NifA Protein Controls the Expression of Hydrogenase Structural Genes in Rhizobium leguminosarum bv. viciae

Abstract

The hydrogenase system from Rhizobium leguminosarum bv. viciae UPM791 allows this strain to recycle hydrogen generated by nitrogenase in symbiosis with peas. In strain UPM791, transcription of hydrogenase structural genes hupSL occurs exclusively under symbiotic conditions, and is controlled by a -24/-12 promoter (hupSp). Previous results had shown that hydrogenase and nitrogenase genes are spatially and temporally co-expressed in pea nodules (Brito et al. 1995), suggesting that their expression responds to similar signals in the nodule. A -173/-88 DNA region of hupSp has been shown to be essential for expression in pea bacteroids. This DNA region does not contain any recognizable binding sequence for known transcriptional activators, including Nif A. However, heterologous expression experiments performed in Klebsiella pneumoniae and Escherichia coli cells have demonstrated that NifA proteins from K. pneumoniae and R. meliloti are able to activate this promoter. The -173/-88 DNA region essential for symbiotic expression was also required for NifA-dependent hupSp induction in heterologous bacterial hosts. In view of the absence of canonical NifA UAS in hupSp, the possibility of that NifA had an indirect effect through activation of an unknown gene had to be ruled out. We were able to demonstrate inhibition of K. pneumoniae nitrogenase activity by multicopy plasmids carrying the hupS promoter DNA region. This inhibition required the presence of the -173/-88 region, suggesting that nitrogenase expression inhibition proceeds through binding of NifA to this region and titration. Experiments with R. leguminosarum nifA mutants were inconclusive, apparently because Fix^- pea nodules are structurally and developmentally aberrant.