Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae

Abstract

The nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae is linked to his, consists of at least 17 genes arranged in seven or eight operons^1–4 (Fig. 1), and its expression is inhibited by oxygen (O₂) and fixed nitrogen (N) including ammonia, nitrate and amino acids (see ref. 5 for review). Previous studies have suggested that the nifA and nifL gene products respectively activate^6–8 and repress^9,10 nif expression. There has also been evidence, from mutants that express nif constitutively in the presence of fixed N, that regulation by fixed N involves the gln (glutamine synthetase) genes^11,12, and that their products act at the promoter of the nifLA operon^13,14. We have recently shown by direct measurement of transcription in vivo that the nifA and nifL gene products are, respectively, an activator and represser of nif transcription intiation from all nif promoters except that of the nifLA operon¹⁵. We now report experiments aimed at clarifying the role of the nifA product and the interaction of the nifLA operon and gln genes in controlling nif expression. Having constructed plasmids that constitutively express the nifA gene and investigated nif expression in a variety of K. neumoniae strains transformed by such plasmids, we show that, in the absence of nifL protein, nifA expression is sufficient to activate nif expression, even in the presence of fixed N and O₂. gln-mediated regulation of nif by fixed N exerts its effect by controlling nifLA expression. We have also confirmed that the nif A protein is temperature sensitive, thus explaining the absence of nif expression at 37 ° C. Our results provide a strategy by which the constitutive expression of nif in the presence of fixed N and O₂ is stabilized and suggest a model for nif transcriptional regulation.