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Molecular Biology > Bacterial Genetics > Flashcards

Bacterial Genetics Flashcards

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1
Q

Antibiotic targets

A
  • cell membrane
  • DNA gyrase
  • RNA polymerase
  • protein synthesis/ribosome inhibitors
  • folic acid biosynthesis
  • cell wall synthesis
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2
Q

Mechanisms of Resistance

A
  • altered critical target enzyme
  • protein degrading drug
  • efflux pump
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3
Q

Vertical Gene Transfer

A
  • passing on genetic information from parent to daughter cells
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4
Q

Horizontal Gene Transfer

A
  • cell passes gene to a new strain of the same species or to a different species
  • allows bacteria to acquire new traits rather than passing on existing one
    1. transformation
    2. transduction
    3. conjugation
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5
Q

Fluctuation Analysis

A
  • Bacteria grown on agar plate to form a lawn and Phages kill bacteria to form plaques. Eventually Phage-resistant colonies will form - but it was unknown if these colonies are spontaneous or induced by phage presence
  • E. coli aliquoted into a series of tubes to create identical independent subcultures each plated on agar with phage
  • If the mutation is caused by the phage the frequency of resistance in each culture should be similar
  • If the mutation is spontaneous the frequency of mutation should be variable
  • Results showed that resistance arises from random mutations (constant rate of random mutation among cells)
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6
Q

Transformation

A

Isolated DNA molecules are taken up from the surroundings and incorporated into the genome

  • solely encoded by recipient bacteria
  • all requires proteins encoded in recipient core genome
  • most transformable bacteria don’t permanently express these proteins
  • competent bacteria undergo transformation
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7
Q

S. Pneumoniae Transformation Experiment

A
  • Mixed live type R (non lethal) and heat killed type S (lethal) in mice and found that the mice died but the bacteria recovered were live type S
  • Therefore, the type R bacteria had been transformed via DNA uptake
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8
Q

Advantages of Transformation

A
  1. nutrition
  2. genome maintenance
    - homologous DNA uptake repairs DNA damage
    - competence systems activated by damage
  3. diversification
    - increase diversity during stress, ie. maximises chances of survival
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9
Q

Disadvantages of Transformation

A
  1. energy cost

2. new genes could be harmful

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10
Q

Mechanism of Transformation

A
  1. bacteria develop competence
  2. cells bind dsDNA in environment
  3. movement of DNA over membrane
  4. conversion to ssDNA
  5. intracellular fate: plasmid maintenance/homologous recombination/degradation
    DNA binding - nuclease digestion - uptake of ssDNA - recombination
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11
Q

Competence Development

A
  • bacteria can become naturally competent
  • usually as a result of stress
  • cells coordinately express a new set of genes (regulon activation)
  • cells synthesize new proteins
  • construct Com pilus and other surface proteins
  • similar to type 4 pili
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12
Q

Gram Positive Species Transformation

A
  1. capture of exogenous DNA by transformation pilus (comGC pilus)
  2. binding of dsDNA by DNA binding protein ComEA
  3. recognition of the dsDNA by the nuclease EndA and conversion to ssDNA
  4. transport of ssDNA strand by ComEC driven by ATP dependent translocase ComFA
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13
Q

Gram Negative Species Transformation

A
  1. PilQ secretome channel enables pilus to cross outer membrane, bind dsDNA (specific sequences) and transport into periplasm
  2. binding of dsDNA by the DNA binding protein (ComE)
  3. transport of DNA strand across inner membrane by ComA
  4. endonuclease probably involved (not identified yet)
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14
Q

Homologous Recombination

A
  • internalised ssDNA recruits RecA protein that polymerises ssDNA
  • promotes homology search along chromosomal DNA
  • used for recombination and DNA repair
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15
Q

Transformation Summary

A
  • transformation is the uptake of foreign DNA
  • involved in creating genetic diversity, repairing DNA, and potentially a nutrient source
  • addition of new genes, deletion of genes, replacement of genetic alleles
  • environmental/cellular cues required for induction
  • gram +/- can be naturally competent and there is broad overlap in the systems
  • natural transformation uses linear ds DNA and artificial transformation uses circular dsDNA
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16
Q

