IHF- and RpoN-dependent regulation of hydrogenase expression in Bradyrhizobium japonicum

Sequence analysis of the Bradyrhizobium japonicum hydrogenase promoter regulatory region indicated the presence of a -24/-12 type promoter, which is recognized by RpoN, and a potential integration host factor (IHF)-binding site. B. japonicum rpoN1-/rpoN2- double mutants were deficient in hydrogen-uptake activity. Using plasmid-borne hup-lacZ fusions, it was shown that the rpoN mutants were also deficient in nickel-dependent transcriptional regulation of hydrogenase. Gel-shift assays of the hydrogenase promoter regulatory region showed that purified IHF from Escherichia coli binds to a 210 bp fragment. DNase footprint analysis revealed a protected region of 31 bp between bases -44 and -75 from the transcription start site. Western analysis with B. japonicum soluble extract and antibodies against E. coli IHF gave two bands equivalent to molecular masses of 12 and 14 kDa approximately. When the IHF-binding area is mutated on a plasmid-borne hup-lacZ fusion, nickel-dependent transcriptional regulation of hydrogenase is still observed, but the transcriptional rates are clearly less than in the parent hup-lacZ fusion plasmid. Like the results with nickel, regulation of hydrogenase by other transcriptional regulators (hydrogen and oxygen) still occurs, but at a diminished level in the IHF-binding-area-mutated construct.