BIO208 Time line for bacterial genetics 1946 Lederberg and Tatum - demonstrated conjugation in E. coli 1950 Cavalli-Sforza - created an E. coli Hfr strain 1952 Lederberg and Zinder - phage and Salmonella, transduction (“filterable agent”) 1953 Hayes - plated exconjugants on strep -> one way transfer during conjugation 1957 Wollman and Jacob - bacterial gene mapping with Hfr strains, interrupted mating 1958 Lederberg wins the Nobel Prize 1959 Adelberg - Hfr -> F’ factor 1961 Jacob and Monod develop the operon model 1965 Jacob, L’Woff, Monod win the Nobel Prize 1966 Gilbert, Muller-Hill isolate the Lac repressor 1969 Delbruck, Hershey, Luria - Nobel prize 1996 Lewis and Lu determine crystal structure of the Lac repressor Bacterial Genetics, key concepts 3 mechanisms for recombination in bacteria: transformation, conjugation, and transduction. All three involve the unidirectional transfer of genetic information to a recipient. Conjugation (Lederberg and Tatum, 1946) 1. The F factor is a plasmid that replicates episomally which allows it to be maintained in the cytoplasm of the F+ cell. It contains ~100,000 base pairs and 19 genes that encode for proteins involved in pili synthesis, DNA transfer, replication, and other functions. 2. Only F+ cells produce a pilus which allows the cell to attach to an F- cell. They are “donor” bacteria. The plasmid contains genes that encode for the pilus among other DNA sequences and genes. 3. One strand of the F factor is transferred to the F- cell (recipient) where the complementary strand is synthesized. A copy of the F factor remains in the F+ cell. Both exconjugate cells now contain the circular, double-stranded F factor (plasmid) and are F+ phenotype. 4. The F factor occasionally integrates randomly into the E. coli chromosome creating an Hfr (high frequency of recombination) cell. Hfr strains have the F factor integrated in a specific location with polarity (direction). Insertion sequences on the bacterial chromosome are required for insertion of the F factor into the chromosome. The Hfr cells no longer contain an episomal plasmid. 5. Hfr cells transfer bacterial genes in linear fashion into an F- cell via a pilus. The F factor is transferred last. Conjugation rarely occurs long enough for F factor to be transferred. Recipient cells remain F-. The DNA transferred may undergo homologous recombination into the recipient DNA. In this way, the donor bacterium has transferred bacterial genes into the recipient bacterium. 6. The interrupted mating technique involves conjugation for specific times and then plating exconjugants on media containing streptomycin to select for F- exconjugants. The use of minimal media +/- nutritional additives is used to determine which genes have been transferred and the order. 7. Mapping genes with Hfr bacteria: The longer cells conjugate, the more donor bacterial chromosome moves into the recipient cell. The pilus is broken at various times by placing bacteria in a blender The F- cell does not become F+ or Hfr because the F factor does not transfer. The F factor can be inserted at different positions in different bacterial chromosomes, the genes move over in the same order but from different starting points in different strains. The F factor can be present in the reverse orientation, so the order with which the genes would move over would be reversed in these strains Transformation 1. A plasmid or other piece of DNA is enters a competent bacterium via receptors on the bacterial cell.BIO208 2. In the lab, bacterial cells are made "competent" by treatment with calcium chloride. A brief heat shock facilitates uptake of DNA into the bacterial cell. 3. The plasmid is maintained extra chromosomally because is has an origin of replication. The genes on the plasmid may confer new traits to the bacterium such as antibiotic resistance. Some can integrate into the bacterial chromosome. It is also maintained extrachromosomally because the laboratory strain of E. coli used is recombination (recA-) deficient. 4. In nature, transformation is a rare event Transduction 1. U-tube experiment showed that bacterial contact is not necessary - transduction is virus-mediated. 2. A bacteriophage infects bacteria and begins lytic cycle (adsorption, injection of DNA, repression of bacterial genes, synthesis of phage components, packaging of phage, lysis of cell) 3. During phage packaging, pieces of chromosomal DNA (bacterial genes) may be incorporated into the phage head in faulty head-stuffing. Up to 1% of the bacterial genome may be incorporated (about 50,000 bp). All bacterial DNA has equal probability of being packaged = generalized transduction. 4. When the host cell lyses, the new viral particles are released and can infect new bacterial cells. Because infection is a property of the phage protein coat, the virus can adsorb to cells and packaged bacterial genes will be injected. 5. The injected DNA may integrate into the host DNA via homologous recombination. 6. In lysogeny, the phage genome integrated into the bacterial genome creating a prophage. The integration is at specific attachment sites in the bacterial chromosome 7. The existence of the integrated prophage prevents superinfection by additional phage 8. In response to bacterial damage or stress, the prophage enters the lytic cycle (genes for phage assembly, packaging, cell lysis are expressed). 9. When the prophage is excised, it may also clip out bacterial genes flanking the prophage attachment sites. This is referred to as specialized transduction. . Lac operon Operon is a set of genes that is coordinately expressed. 1. I gene encodes the repressor protein which binds to the operator in the absence of lactose sugar. When the repressor is bound to the operator, RNA polymerase, which normally binds the promoter, cannot transcribe structural genes, Z,Y,A 2. Lactose binds repressor molecules which undergo a conformational change. The change in shape prevents repressor binding to the operator DNA. Transcription of the structural genes, Z, Y, A, is now de-repressed. Lactose is referred to an "inducer" of the operon. 3. Structural gene Z encodes the enzyme, betagalactosidase, which cleaves lactose into glucose + galactose. Structural genes Y and A encode enzymes also involved in lactose metabolism 4. Various lac operon mutants have been used to elucidate control of operon expression 5. A constitutive