INTRODUCTION OF A GENE FROM ESCHERICHIA COLI B INTO HFR AND F ^- STRAINS OF ESCHERICHIA COLI K-12

Abstract

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.1We 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. coli, 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-.2We adopted the following steps for the transfer of the arg6allele 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.3The resulting arg5-Flac+ strain was mated with an Hfr strain of K-12, which had been converted by prolonged aeration into a tem- porary F-phenocopy.Hfr recombinants were isolated, some of which were args-.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 Flac+ 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.