CHAPTER 8 GENETIC ANALYSIS AND MAPPING IN BACTERIA AND BACTERIOPHAGES 8 1 Bacteria Mutate Spontaneously and Are Easily Cultured The Adaptation Hypothesis proposes that interaction of bacteriophage and bacterium is essential to the bacterium s acquisition of immunity to the phage Exposure to the phage induces resistance in the bacteria A prototrophs a bacteria that can synthesize all essential organic compounds and therefore can be grown on minimal medium o A minimal medium is one that is composed of an inorganic carbon source and a variety of ions Na K Mg2 Through mutation an auxotroph has lost the ability to synthesize one or more essential compounds It must be provided with them in the medium if it is to grow o Typically result from loss of function type mutations o Multiple Auxotroph s means multiple mutations Bacteria mutate spontaneously and grow at an exponential rate 8 2 Genetic Recombination Occurs in Bacteria Genetic information can be transferred from one bacterium to another resulting in an altered genotype Three processes result in this transfer of genetic information o Conjugation bacterial sex F fertility factor mediated Fig Genetic recombination of two auxotrophic strains producing prototrophs Genetic information is transferred from one bacterium to another It recombines with the second bacterium s DNA o Transformation Direct uptake of naked DNA by 1 cell o Transduction Viral transfer of genetic material between bacteria Vertical Gene Transfer When transfer of genetic information occurs between members of the same species Horizontal Gene transfer When transfer occurs between members of related but distinct bacterial species Cells serving as donors of parts of their chromosomes are designated F cells F for fertility F cells Recipient bacteria receive the donor DNA from the F cells and recombine it with part of their own chromosome If there is no direct contact auxotrophs F and F cells do not exchange genetic material and no prototrophs are formed o Physical contact is the first step in conjugation which is established by o Bernard David showed this using the Davis U tube for growing F and the F pilus F cells F cells contain fertility factor F factor that confers the ability to donate part of their chromosome during conjugation o F factor is mobile and consists of a circular double stranded DNA molecule containing 19 genes o One strand of the double helix moves into recipient cell via the sex pilus and the other one remains Both re form their double helix and become F Figure How an F cell is converted to an F cell during conjugation F X F produces all F cells E coli may or may not contain the F factor o When the F factor is present the cell is able to form a sex pilus and serve as a donor of genetic information o During conjugation a copy of the F factor is transferred from the F cell to the F recipient converting it to the F state The F factor is an autosomous genetic unit called a plasmid Hfr high frequency recombination cells behave as donors and are a An Hfr strain can donate genetic information to an F cell but the recipient special class of F cells does not become F o F X F recipient becomes F low rate recombination o Hfr X F recipient remains F high rate of recombination Interrupted matings technique demonstrated that the genes of a given Hfr strain are transferred and recombined sooner than others Figure Wollman and Jacob demonstrated an order of transfer of genes that correlates with the length of time conjugation proceeded An ordered linear transfer of genes is correlated with the length of time conjugation proceeded o The gene order and distance between genes could be predicted o Basis for first genetic map in bacteria E Coli chrmosome is circular and the point of origin O varies from strain to strain o The F factor integrates into the chromosome at different points and its position determines the O site The direction of transfer is determined by the orientation of the F factor as it integrates When F and F cells are mixed conjugation occurs readily and each F cell involved in conjugation with an F cell receives a copy of the F factor but no genetic recombination occurs At an extremely low frequency in a population of F cells the F factor integrates spontaneously from the cytoplasm to a random point in the bacterial chromosome converting the F cells to the Hfr state as in the Figure In F F mating s the low frequency of genetic recombination is attributed to the rare newly formed Hfr cells which undergo conjugation with F cells Because the point of integration of the F factor is random The gene or genes that are transferred by any newly formed Hfr donor will also appear to be random the larger F F population o The recipient bacteria is a recombinant In some cases the F factor is removed from the chromosome of an Hfr strain and reverts to the F state FIGURE This condition is labeled F to distinguish it from F and Hfr F is a conversion from Hfr whereas Hfr is a conversion from F Transfer of an F to an F cell results in a partially diploid cell called a merozygote but will remain F upon conjugation with an F cell will become F 8 3 Rec Proteins are Essential to Bacterial Recombination RecA protein plays an important role in recombination involving either a single stranded DNA molecule or the linear end of a double stranded molecule that has unwound Single stranded displacement is a common form of recombination in many bacterial species Double stranded DNA entering a recipient cell undergoes one strand degradation o The remaining strand joins the homologous region along the host chromosome and once it does RecA protein facilitates recombination The RecBCD protein is important for unwinding a double stranded DNA molecule that serves as the source for genetic recombination o RecBCD unwinds the helix facilitating recombination that involves RecA to eukaryotes o This model of recombination based on the rec discoveries also applies o Rec A homologs in eukaryotes include meiosis specific enzymes that carryout the recombination associated with crossovers 8 4 The F Factor is an Example of a Plasmid Plasmids are composed of a double stranded closed circle of DNA o Exist in multiple copies in the cytoplasm o May contain one or more genes o Use the same replication enzymes as host o Are distributed to daughter cells o Replicate independently of the bacterial chromosome F factor plasmids confer fertility and contain genes for sex pilus formation on which genetic
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