Artificial Transformation

A
  • many bacteria able to be transformed artificially
  • uses circular dsDNA
  • relatively inefficient
  • artificial induced competence
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17
Q

Methods of Artificial Transformation

A

Electroporation: electric field changes membrane permeability properties so dsDNA enters cell
- bacterial membrane repair restores natural permeability
Chemical Transformation: CaCl2 treatment of cells at low temp. allows membrane permeability change
- plasmid entry possible

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18
Q

Determinants of Transformation Efficiency

A
  1. genetic background of transformants
    - no restriction system
  2. preparation of artificially competent bacteria
    - alteration of membrane permeability
  3. quality of transforming DNA
    - ion chelators/salt contaminants/DNA damage
  4. size of transforming DNA
    - large plasmids taken up less efficiently
    * usually very inefficient*
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19
Q

Gene Cloning

A

Selection: used to select for transformants (taken up plasmid) with an antibiotic resistance gene in the plasmid vector
Screening: transformants will contain either vector alone or vector plus insert so replica plating allows distinction

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20
Q

Steps of Artificial Transformation

A
  1. add DNA and incubate on ice to allow DNA binding to bacterial surface
  2. heat shock DNA and cell mixture to allows uptake and change membrane permeability/or electroshock
  3. put on ice for 30 minutes to restore normal permeability of membrane
  4. add rich broth to cells and incubate to allow expression of selective marker
  5. plate on selection/screening medium
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21
Q

Phage Transduction

A
  • allows bacteria to gain genetic diversity
  • genes transferred from one bacterium to another via bacteriophages and can be inserted into the chromosome of the recipient cell (lysogeny)
22
Q

Phage T4

A

Structure: head, neck/collar, core, sheath, end plate, fibers
- basically composed of protein and nucleic acid

23
Q

U Tube Experiment

A

Phage mediated transfer of genetic material
- Mixed two auxotrophic mutants together to obtain a protrophic strain
- repeated with a u-tube with filter (large pore size) and obtained protrophic recombinants
- repeated with a u-tube with filter (small pore size) and did not obtain protrophic recombinants
- filters used prevented cell:cell contact, preventing conjugation
- concluded genetic transfer was mediated by bacteriophage
Conclusion: recombination was occurring by the passage of an agent through the larger pores. smaller pores are around the size of a phage.
It has been shown that a lysate of phage particles from samonella could mediate gene transfer to recipient strain

24
Q

Phage Life Cycle

A
  1. absorption to bacterium
  2. production of phage specific mRNA; translation to produce nucleases to degrade host chromosome
  3. replication of viral DNA
  4. coat proteins encoded by virus appear
  5. coat proteins condense around phage DNA and phage makes lysozyme
  6. lysis of host cell; 100s of progeny viruses released
  7. infectious virus contains genetic material surrounded by a complex protein head; protein tail required for absorption to host cell
25
Q

Phage Types

A

Virulent: only undergo lytic cycle, always kill host
Temperate: can undergo lytic/lysogenic cycle (replication only), can exist in host for long periods as prophage, can introduce genetic information to host

26
Q

Lysogenic

A

The lysogenic cycle, sometimes referred to as temperate or non-virulent infection, does not kill the host cell, instead using it as a refuge where it exists in a dormant state. Following the injection of the phage DNA into the host cell, it integrates itself into the host genome, with the help of phage-encoded integrases, where it is then termed a prophage. The prophage genome is then replicated passively along with the host genome as the host cell divides for as long as it remains there and does not form the proteins required to produce progeny. As the phage genome is generally comparatively small, the bacterial hosts are normally relatively unharmed by this process.

