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Micro > 4/5 bacterial genome and gene transfer > Flashcards

4/5 bacterial genome and gene transfer Flashcards

1
Q

chromosomes

A
  • one or few
  • essential genes
  • .5-10Mbp (500-10,000kb pairs)
  • 1 gene/kb
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2
Q

plasmid

A
  • do NOT encode essential functions
  • autonomously replicating nuecleic acid molecules
  • sizes range
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3
Q

conformations of plasmids and chromosomes

A

most are circular and supercoiled, some are linear

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4
Q

plasmid sizes

A

mobilizable are smallest, and non transmissable

conjugative are biggest

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5
Q

copy number of plasmids

A
  • if small, usually high & no partition mech. encoded

- if large plasmid, usually low copy number and encode PARTITION MECHANISM

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6
Q

partition mechanisms

A

for low copy number plasmids, ensure plasmid is passed on, simialr to centromeres in euks, VERTICAL TRANSMISSION stability

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7
Q

poison/antidote = addiction system

A
  • 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
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8
Q

what can ensure vertical transmission in plasmids with low copy number

A
  1. partition mech

2. addiction system (poison/antidote)

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9
Q

transmissibility of plasmid

A
  • horizontal transmission
  • from themselves to another cell
  • self-transmissable (conjugative) plasmids
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10
Q

“selfish DNA” of plasmids

A
  • selection at DNA Level, not cell level

- ex: poison/antidote (benefits plasmid, but not cell (at least in short term))

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11
Q

population level plasmid selection

A
  • 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
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12
Q

accessory traits

A

-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
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13
Q

bacteriophage

A
  • phage = virus that infects bacteria
  • reproduce by lysing bacteria (lytic) OR by integrating themselves stably into bacterial chromosome as prophage (lysogenic)
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14
Q

lysogenic pathway, bacteriophage infections

A
  • phage insterts its genome into bacteria
  • bacterial chromosome takes it up
  • stably integrates into bacterial DNA and confer new properties on host
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15
Q

transposable genetic elements

A

move between sites on on DNA via NON-HOMOLOGOUS RECOMBINATION (between sequences that lack similarity)

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16
Q

structure of transposable element structure

A
  • 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
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17
Q

2 mechanisms of tranposing a transposable element

A
  1. non replicative: cut and paste

2. replicative: copy and paste, original stays adn a copt moves (often end up with many copies)

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18
Q

parts of transposon

A
  1. insertion sequences = inverted repeats, boundaries (transposase cuts here)
  2. transposase gene
  3. additional gene (like abx-res)
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19
Q

Southern blots of replicative transposition

A

each element produces one or two bands, so lots of bands means lots of copies of that element

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20
Q

how does a transposon replicate?

A

relies on host (cannot do autonomous replication)

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21
Q

where can transposons move

A

anywhere. from chromosome to plasmid, plasmid to chromosome, p to p, to a different site on the same P or C

22
Q

2 mechanisms of transposons acquiring another fxnal gene

A
  1. 2 related transposons insert near each other and transposase only cuts outer inverted repeats on them, so the 2 are cut out as one
  2. one transposon inserts into another
23
Q

consequence of transposition

A
  • facilitates rapid spread of resistance to many abx

- effect is greater when transposons integrate into plasmids

24
Q

how do scientists use plasmids

A
  • recombinant DNA/molecular cloning
  • isolate/overproduce DNA for recombinant proteins
  • insulin
  • DNA vaccines
  • gene therapy
  • sequence analysis
25
Q

microbiome vs metagenome

A

microbiome = entire colelction of microbes present in particular habitat

metagenome = entire collection of microbial genes/genomes in particular habitat

26
Q

16s rRNA sequencing

A
  • 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
27
Q

16s rRNA outcome

A

determine bacteria present in clinical setting w/o having to grow them

28
Q

16s rRNA limitations

A
  • 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
29
Q

bacterial conjugation

A
  • cell to cell contact DNA transfer between all bacteria

- conjugative genes in donor, usually on plasmid (or plasmid integrated in chrom.)

30
Q

E Coli F plasmid model of conjugation

A

F = fertility factor; F plasmid is conjugative

donor = F+ and donates to F- recipient

donor has pili, nicking activity, DNA transfer machinery

31
Q

process of conjugation, 6 steps

A
  1. cells drawn togetehr by pilus retraction
  2. mating aggregates form
  3. cytoplasmic bridge formed
  4. F-encoded endonuclease makes SINGLE strand cut at oriT on F
  5. SS DNA passes to F-cell
  6. both cells make complementary strand & cells separate and are now both F+ donors
32
Q

experimental results of mixing F+ and F- cells togetehr

A

get F+ recipients

33
Q

Hfr formation (high frequency recombination)

A
  • F integrates into chromosome
  • single cross over event occurs between HOMOLOGOUS sequences on F and chromosome
  • this happens within a single cell
34
Q

Hfr = high freq recombination

what happens in Hfr transfer?

A
  • 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
35
Q

Hfr transfer experimental results, mix Hfr and F-

A

get F- recipients (they got new genes, but they did not get F = fertility genes of tra operon, needed to become a donor)

36
Q

F’ formation (happens within a single cell)

A

-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’)

37
Q

experimental results, conjugation:

F+ & F-
Hfr & F-
F’ & F-

A

F+ & F-: get F+
Hfr & F-: get F-
F’ & F-: get F-

38
Q

mobilization

A

(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
39
Q

what is one requirement for plasmid to be mobilizatble

A

oriT

40
Q

transformation

A
  • uptake and incorporation of free DNA from environment taken up by genetically competent cells
  • siurce of DNA = lysed bacterial celsl
41
Q

transformed DNA coming in can be of 2 types:

A
  1. plasmid DNA
  2. chromosomal: must integrate into chromosome, must have homology to recipient genome, does not enter in one piece (too big) but as fragments
42
Q

what happens to recipient chromosome when chr. DNA transformed

A
  • homology, lines up
  • synapsis
  • replacement of recipient DNA with homologous donor DNA
43
Q

griffith experiment

A
  1. rough cells, mice live
  2. smooth cells (capsule = virulence), mice die
  3. heat killed (lysed) smooth cells, live
  4. heat killed (lysed) smooth + live rough cells = DEAD because transformation
44
Q

competent cell (transformation)

A

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)

45
Q

transformation and cell contact

A

not needed. you can take the centrifuge supernatant (above the cell pellet) aka filtrate and that works when you put it with recipeint cells

46
Q

DNase and transformation

A

blocks it. DNA straight up not protected and DNase tears it to free n.tides/oligonuc.tides

47
Q

lytic pathway gone wrong and its role in transduction

A
  • 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
48
Q

transduction, experiment

A
  • cell contact not needed
  • filtrate/supernatant will work
  • Dnase has no effect because the DNA is protected in phage head
49
Q

lysogenic conversion

A

-lysogenic phage carriees genes that confer new properties on host and are NORMAL constituents of phage genome (NOT chrom. genes!!)

50
Q

examples of lysogenic conversion

A
  • diptheria toxin gene
  • cholera toxin gene
  • superantigen genes

bacteria get virulence factors that are from a virus; they start making whatever it is

51
Q

transduction

A

uses lytic pathway, errors in packaging, requires HEAD FULL mechanism. to get chrom.DNA

or lysogenic pathway to get viral DNA

52
Q

transformation

A

uptake of free DNA by competent cells

Micro (7 decks)

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