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BIOL253: Genetics > Bacterial genetics > Flashcards

Bacterial genetics Flashcards

1
Q

What are the three mechanisms used by bacteria to achieve genetic exchange?

A

Transformation (taking up naked DNA from environment)

Transduction (use of bacteriophage)

Conjugation (use of plasmids)

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2
Q

What type of DNA sequence is predominant in bacterial genome?

A

unique (very little repetitive DNA sequences)

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3
Q

What are the three distinct types of plasmid?

A

col plasmids
R plasmids (resistance)
Sex (E.g. F) plasmids

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4
Q

Describe the bacterial Sex plasmids

A

E.g. F plasmid of E.coli = unidirectional transfer of genetic information from donor to recipient cell

  • relatively LARGE (~100 kpb)
  • precise replication (1-2 COPY NUMBER)
  • SELF MOBILE
  • about 35% encodes for FUNCTIONS PERMITTING TRANSFER between bacteria
  • remaining sequence contains four TRANSPOSONS (insertion sequence)
  • mediate transfer of bacterial genes by CONJUGATION
  • an EPISOME = free circular plasmid or integrated into chromosome
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5
Q

Describe bacterial R plasmids

A
  • variable size but relatively large
  • self mobile - can transfer between unrelated species
  • encodes resistance to antimicrobials, heavy metals and toxins
  • evolved parallel with use of widespread antibiotics
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6
Q

Describe bacterial col plasmids

A
  • variable size but small (roughly 10 kbp)
  • encode biological factors (e.g. colicin)
  • relaxed replication (~30 COPY NUMBER)
  • do not encode functions permitting transfer
  • may be transferred if F or R plasmids present that encode functions required for transfer
  • extensively manipulated by molecular biologist to generate cloning vectors.
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7
Q

Describe the experiment that discovered the F plasmid mediated conjugation in E. coli

A

Lederberg and Tatum

  • mixed two multiply auxotrophic strains of E.coli (A and B - what the A strain was auxotrophic for, the B strain was wildtype and vice versa) and plated out Strain A, strain B and the mixture of both strains onto minimal media
  • observed some phototrophic colonies on the minimal media with the mixture which resulted from genetic exchange between the A and B strains.
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8
Q

What was the experiment that proved transfer of genetic material was being observed in the Lederberg and Tatum experiment?

A

Some people believed that the reason for colony growth when the two strains were mixed was due to the production of substances in the medium by the two strains that permitted growth.

Therefore: the U-tube experiment was performed to prove that this was not the case:

  • the two strains were placed into either end of a U tube with a fine pored filter between the strains at the base of the tube, which allowed medium to move between the two sides, but not bacteria.
  • then separated out the two strains and plated then on minimal media
  • saw no growth which proved that the bacterial cells required contact to transfer genetic material and facilitate growth of prototrophic colonies
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9
Q

Describe the process of bacterial conjugation in F+ x F- matings

A

1) F+ cell contains an F plasmid which contains the tra genes, such as that encoding pilin required for the formation of the F-pilus (tra genes encode contact and DNA transfer functions)
2) the F-pilus contacts an F- cell, forming a bridge
3) transfer intitiated by introducing a nick at OriT (Origin of transfer) and rolling circle replication occurs from the 3’ OH, displacing the 5’ end of the strand, which is then transferred into the recipient through the cytoplasmic bridge (the 5’ end of ssDNA leads into recipient cell)

4) As the DNA moves into the recipient cell, synthesis of the complementary strand occurs in a 5’-3’ direction
- the results are two F+ cells

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10
Q

Describe the process of bacterial conjugation in Hfr x F- matings

A

1) rare, low frequency generation of a Hfr strain by F plasmid integration into the bacterial chromosome through recombination between specific transposons on the F plasmid and the chromosome - reversible insertion
2) F plasmid encodes for transfer functions such as formation of the cytoplasmic bridge
2) OriT is nicked and rolling circle DNA synthesis occurs from the 3’ OH, displacing the 5’ end causing it to lead into the recipient cell - the F plasmid is transferred into the recipient, followed by bacterial chromosome.
3) within the recipient cell the complementary strand is synthesised in 5’ to 3’ direction and homologous recombination can occur between the transferred chromosomal DNA and the recipient chromosome

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11
Q

Why is it unusual to get transfer of the entire chromosome or F plasmid?

A

the cytoplasmic bridge is fragile so it usually breaks before compete transfer is achieved

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12
Q

What is horizontal gene transfer? why does conjugation permit this?

