Derivation and properties of F-prime factors in Escherichia coli carrying nitrogen fixation genes from Klebsiella pneumoniae.

Abstract

A His+ Nif+ Escherichia coli K12, Hfr strain (UNF43) was constructed by an intergeneric mating between a Klebsiella pneumoniae donor strain (HF3) and a his-HFR E. coli strain (SBI824) which transfers his as an early marker. An F-prime nif plasmid, FN39, carrying genes which correspond to the E. coli chromosomal region, metG gnd his shiA, but excluding purF and aroD, was isolated from UNF43. Translocation of carbenicillin resistance genes from a P-type R-factor, R68, to FN39 increased the stability of his and nif on the derivative F-prime, FN68. Sedimentation analysis of both F-primes in sucrose gradients revealed our covalently closed circular(CCC) DNA species of molecular weights 279 +/- 9, 136 +/- 3, 90 +/- 1 and 44 +/- 1 megadaltons. It is suggested that the two smallest CCC-DNA species are component replicons of the composite F-primes of molecular weight 136 +/- 3 megadaltons, and that the molecules of 279 +/- 9 megadaltons are CCC-dimers. FN68 was transferable in intergeneric matings to Klebsiella aerogenes, K. pneumoniae and Salmonella typhimurium but not to Proteus mirabilis; only carbenicillin resistance and sex factor activity were transferred to Erwinia herbicola. nif genes on FN68 were expressed in a Nif- mutant of K. pneumoniae and also in S. typhimurium, which in conventional tests is naturally non-nitrogen-fixing; expression of the his determinant of FN68 became temperature-sensitive in S. typhimurium.