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.
Black
L K
LK
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Maier
R J
RJ
D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D004790:Enzyme Induction; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006864:Hydrogenase / Q000096:biosynthesis* / Q000235:genetics; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D012230:Rhizobiaceae / Q000235:genetics*; D012808:Sigma Factor / Q000502:physiology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; D012808:Sigma Factor; C497751:rpoN protein, E coli; D009532:Nickel; D006864:Hydrogenase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54