A

transfer between species

conjugation permits this because it is not species specific

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13
Q

Describe the experiment that used conjugation to map bacterial genes

A

Jacob and Wollman = interrupted mating experiment

Donor HfrH - phototrophic, resistant to sodium azide, resistant to infection with bacteriophage T1, sensitive to streptomycin

Recipient F- - auxotrophic for Leu and Thr, sensitive to sodium azide, sensitive to bacteriphage T1 infection, resistant to streptomycin

  • Bacteria mixed and vortex at various times after mating to break the cytoplasmic bridge and prevent transfer of anymore genetic material
  • plate on selective media and determine which genes have been transferred from Hfr to F- stain = shows the order of genes relative to OriT
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14
Q

What did interrupted mating experiments with a variety of Hfr strains (different sites of insertion) show about the E.coli chromosome?

A
  • showed it was circular
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15
Q

What are F’ plasmids and how are F’ plasmids formed?

A

F’ plasmids are free circular F plasmids that carry bacterial genes

F’ plasmids are formed by imprecise excision of the F factor from the bacterial chromosome resulting in adjacent genes being taken with it

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16
Q

What can be the result of conjugation involving an F’ plasmid?

A
  • full F’ plasmid is transferred into the recipient cell, which means that the recipient cell now become a PARTIAL DIPLOID for the specific gene being carried (as it contains one in its chromosome, and one in the F’ plasmid)
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17
Q

Describe the process of gene transfer by generalised transduction (using bacteriophage P1 and E.coli as an example)

A
  • virulent P1 bacteriophage infects E.coli
  • P1 contains genes that encode for proteins that digests the bacterial chromosome and allow phage genome replication, translation and assembly (some E.coli genome may be packed into a P1 phage head to produce a TRANSDUCING PHAGE)
  • phage produces lysozyme to destroy the bacterial cell wall and cause lysis and release of bacteriophage into the surroundings

The transducing phage can then infect another E.coli cell and there may be recombination between the bacterial DNA carried by the phage and the chromosome of the new host. The recipient allele is degraded and there is a PHENOTYPE CHANGE.

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18
Q

Give an example of an F’ plasmid?

A

F’lac = critical in understanding the lac operon

- used in sexduction = transfer F’ into a recipient cell

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19
Q

What is cotransduction?

A

Cotransduction is the use of bacteriophage to observe the location of genes within the chromosome:

  • genes closer together are cotransduced more frequently
  • frequency with which genes are cotransduced allows determination of gene order
20
Q

What is the limit of cotransduction?

A

approximately 100 kbp (size of the P1 genome)

21
Q

What is the difference between generalised and specialised transduction?

A

generalised = bacteriophages can pick up any portion of the host’s genome.

Specialised = bacteriophages pick up only specific portions of the host’s DNA

22
Q

What type of bacteriophage mediates generalised transduction and what type of bacteriophage mediates specialised transduction?

A

Generalised = virulent phage (lytic lifecycle)

Specialised = temperate phage (can adopt lytic or lysogenic lifecycle depending on genes expressed)

23
Q

Describe the lysogenic lifecycle of a bacteriophage

A

1) genome inserted into bacterial chromosome as a prophage and replicates along with the bacterial chromosome
2) occasionally, the prophage is excised and the phage enters the lytic lifecycle (this may occur as a part of a DNA damage response)

24
Q

Describe the different lifecycles adopted by the temperate phage lambda

A

(1) lytic pathway:
- circularised lambda DNA is replicated by rolling-circle mechanism to produce a concatamer of linear DNA.
- concatamer is cut at cos sequence by Ter enzyme to produce lambda size genome molecules with overhangs that can allow circularisation and ligation - these are then packaged into phage heads.

(2) Lysogenic pathway:
- circular Lambda DNA integrates into bacterial chromosome using SITE SPECIFIC RECOMBINATION within the O sequence common to both attB and attP sites
- -> recombination between bacterial attB site and Lambda DNA attP site (both sites have a central pacer region (O) that is flanked by different integrase binding sites - C and C’ in attP, B and B’ in attB)
- following integration of prophage, the prophage is flanked by the attL (left) and attR (right) sequences which are composites of the attP and attB sites
- integrated prophage excised by site-specific recombination between core O sequences of attL and attR.

25
Q

What is a concatamer?

A

long DNA molecule with multiple end to end copies of the lambda DNA

26
Q

Describe the process of gene transfer by Specialised transduction (using bacteriophage lambda and gal gene as an example)

A

On rare occasions, prophage is excised aberrantly:

  • some of the prophage remains in the bacterial chromosome
  • excised prophage lacks some phage genome (d defective - contains attR or attL site instead of attP) but carries some bacterial chromosome
  • resulting in mixed phage lysate (contains wildtype lambda phage and lambda d gal+ phage)
  • defective gal+ phage cannot integrate at att site as it lacks attP
  • defective phage also lacks genes required for replication so is unable to replicate and enter the lytic cycle
  • defective phage integrates by homologous recombination at the gal gene = can result in phenotype change.
27
Q

what is the limitation of specialised transduction to map bacterial genes?

A

can only transduce genes close to attB site

28
Q

Describe the process of gene transfer by transformation

A

1) uptake of naked DNA

2) recombination between transformed DNA and bacterial chromosome

29
Q

What is cotransformation?

