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

Abstract

ABSTRACT 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.