27
Q

Lytic

A

In the lytic cycle, sometimes referred to as virulent infection, the infecting phage ultimately kill the host cell to produce many of their own progeny. Immediately following injection into the host cell, the phage genome synthesizes early proteins that break down the host DNA, allowing the phage to take control of the cellular machinery. The phage then uses the host cell to synthesize the remaining proteins required to build new phage particles. The heads and sheaths are assembled separately, the new genetic material packed into the head and new daughter phage particles constructed. During this process, the host cells gradually become weakened by phage enzymes and eventually burst, releasing on average 100-200 new phage progeny into the surrounding environment

28
Q

Transition between Lytic/Lysogenic Cycles

A

If a bacterium containing prophage is exposed to stressors, such as UV light, low nutrient conditions, or chemicals like mitomycin C, prophage may spontaneously extract themselves from the host genome and enter the lytic cycle in a process called induction.

This process, however, is not perfect and prophage may sometimes leave portions of their DNA behind or take portions of host DNA with them when they re-circularize. If they then infect a new host cell, they may transport bacterial genes from one strain to another in a process called transduction. This is one method by which antibiotic resistance genes, toxin and superantigen-encoding genes and other virulence traits may spread through a bacterial population

29
Q

2 Types of Transduction

A
  1. generalised: any part of bacterial chromosome, occurs during lytic cycle, random packing of host DNA into phage
  2. specialised: only transfer DNA adjacent to prophage insertion, occurs during lysogenic to lytic conversion, inaccurate excision of prophage from host chromosome
30
Q

Phage Insertion

A
  • reciprocal recombination takes place between a specific attachment site on the circular DNA and a specific region called the attachment site on the E Coli chromosome between the gal and bio genes
31
Q

Generalised Transduction

A
  • bacteriophage must digest host DNA with nuclease
  • one in 1,000 phages carry host DNA (transducing particles)
  • homologous recombination using host RecA enzyme
  • provides linkage information
  • co transduction of genes depends on distance (higher transduction frequency)
  • Phages pick up DNA during lytic cycle
  • Degradation and packaging/Random DNA
32
Q

Abortive Transduction

A
  • circularised DNA fragment doesn’t integrate into chromosome, unable to replicate autonomously, cells divide and dilute out single cell with transduced DNA fragment
33
Q

Co-transduction Frequency

A
  • two genetic markers packaged onto single generalised fragment
  • phenotype of one marker selected
  • transductants inherited one marker will be screened for second marker
    frequency = ratio of transductant that coinherited / total number of A transductants
34
Q

Wu Formula

A

Links distance to co-transductions

C = 1 - (d/L)^3

35
Q

Specialised Transduction

A
  • Donor bacterial genes picked up during transition from lysogenic to lytic cycle
  • Only genes near site of prophage integration
    Repression relieved:
    • Lytic cycle kicks off
    • Prophage excision
      Excision of phage from chromosome:
      • via a site-specific recombination process
      • ‘integrase’ and ‘excisionase’
      • 1 excision per 106 is incorrect and takes with it some host DNA
        Result is ‘specialised transducing phage’
    • can still infect
    • may have a defective genome
    • might need a wild-type helper to infect
    • carries genes adjacent to original site of integration

Only specific portions of bacterial
chromosome are transduced

36
Q

Bacterial Conjugation

A
  • bacterial mating
  • mostly in gram - bacteria
  • advantageous to species
  • usually transfers plasmids
  • transfer is unidirectional
37
Q

Conjugation Experiment

A

** see news **

38
Q

F factor

A
  • fertility factor
  • strains found to transfer genes by conjugation
  • confers ability to donate DNA
  • can be lost and regained easily
  • F strains are donors (F+)
  • F strains are recipients (F-)
  • F is conjugative plasmid which encodes the machinery for conjugation
39
Q

F Pilus

A
  • F pilus connects donor and recipient
  • synthesized by the donor cell by the type IV secretion machinery
  • once attached to a recipient, the cells come together
  • DNA transfer then occurs
40
Q

F Plasmid

A
  • type 4 secretion system, synthesizes the pilus (F pilus) ‘surface exclusion’
  • stabilisation of mating pairs
  • DNA transfer
  • regulation
41
Q

