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Bacteriophages Help (page 3)

By — McGraw-Hill Professional
Updated on Aug 23, 2011

Transduction

Transduction is the virus-mediated transfer of DNA from a donor bacterial cell to a recipient cell. There are two types of transduction: specialized and generalized. In both cases, bacterial DNA is incorporated into the genome of a mature virus that then goes on to infect a different bacterial host. Generally, specialized or generalized tranducing phage are defective due to the integration of additional DNA into their genomes. However, they must be normal enough to infect a new cell.

  1. Specialized Transduction. Specialized transduction occurs when a specific region of the bacterial chromosome becomes integrated into a mature viral particle. There are four distinguishing characteristics of specialized transduction: (1) the only bacterial genes that can be transduced are those very near the site at which the prophage is integrated, (2) only λ-type prophage are involved, (3) it results from defective excision of the prophage from the host chromosome, and (4) recombinant progeny bacteria may be partial diploids. The only site at which lambda phage integrates into the host chromosome (Fig. 11.4) is between the genes for galactose fermentation (gal) and biotin synthesis (bio). The head of the phage can only contain a limited amount of DNA, so if the prophage deintegrates abnormally from the host chromosome (taking some bacterial DNA in place of its own DNA), only the gal or bio genes could be transduced. Thus, all transducing lambda phages are defective in part of their own genome and cannot replicate on their own. A lambda phage that transduces the galactose genes is therefore called λgal or λdg (d = defective; g = galactose). If a gal cell is infected by λdg (bearing the gene gal+), integration of the defective prophage into the host chromosome produces (for the gal locus) a partial diploid recombinant chromosome. Aberrant excision of the prophage is usually a rare event, so restricted transduction is an event of low frequency in nature. However, high-frequency transduction can be attained under laboratory conditions. If a bacterial cell is doubly infected with a wild-type lambda phage and a λdg phage, the wild-type phage can supply the functions missing in the defective phage, and the progeny will contain about equal numbers of both types. When the lysate is used for transduction, the process is referred to as high-frequency transduction. In many cases, because of its defective genome, λdg fails to be integrated into the host chromosome and therefore is not replicated. At each division, only one of the two progeny cells contains the defective phage genome; this process is called abortive transduction.
  2. Bacteriophage Life Cycles

  3. Generalized Transduction. This type of transduction occurs when any region of bacterial DNA is incorporated into the genome of a mature virus particle. The hallmarks of this type of transduction have been extensively studied in the P phages (P1 of E. coli and P22 of Salmonella typhimurium) and are as follows: (1) any bacterial gene can be transduced, (2) the transduction results from a packaging error during phage maturation, and (3) haploid recombinants are produced. Since there is no homology between DNA sequences of the phage and the sequences of their host, there is no preferential site at which the prophage integrates. Any gene can be transduced because the head of the phage can package an entire headful of bacterial DNA. Cotransduction is the process of transducing two or more genes via the same defective phage. Reciprocal crossing over is required to integrate the transduced genes, so recombinant bacteria tend to be haploid rather than diploid. The endogenote segment replaced by the exogenote fails to replicate for lack of an ori (origin of replication) site and becomes lost in the culture through dilution or digestion.

Fine-Structure Mapping of Phage Genes

  1. Complementation Mapping. Because so little of the phage or bacterial genome consists of nonfunctional sequences, virtually all crossing over occurs within, rather than between, genes. Before the discovery that DNA is genetic material, the gene was thought to be the smallest genetic unit by three criteria: mutation, recombination, and function. In 1954, Semour Benzer set out to determine the limits of these operational units by performing the most definitive fine-structure mapping ever performed on a phage gene. He chose to investigate the rII region of phage T4.When wild-type (r+) T4 infects its host E. coli, it produces relatively small plaques. Many mutations result in a shorter life cycle than the wild type, thus yielding larger plaques. These "rapid lysing" mutants may be classified into three phenotypic groups, depending on their ability to lyse three strains of E. coli (Table 11.1). The sites of rI, rII, and rIII map at noncontiguous locations in the T4 genome. The rII region is about 8 recombination units long, representing approximately 1 % of the phage DNA. Complementation tests were used to determine that the rII region consists of two genes.
  2. EXAMPLE 11.5 If two rII mutants are added to strain K12 in sufficient numbers to ensure that each cell is infected with at least one of each mutant, one of two results is observed. If all the E. coli K12 cells lyse after one normal propagation cycle (20–30 min), we may infer that the mutants were in different functional units (genes). Each mutant was making a different polypeptide chain, and the two chains ''cooperated'' to allow a normal-sized burst to occur. On the other hand, if the two rII phages contain a mutation in the same gene, progeny phage can only be produced by genetic recombination with a frequency dependent upon how closely the two point mutations are linked. In any event, only a few of the cells would be expected to lyse by this mechanism in the same period of time. Thus, the results of complementation are easily distinguished from those of recombination. In 1954 Benzer found that all of the point mutations in the rII region mapped into two genes (A and B).
  3. Deletion Mapping. Benzer also found that about 10% of his more than 2000 rII mutants did not backmutate to wild type because they were deletions of various lengths. By infecting cells with two different deletions, wild-type recombinants could be produced if the deletions did not overlap. Wild-type recombinants cannot be produced if the two deletions overlap to any extent. Thus, by a series of crosses Benzer was able to draw a topological map in which deletions were shown to overlap or not overlap. The lengths of the deletions or the degree of overlap or non-overlap is arbitrary at this point, although they can be determined by crosses with point mutations. Having obtained a topological map, it was then possible to assign one or more point mutations to a relatively small segment of a gene by crossing them with deletion mutants because a point mutation cannot recombine with a deletion at the same site. This principle allowed Benzer to group mutants within relatively small regions of each gene. This was accomplished by doubly infecting strain B with a pair of rII point mutants (e.g., rIIa and rIIb) in broth, and then allowing them to lyse the culture. The total number of progeny phage can be estimated by plating dilutions of this lysate on E. coli B and counting the resulting plaques. Wild-type recombinants are scored by plating the lysate on strain K. For every wild-type (r+IIa; r+ IIb) plaque counted on strain K, we assume that an undetected double mutant (rIIa, rIIb) reciprocal recombinant was formed.

The smallest reproducible recombination frequency that Benzer observed between two sites in the rII region was about 0.02%, corresponding to approximately 1/400 of a genetic region whose total length is only 8 recombination units. Thus, it was concluded that the genes of the rII region each contained hundreds of possible mutation sites, and that recombination could occur between the closest of these mutant sites. Benzer reasoned that the smallest distance within which recombination occurs might be as small as adjacent nucleotide pairs. The smallest segment of DNA that mutated could cause a phenotypic effect was found to be as small as five nucleotides or smaller. We now know that a mutation in a single nucleotide base pair is sufficient to cause a mutant phenotype.

A surprising finding of this work was that the point mutants were not randomly located in the rII region; a few locations (called hot spots) in both genes had many more mutations than elsewhere (over a hundred in a couple of positions vs. about 1 to 10 elsewhere).

Practice problems for these concepts can be found at:

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