but called F’)
experimental results, conjugation:
F+ & F-
Hfr & F-
F’ & F-
F+ & F-: get F+
Hfr & F-: get F-
F’ & F-: get F-
mobilization
(hitchhiking)
- plasmids transferred from one to cell to another through action of conjugative plasmids
- conjugation machinery encoded by another plasmid acts on oriT region of mobilizable plasmid to effect transfer
what is one requirement for plasmid to be mobilizatble
oriT
transformation
- uptake and incorporation of free DNA from environment taken up by genetically competent cells
- siurce of DNA = lysed bacterial celsl
transformed DNA coming in can be of 2 types:
- plasmid DNA
- chromosomal: must integrate into chromosome, must have homology to recipient genome, does not enter in one piece (too big) but as fragments
what happens to recipient chromosome when chr. DNA transformed
- homology, lines up
- synapsis
- replacement of recipient DNA with homologous donor DNA
griffith experiment
- rough cells, mice live
- smooth cells (capsule = virulence), mice die
- heat killed (lysed) smooth cells, live
- heat killed (lysed) smooth + live rough cells = DEAD because transformation
competent cell (transformation)
when a cell can do it…E coli are not always competent, but at times can be induced to do transformation
some are always competent (G pos adn G neg)
transformation and cell contact
not needed. you can take the centrifuge supernatant (above the cell pellet) aka filtrate and that works when you put it with recipeint cells
DNase and transformation
blocks it. DNA straight up not protected and DNase tears it to free n.tides/oligonuc.tides
lytic pathway gone wrong and its role in transduction
- lytic pathway of viruses; virus accidentally packages DNA of the host cell and then bacterial cell is lysed and virus goes to infect another bacteria, but its phage head is full of DNA from a bacteria, not viral DNA
- that bacterial DNA is incorporated into recipient chromsome
transduction, experiment
- cell contact not needed
- filtrate/supernatant will work
- Dnase has no effect because the DNA is protected in phage head
lysogenic conversion
-lysogenic phage carriees genes that confer new properties on host and are NORMAL constituents of phage genome (NOT chrom. genes!!)
examples of lysogenic conversion
- diptheria toxin gene
- cholera toxin gene
- superantigen genes
bacteria get virulence factors that are from a virus; they start making whatever it is
transduction
uses lytic pathway, errors in packaging, requires HEAD FULL mechanism. to get chrom.DNA
or lysogenic pathway to get viral DNA
transformation
uptake of free DNA by competent cells
