A HYDROGEN-SENSING SYSTEM IN TRANSCRIPTIONAL REGULATION OF HYDROGENASE GENE-EXPRESSION IN ALCALIGENES SPECIES

Heterologous complementation studies using Alcaligenes eutrophus H16 a s a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophi lus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of two-component regulatory systems present in a number of H-2-oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, re spectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyr hizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine prot ein kinases. Introduction of the complete set of genes on a broad-host -range plasmid into A. eutrophus H16 caused severe repression of solub le and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H-2. This repression was released by truncation of hoxJ. H-2-dependent hydrogenase gene transcription is a typical featur e of A. hydrogenophilus and differs from the energy and carbon source- responding, H-2-independent mode of control characteristic of A. eutro phus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in fram e deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H -2 in response to the availability of the carbon and energy source. RN A dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinas e HoxJ is involved in sensing molecular hydrogen, possibly in conjunct ion with the hydrogenase-like polypeptides HoxB and HoxC.