Conjugation Mechanism

A
  • Pilus retraction, cells are brought together
  • Tra proteins form a pore complex - “mating bridge” -between two cells
  • DNA relaxase nicks one strand of F plasmid at oriT site and attaches to the 5’P
  • relaxase/ ssDNA migrates to Tra pore complex and transfers to recipient cell
  • donor strand is resynthesised by DNA polymerase
  • re-synthesis of F plasmid is completed in donor cell
  • DNA transfer completed and ssDNA converted to ds circular DNA in recipient cell
  • Cells separate
  • Both cells now have F plasmid and can make F pilus
  • Both can act as donors to new recipient cells
  • Neither cell can be a recipient because of surface exclusion
42
Q

Which cells can be recipients?

A

The host range is defined by several factors:

  1. Recognition of recipient by donor
  2. Successful completion of DNA transfer
  3. Replication of transferred plasmid
43
Q

R factor

A
  • resistance factor is a plasmid encoding multiple antibiotic resistance
  • not conjugative plasmids but many are
44
Q

HFr Strains

A
  • F plasmid integrated into chromosome
  • occurs due to homologous recombination between insertional sequences on plasmid and chromosome
  • genome has multiple IS
45
Q

HFr Strain Properties

A

F-plasmid integration:
- can still initiate conjugation
- can result in bacterial chromosome mobilisation
(transfer of chromosomal genes to the recipient cell)
- initiated at oriT site
- rolling circle replication

Transfer machinery:
- still operates; does not distinguish between F plasmid alone or F plasmid integrated within bacterial chromosome

Integrated F-plasmid and chromosomal genes
are transferred

46
Q

HFr Conjugation

A
  • Conjugation and Gene transfer between Hfr donor and F- recipients
  • just like described earlier with F plasmid
  • but with bacterial chromosome section included
  • recombination between chromosomal genes may occur if homology present
  • the DNA strand transferred is very long and often breaks; the F plasmid would be the last piece transferred, therefore recipients do not become F+
47
Q

HFr Transport

A
  • Linear transport of Hfr DNA- directional - can use this to map gene order- based on transfer frequency to recipient
  • Genes proximal and downstream of ‘origin of transfer’
  • more likely to be transferred
  • transfer of proximal locus occurs more frequently relative to distal loci
48
Q

Interrupted Mating Experiment

A

A genetic experiment in which the manner of gene transfer between conjugating bacteria is studied by withdrawing samples at various times and subjecting them to a strong shearing force in an electric blender

  • One must start with Hfr bacteria from the same strain (having the same site and orientation of F factor integration).
  • Individual pairs of Hfr and F- bacteria must begin conjugation nearly simultaneously.
  • One must be able to select against Hfr cells following conjugation.
  • One must be able to remove aliquots of the conjugating bacteria and stop the mating at different time points.
  • All of the above are essential when attempting to map the bacterial chromosome in a single interrupted mating experiment.
49
Q

Time of Entry Mapping

A

Time of entry mapping can be used to order genes for a particular Hfr strain. By combining results from multiple Hfr strains it is possible to construct a detailed map ordering the genes.
- composite map obtained by combining the Hfr maps

50
Q

Conjugation Summary

A

Hfr conjugation

- Hfr strain formed via F+ integration within bacterial chromosome
- can transfer host chromosome
- Transfer originates at oriT of inserted F plasmid
- Transfer of entire chromosome is very rare (requires 100 minutes)
- The acceptor strain remains F-
- Was very useful in establishing a genetic map of  E. coli genome	 (interrupted mating)

F’ strains

- arise, via illegitimate recombination, from excision of integrated F plasmid
- flanking host sequences are included in the resulting F’ plasmid
- can replicate and transfer DNA just like F+
- Transferred DNA is a plasmid in recipient, but carries a portion of the donor 	genome 
- F’ x F - = F’ and F’

Molecular Biology (11 decks)

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