Genetic Analysis and Mapping in bacteria and bacteriophage Bacteria and Bacteriophage modern molecular genetics is founded on bacterial and bacteriophage studies they have extremely short reproductive cycles can be studied in pure cultures not mixed all individuals are the same pure cultures exhibit heritable variation origin of this variation was once hotly Complex processes have evolved for the transfer of genes between individual debated cells in populations of bacteria Mutation in bacteria 98 of life on earth is microscopic Fluctuation test by Luria and Delbruck showed that spontaneous mutation is the primary source of genetic variation in bacteria Prototrophs can synthesize all essential organic compounds for growth from a minimal medium single carbon and nitrogen sources and inorganic ions Auxotrophs have lost through mutation the ability to make at least one Culture growth middle of exponential growth is the ONLY reproducible essential organic component physiological state for bacteria typical bacterial population growth curve showing the initial lag phase the subsequent log phase where exponential growth occurs and the stationary phase that occurs when nutrients are exhausted Dilution technique to enumerate bacterial population 10 fold serial dilutions of bacteria are plated on nutrient medium on the first plate 1 mL of a 103 fold dilution of cells was spread On the second and third 1 mL of 1 in 104 and 105 was spread What was the original concentration of cells plate 3 has 15 colonies so 15 x 105 cells per mL of undiluted cells Genetic recombination in bacteria already known that naked DNA can be taken up by bacteria genetic transformation Lederberg and Tatum 1946 found bacteria undergo a parasexual process whereby genetic material is passed from one bacterium to another conjugation demonstrated by mixing two multiple auxotrophs and obtaining prototrophs cells that donated genetic information dubbed fertile or F those that received genetic information recipients dubbed F direct contact between donor and recipient is required Davis U tube experiment via a sex pilus when a strain A and B auxotrophs are grown in a common medium but separated by a filter no genetic recombination occurs and not prototrophs are produced mixed auxotrophs produce prototrophs evidence for genetic recombination F factor found to be mobile and when passed to a recipient then the recipient became F now know to be a 100 kb plasmid with at least 19 genes involved in formation of the sex pilus and conjugative process F factor is replicated during transfer to recipient Hfr strains high frequency of recombination certain genes are transferred from a donor strain at a high frequency and a non random fashion A Hfr strain has an F factor plasmid integrated into the host chromosome Occasionally this will recombinant back out of the chromosome and frequently this will be imprecise and the new F factor will carry adjacent genes this is called F F prime When F factor is transferred to another cell by conjugation there will be two copies of the genes on the F factor a partial diploid or Merozygote Ex Hfr Bacteria 1 Conjugation occurs between F and F cell 2 One strand of the F factor in nicked by an endonuclease and moves across the conjugation tube 3 The DNA complements is synthesized on both single strands 4 Movement across conjugation tube is completed DNA synthesis is completed 5 Ligase closes circles conjugants separate the end result is that the F donor cell transmits the F plasmid and the F recipient not becomes F fertile also Hfr bacteria and chromosome mapping 1 F factor is integrated into the bacterial chromosome and the cell becomes an Hfr cell A Hfr strain has an integrated F factor plasmid Conversion of F to an Hfr state occurs by integration the F factor into the bacterial chromosome the point of integration determines the origin O of transfer 2 Conjugation occurs between an Hfr and F cell The F factor is nicked by and enzyme creating the origin of transfer of the chromosome O 3 Chromosome transfer across the conjugation tube begins The Hfr chromosome rotates clockwise During conjugation an enzyme nicks the F factor not integrated into the host chromosome initiating transfer of the chromosome at that point Conjugation is usually interrupted prior to complete transfer 4 Replication begins on both strands as chromosome transfer continues The F factor is now on the end of the chromosome adjacent to the origin 5 Conjugation is usually interrupted before the chromosome transfer is Only the A and B genes are transferred to the F cell which may recombine with the host chromosome complete Figure 8 5 Minutes of conjugation vs relative frequency of recombination graph The progressive transfer during conjugation of various genes from a specific Hfr strain of E coli to an F strain Certain genes transfer more quickly than other and recombine Others take longer to transfer and recombinants are found at a more frequently lower frequency Time map of genes studied in the experiment by recording the time interval between the transfer of each trait to recipients the relative distance between the genes can be estimated in min The F state and Merozygotes Partial diploids 1 Excision of the F factor from the chromosome begins During excision the F factor sometimes carries with it part of the chromosome 2 Excision is complete during excision the A and E regions of the chromosome are retained in the F factor The cell is converted to F occasionally the Hfr plasmid will recombine back out of the chromosome and frequently this will be imprecise and the new F factor will carry adjacent genes this is called F F prime 4 The F factor replicated as one strand is transferred following conjugation with an ordinary F cell the recipient cell becomes partially diploid and is called a merozygote it also behaves as an F donor cell 5 Replication and transfer of the F factor is complete the F recipient has become partially diploid and is called a merozygote it is also F when an F factor is transferred to another cell by conjugation there will be two copies of the genes on the F factor a partial diploid of merozygote By looking for genes that reduced the frequency of integration of F plasmid genes called recA recB recC and recD were discovered which diminish recombination about 1000 fold similar proteins are found in Eukaryotes RecA plays a role in recombination between single stranded DNA ssDNA and its homolog on the host chromosome