The FixK(2) Protein Is Involved in Regulation of Symbiotic Hydrogenase Expression in Bradyrhizobium japonicum

The roles of the nitrogen fixation regulatory proteins NifA, FixK(1), and FixK(2) in the symbiotic regulation of hydrogenase structural gene expression in Bradyrhizobium japonicum have been investigated. Bacteroids from FixJ and FixK(2) mutants have little or no hydrogenase activity, and extracts from these mutant bacteroids contain no hydrogenase protein. Bacteroids from a FixK(1) mutant exhibit wild-type levels of hydrogenase activity. In β-galactosidase transcriptional assays with NifA and FixK(2) expression plasmids, the FixK(2) protein induces transcription from the hup promoter to levels similar to those induced by HoxA, the transcriptional activator of free-living hydrogenase expression. The NifA protein does not activate transcription at the hydrogenase promoter. Therefore, FixK(2) is involved in the transcriptional activation of symbiotic hydrogenase expression. By using β-galactosidase transcriptional fusion constructs containing successive truncations of the hup promoter, the region of the hup promoter required for regulation by FixK(2) was determined to be between 29 and 44 bp upstream of the transcription start site.