30. Genetics and recombination in bacteria Lecture Outline ...

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30. Genetics and recombination inbacteria Lecture Outline 11/16/05

• Replication in bacteria• Types of recombination in bacteria

– Transduction by phage– Conjugation (“mating”)

• F+ plasmids• Hfr strains

– Transformation of raw DNA• Evidence for recombination in nature

– Resistance plasmids

The Bacterial Genome and ItsReplication

• The bacterial chromosome– Is usually a circular DNA molecule with few

associated proteins• In addition to the chromosome

– Many bacteria have plasmids, smaller circular DNAmolecules that can replicate independently of thebacterial chromosome

Genetics fo Bacteria• Use huge numbers of

individuals (billions) To findvery rare events

• Few morphological traits– Antibiotic resistance– “Auxotrophs” cannot synthesize

essential nutrients (arg- or trp-)– “Prototrophs” have normal

synthesis (arg+, trp+)

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Replication of the circular chromosome

Replicationfork

Origin of replication

Termination of replication

Figure 18.14

Replication alwaysstarts at a certain place

Normal replicationfork for DNAsynthesis

Mutantstrainarg− trp+

EXPERIMENT

Figure 18.15

Mix two mutant strains: Arg+ Trp- and Arg- Trp+.Grown them on complete media. After a short while, test themon culture medium without Trp or Arg.

Mutantstrainarg+ trp–

Mixture

Bacterial cells usually divide asexuallyby binary fission

but they can occasionally exchange genes:

Coloniesgrew

Mutantstrainarg+ trp–

Mutantstrainarg– trp+

No colonies(control)

No colonies(control)

Mixture

To grow on minimal medium, the cell must be able tomake both Arginine and Tryptophan (Arg+, Trp+).

--> Evidence for genetic transfer of one of those genes tothe other strain.

CONCLUSION

RESULTS

Now test them on minimal culture mediumWhy do they needthe control plates?

Four ways bacteria can exchange genes

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1. Transduction

Phage can transferbacterial genesbetween cells

1. Phage virus infectsA+B+ cell

2. Reproductionand lysis

Once in a while hostDNA is mistakenlypackaged in a capsid

3. Transfer of a+ DNA from phage to new cell

2. Conjugation– direct transfer of genetic material between bacterial

cells that are temporarily joined

Figure 18.17 Sex pilus 1 µm

Recipient cellis F-(has no plasmid)

Donor cellcontains F+plasmid

One way transfer

Conjugation and transfer of an F plasmidfrom an F+ donor to an F− recipient

Figure 18.18a

F+ cell can form amating bridge with an F– celland transfer its F plasmid.

Single strand of the F plasmid breaks at a specific point and moves into the recipient cell.

Both cells are now F+.

Bacterial chromosomes

F+ cell

F+ cell

F+ cell

Mating bridge

F– cell

F Plasmid

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Donor F+ cell

Synthesis ofcomplementarystrand in recipient

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Structure of F plasmid

These genes play a role in the transfer of DNAThey are thus designated tra and trb followed by a capital letter

3. High Frequency Recombination(Hfr cells)

F factor can sometimes become integrated in to abacterial chromosome.

Cell is F+ because ithas all of the F factorgenes

F+

MUCH more likely to transfer chromosomalgenes to F- cell during conjugation

Usually carries some chromosomal DNA along with itwhen it is transferred to an F– cell

See this in action

Conjugation ofHfr cell withF- cell)

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Conjugation and transfer of part of the bacterialchromosome from an Hfr donor to an F– recipient

F+ cell Hfr cell

F factorThe circular F plasmid in an F+ cellcan be integrated into the circularchromosome by a single crossoverevent (dotted line).

1The resulting cell is called an Hfr cell (for High frequency of recombination).

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Since an Hfr cell has all the F-factor genes, it can form a mating bridge with an F– cell and transfer DNA.

3 A single strand of the F factorbreaks and begins to move through the bridge. DNA replication occurs in both donor and recipient cells, resulting in double-stranded DNA

4 The location and orientation of the F factor in the donor chromosome determine the sequence of gene transfer during conjugation. In this example, the transfer sequence for four genes is A-B-C-D.

5 The mating bridgeusually breaks well before the entire chromosome andthe rest of the F factor are transferred.

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Two crossovers can result in the exchange of similar (homologous) genes between the transferred chromosome fragment (brown) and the recipient cell’s chromosome (green).

7 The piece of DNA ending up outside thebacterial chromosome will eventually be degraded by the cell’s enzymes. The recipient cell now contains a new combination of genes but no F factor; it is a recombinant F– cell.

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Temporarypartialdiploid

Recombinant F–

bacterium

A+B+ C+

D+

F– cell A–B–

C–D–

A–B–

C–D– D–

A–C–

B–

A+B+C+D+A+B+

D+C+

A+

A+

B+

A–B–

C–D–

A–B+

C–D–

A+B+ B–

A+

Hfr cell

D–A–C–

B–

A+B+C+D+

A+B+

Figure 18.18b

Integration of F+ plasmid into achromosome

Transformation

• Transformation– uptake of naked, foreign DNA from the

surrounding environment

• Remember Griffith’s experiment with heat killedbacteria and mice?

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Does this happen in nature?

• In E. coli and Salmonella, roughly 17% oftheir genes have been acquired from otherspecies (over 100 million years . . . )

• Such “horizontal transfer” is an importantissue for the spread of antibiotic resistance

Spread of Atrizine decomposingbacteria

• A few bacterial species are capable ofmetabolizing the synthetic herbicideAtrizine

• All have nearly identical genes.

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Atrizine catabolism plasmid

Transposonsflank thesegenes

Dispersed atrizinecatabolism genes(ABC) acquiredseparately?

Genes DEF in an operon

Martinez et al. J Bact. Oct 2001

Resistance mechanismsAntibiotic Method of resistance------------------------------------------------------------------------

Chloramphenicol reduced uptake into cellTetracycline active efflux from the cellB-lactams, Erythromycin, eliminates or reduces binding of antibiotic to targetB-lactams, Erythromycin hydrolysisAminoglycosides, Chloramphenicol, inactivation of antibiotic by enzymatic modificationB-lactams, Fusidic Acid sequestering of the antibiotic by protein bindingSulfonamides, Trimethoprim metabolic bypass of inhibited reactionSulfonamides, Trimethoprim overproduction of antibiotic target (titration)Bleomycin binding of specific immunity protein to antibiotic

http://www.bioteach.ubc.ca/Biodiversity/AttackOfTheSuperbugs/