Introduction of a gene from Escherichia coli B into HFR and F-strains of Escherichia coli K-12.

In this paper, we shall describe a general method for transferring genes from E. coli B into Hfr and F- strains of E. coli K-12. This method was developed during our studies on the mechanism of repression in the biosynthesis of arginine.l We had used for this work strains of K-12 which permitted us, because of the availability of mating types, to study the genetics of the arginine repression system. At one point, it became necessary to have mutants blocked in the formation of one of the arginine enzymes, ornithine transcarbarmylase (arg5-) both in Hfr and F- strains. Such mutants had never been isolated in K-12, despite repeated attempts, although they had been found in other strains of E. colli, such as the B strain. We therefore undertook to transfer this gene from E. coli B to Hfr and F- strains of K-12. E. coli B has been reported to be F-. 2 We adopted the following steps for the transfer of the arg5- allele into K-12. The arg5- mutant of B was converted into a genetic donor by introducing into it an Flac+ episome through mating with an Flac+ strain. Such Flac+ strains transfer the Flac+ particle with very high frequency and their chromosomal genes with a frequency somewhat lower than that of ordinary Hfr strains.3 The resulting arg5- Flac+ strain was mated with an Hfr strain of K-12, which had been converted by prolonged aeration into a temporary F- phenocopy. Hfr recombinants were isolated, some of which were arg. -. From these hybrids, the args- allele could easily be introduced into F- strains of K-12 by mating. We shall describe in detail the steps involved in this transfer and also the mapping of the arg5 gene. During these studies, we discovered an incompatibility phenomenon which brought to light a new type of regulatory system in the cell. In crosses between arg5- Flac+ and arg5+ Hfr strains, only non-Hfr recombinants carried the IFlac+ episome, whereas Flac + was excluded from Hfr recombinants. Besides its intrinsic interest, this exclusion phenomenon proved to be instrumental for the effectiveness of our transfer technique. Materials and Methods. -Media: The minimal medium used was medium A,4 with 0.2 per cent of the sugar specified as carbon source. Other additions were made as indicated. Amino acids were used at a concentration of 100 ug per ml and thiamin at one ug per ml. Enriched medium was either Difco neopeptone broth (NPB), or NY medium, the latter being prepared by supplementing medium A with 0.2 per cent glucose, 0.2 per cent hydrolyzed casein (NZ Case, Sheffield), and 0.2 per cent yeast extract. For solid media, agar was added to a concentration of 2 per cent. Bacto MacConkey Agar ('Difco) served as indicator agar for lactose fermentation. Strains: The pertinent properties of the strains used in the present paper are summarized in Table 1. Mating conditions: Exponentially growing cultures on NPB medium, at 109 cells/ml, were mixed as follows: into a 125-ml Erlenmeyer flask were added 4.5 iml of the female culture, 0.5 of the male culture, and 5 ml of NPB. Since the male was the minority parent, results are expressed in terms of per cent male input. The mating mixture was incubated at 37? with gentle shaking. In interrupted mating experiments, samples were withdrawn at the times specified, diluted in medium A-N (medium A without ammonium sulfate), and plated on the appropriate selective media. Whenever the frequency of recombination was so low that a dilution of less than 103 1887