The wild-type gene for glutamine synthetase restores ammonia control of nitrogen fixation to Gln- (glnA) mutants of Rhodopseudomonas capsulata

Abstract

The wild-type glnA gene, coding for glutamine synthetase, was cloned from the photosynthetic bacterium Rhodopseudomonas capsulata by using a cosmid library to complement the Gln- phenotype of an Escherichia coli glnA deletion strain. The original cosmid plasmid contained 37 kilobase pairs (kbp) of R. capsulata DNA, of which only 2 kbp was necessary for Gln complementation in E. coli. A plasmid containing this 2-kbp insert was mobilized into G29, a Gln- mutant of R. capsulata which is also unable to repress nitrogenase in ammonia-containing media (Nifc phenotype). The 2-kbp fragment restored glutamine-independent growth and ammonia repression of nitrogenase, indicating that in R. capsulata, production of the signal for nitrogen repression of nif depends on the activity of the glnA gene. Repression of nitrogenase was shown, by hybridization of RNA to cloned nif DNA, to occur at the level of transcription in the wild-type and the complemented G29 strains.