A

Transformed bacterial DNA containing more than one gene taken up by a bacterium

30
Q

What is the use of co-transformation analysis?

A

observe co-transformation frequencies of different genes to map their order in the genome
- genes close together more likely to be on the same DNA fragment so will have a higher co-transduction frequency

31
Q

What is the definition of a transposon?

A

pieces of DNA that can move around the genome and insert at target sites by transposition - can move between chromosomal DNA and extrachromosomal DNA within the same cell (comprise large parts of genome in all organisms)

32
Q

Why are transposons are major source of mutation?

A

insertion of transposon in a gene will knock-out its function

33
Q

What are the effects of transposon insertion? (5 things)

A 
affect gene regulation and expression (insert into a promoter)
affect chromatin structure
affect genome stability
cause mutation
affect evolution
34
Q

What two ways can transposons be moved?

A
  • excision and integration

- replication

35
Q

Why does transposon insertion result in target site duplications?

A

due to DNA repair mechanisms results in duplication of target site either side of the transposon - amount of this duplication is characteristic of the particular transposon

36
Q

What are the three main types of transposon? and which organisms they are found in (bacteria/eukaryotes)?

A

1) DNA-only cut and paste elements (all organisms)
2) Long terminal repeat (LTR) elements (eukaryotes)
3) Non-LTR elements (eukaryotes)

37
Q

Describe the structure and features of a DNA-only cut and paste element

A
  • have terminal inverted repeats (TIRs)
  • autonomous (contains a transposase between TIRs that encode proteins needed to move DNA)
  • non-autonomous (no transposase so relies on proteins made by an autonomous element)
  • widespread in bacteria and eukaryotes
  • only transposon found in bacteria
  • requires no homology for insertion
38
Q

What are the only type of transposon found in bacteria?

A

DNA-only cut and paste elements

39
Q

What are the three subdivisions of DNA-only transposons in bacteria? give examples

A

1) insertion sequence (IS) elements (TIRs flank transposase)
example: IS10

2) composite transposons (pair of insertion sequences flanking gene(s) encoding antibiotic resistance, IS elements may be in same or inverted orientation)
Example: IS elements in same orientation (Tn9), IS elements in inverted orientation (Tn10, Tn5)
- transcription either of entire transposon or just the IS element

3) non-composite transposon (TIRs flank number of genes encoding drug resistance and transposases, large, 5 bp target site duplication)
Example: TnA family - Tn3, Tn1000

40
Q

What are the typical structure of eukaryotic DNA-only transposons?

A

Transposase flanked by TIRs

41
Q

Describe a long terminal repeat (LTR) element

A
  • are retroviruses or similar to retroviruses
    = move around genome using reverse transcriptase to produce mRNA copy, which is reverse transcribed into DNA copy, which moves around the genome
42
Q

Describe a non-LTR element

A

can be autonomous or non-autonomous:

  • Long interspersed elements (LINEs) = autonomous - encode proteins to mediate their own transposition (common in human genome)
  • short interspersed elements (SINEs) = non-autonomous - rely on proteins encoded by LINEs
43
Q

Briefly describe the two potential mechanisms of transposition by DNA-only transposons

A

1) cut-and-past mechanism: most common
- element excises completely and inserts into target site
- small amount of replication repairs the join sites (duplication of target site)

2) nick-and-paste: bacteria only
- transposon remains attached to donor DNA and is joined to the target site of donor DNA - resolution produces two molecules that each have a copy of the transposon.

44
Q

Describe the mechanism of transposition of DNA-only elements by cut-and-paste mechanisms

A

1) transposon encodes a transposase that recognises and binds to the TIRs
2) transposase dimerises, bringing the two ends of the transposon together and activation of transposon cleavage from the donor site from transposase bound at opposite end
3) Transposase/transposon complex binds to target site on recipient DNA and the free 3’ OH ends attack the target site, introducing a staggered cut with overhang (characteristic of particular transposons)
4) the 3’ ends of the transposon binds to the exposed 5’ ends of the target site (at the end of each overhang)
5) host DNA synthesis repairs the DNA opposite the overhangs in a 3’ to 5’ direction resulting in duplication of target site either side of transposon

45
Q

If the transposon leaves the insertion site cleanly, what is left in the recipient DNA?

A

repeat of the target site (short repeat sequence)- footprint of where a transposon has been

46
Q

What are the importance of transposons in bacterial genetics? give examples

A

Episomes/plasmids:

  • E. coli F factor that allow recombination with the same transposons in the bacterial chromosome required to generate Hfr strains
  • R plasmid contains composite transposons - antibiotic resistance = can transfer between donor and recipient where the transposons can move out of the plasmid and into the bacterial genome
47
Q

What are the main reasons for working with bacteria?

A
  • well-defined genetic systems
  • easy to grow
  • haploid so can see phenotypes

BIOL253: Genetics (6 decks)

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