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Chapter 15 - Genetics of Bacteria and Bacteriophages : Mapping bacteria, 3 different methods : Conjugation Transformation Transduction Bacteriophage mapping : Bacteriophage gene mapping Cis -trans complementation test. Bacteria transfer (or receive) genetic material 3 different ways :
Mapping bacteria, 3 different methods:
Discovered by Joshua Lederberg and Edward Tatum in 1946.
Unidirectional transfer of genetic material between donor and recipient bacteria cells by direct contact.
Segment (rarely all) of the donor’s chromosome recombines with the homologous recipient chromosome.
Recipients containing donor DNA are called transconjugants.
William Hayes (1953) demonstrated that genetic exchange in E. coli occurs in only one direction.
Genetic transfer is mediated by sex factor F.
Donor is F+ and recipient is F-.
F is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome).
F plasmid contains an origin sequence (O), which initiates DNA transfer. It also contains genes for hair-like cell surface (F-pili or sex-pili), which aid in contact between cells.
No conjugation can occur between cells of the same mating type.
Conjugation begins when the F plasmid is nicked at the origin, and a single strand is transferred using the rolling circle mechanism.
When transfer is complete, both cells are F+ double-stranded.
Transfer of the F factor
No chromosomal DNA is transferred by standard sex factor F.
Transfer of chromosome DNA is facilitated by special strains of F+ integrated into the bacteria chromosome by crossing over.
Hfr strains = high frequency recombination strains.
Discovered by William Hayes and Luca Cavalli-Sforza.
Hfr strains replicate F factor as part of their main chromosome.
Conjugation in Hfr strains begins when F+ is nicked at the origin, and F+ and bacteria chromosomal DNA are transferred using the rolling circle mechanism.
Complete F+ sequence (or complete chromosomal DNA) is rarely transferred (1/10,000) because bacteria separate randomly before DNA synthesis completes.
Recombinants are produced by crossover of the recipient chromosome and donor DNA containing F+.
Transfer of the HfrF+ factor
Excision of the F+ factor also occurs spontaneously at low frequency.
Begin with Hfr cell containing F+.
Small section of host chromosome also may be excised, creating an F’ plasmid.
F’plasmid is named for the gene it carries, e.g., F’ (lac)
F-thr leu aziS tons lac gal strS
Interrupted mating experiment
Location and orientation of the HfrF+ in the circular chromosome varies from strain to strain.
Overlap in transfer maps from different strains allow generation of a complete chromosomal map.
Total map units = 100 minutes
Time required for E. coli chromosome to replicate at 37°C.
Unidirectional transfer of extracellular DNA into cells, resulting in a phenotypic change in the recipient.
First discovered by Frederick Griffith (1928).
DNA from a donor bacteria is extracted and purified, broken into fragments, and added to a recipient strain.
Donor and recipient have different phenotypes and genotypes.
If recombination occurs, new recombinant phenotypes appear.
Bacteria vary in their ability to take up DNA.
Bacteria such as Bacillus subtilis take up DNA naturally.
Other strains are engineered (i.e., competent cells).
Competent cells are electroporated or treated chemically to induce E. coli to take up extracellular DNA.
p+ q+ o+ x p q o
Life cycle of phage
Generalized transduction of E. coli by phage P1
Infect bacteria with phages of different genotypes using two-, three-, or four-gene crosses crossover.
Count recombinant phage phenotypes by determining differences in cleared areas (no bacteria growth) on a bacterial lawn.
Different phage genes induce different types of clearing (small/large clearings with fuzzy/distinct borders).
Same principles of intergenic mapping also can be used to map mutation sites within the same gene, intragenic mapping.
First evidence that the gene is sub-divisible came from C. P. Oliver ‘s (~1940) work on Drosophila.
Seymour Benzer’s (1950-60s) study of the rII region of bacteriophage T4.
Benzer’s deletion analysis of the rII region of T4:
No recombinants can be produced if mutant strain lacks the region containing the mutation.
Small squares indicate point mutations mapping to a given site.
Used to determine the number of functional units (genes) defined by a given set of mutations, and whether two mutations occur on the same unit or different units.
If two mutants carrying a mutation of different genes combine to create a wild type function, two mutations compliment.
If two mutants carrying a mutation of the same gene create a mutant phenotype, mutations do not compliment.