Localized Mutagenesis of Any Specific Small Region of the Bacterial Chromosome

Abstract

A method, which we call localized mutagenesis, is described for the isolation of temperaturesensitive and other types of mutations in any specific small region (about 1%) of the bacterial chromosome. The principle of this method is to mutate the transducing DNA rather than the bacterial DNA. One can select for the introduction of this mutated DNA into any particular region of the bacterial chromosome by transducing an auxotrophic marker in that region to prototrophy, thereby introducing new mutations in the neighborhood. We have used this method to isolate many different temperaturesensitive mutations in genes of unknown function in particular regions of the chromosome. Since the method is very simple, it can be used to saturate any region of the map with mutations in essential genes, or for various types of genetic manipulations. Although we have used hydroxylamine-mutagenized phage P22 and Salmonella typhimurium, the method should be applicable to other mutagens and bacteria and transducing phage.Temperature-sensitive (ts) mutations in bacteria are extremely useful because they can be obtained in genes that code for proteins that are essential for the growth of the cell. Since the pioneering work of Horowitz and Leupold (1), temperature-sensitive mutants in bacteria have been used to study activating enzymes (2), ribosomes (3-5), DNA synthesis (6), and other components and aspects of macromolecular syntheses.A number of workers have isolated ts mutants by mutagenizing Escherichia coli or Salmonella typhimurium. Although the isolation of these mutants is easy, the mapping of them involves a considerable amount of work since they may map anywhere on the chromosome. We have developed a simple method, which we call localized mutagenesis, for the easy isolation of ts and other mutants in any specific small region of the chromosome. MATERIALS AND METHODSBacterial Strains. All strains were derived from S. typhimurium strain LT-2.Phage. Phage used for mutagenesis and transduction was P22-int-4, a nonintegrating mutant of P22 (7). The phage sensitivity test was done with P22-H5, a clear (c2) mutant of P22. Phage were stored in T2 buffer (8).Media. Nutrient broth (Difco) was used as maximally supplemented medium. E medium (9) (made 0.4% in glucose) Abbreviation: ts, (i.e., heat-sensitive).was used as minimal-salts medium. Solid medium contained 1.5% agar.Transducing Phage Mutagenesis. Phage (P22-int-4) was grown on wild-type strain LT-2 at 370C with shaking in a medium composed of 100 parts of nutrient broth, 3.5 parts of 40% glucose, and 1.3 parts of concentrated (50X) medium E. The phage, a mixture of phage and bacterial DNA in phage heads, usually had a titer of 1-3 X 1011/ml. Concentrated phage (1012/ml) in 0.9% saline was mutagenized at 370C with hydroxylamine (10). To 1 part of phage in saline were added 5 parts of 0.1 M sodium phosphate buffer (pH 6.0) containing 1 mM EDTA, and 4 parts of 1 M hydroxylamine * HCl (adjusted to pH 6.0 with NaOH) containing 1 mM EDTA. At the desired time the phage were collec...

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