The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus M R l l into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7-4 kbp Asnl fragment of the linear plasmid pHG2Ol via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hoxF, -U, -Y, -H, -Wand ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hoxF, -U, -Y and -H encode the subunits a, 7, 6 and B, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the / 3 subunit. Finally, ORF7 encodes a protein which has similarities to CAMP-and cGMP-binding protein kinases, but its function is not known. ORFI, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus M R l l indicated that the hydrogenase genes are under control of a like promoter located at the right end of ORFI and are even transcribed under heterotrophic conditions a t a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHYl harbouring the 7.4 kbp Asnl fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.Keywords : Rhodococcus opacus MR11, NAD-reducing hydrogenase, box genes INTRODUCTIONRhodococcus opacus strain M R l l (formerly Nocardia opaca 1 b) is a Gram-positive, facultative chemolithoautotrophic bacterium which can grow on carbon dioxide and gaseous H, as the sole carbon and energy sources. Physiologically, it belongs to the knallgas bacteria, a group which is composed of phylogenetically diverse organisms. For the activation of H,, all knallgas bacteria contain hydrogenases, which are of two basic types : the membrane-bound hydrogenases (MBHs) , which are coupled to the electron transfer chain and are The GenBank accession number for the nucleotide sequence reported in this paper is U70364.not capable of reducing NAD, and the soluble hydrogenases (SHs), which are localized in the cytoplasm and catalyse the transfer of electrons directly to NAD (Aragno & Schlegel, 1992;Schneider et al., 1984a;Zaborosch et al., 1989; Schink & Schlegel, 1979). The MBHs belong to the more common group of NiFehydrogenases consisting of one large and one small subunit, whereas the SHs belong to a family of less abundant multimeric NiFe-hydrogenases (Friedrich & Schwartz, 1993 ;Wu & Mandrand, 1993). The majority of the knallgas bacteria contain only the MBH, but a few, ...

PL promoters direct the transcription of the duplicatedcbb operons from the facultative chemoautotrophRalstonia eutropha H16. The operons encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle required for CO2 assimilation. Their transcription depends on the activator protein CbbR. Structure-function relationships in the cloned chromosomal promoter region were analyzed by site-directed mutagenesis. PL was altered in its presumed hexameric −35 and/or −10 box or in the spacer region between the boxes to achieve a greater or lesser resemblance to the structure of the ς70 consensus promoter of Escherichia coli. PL::lacZ transcriptional fusions of various promoter variants were assayed in transconjugant strains ofR. eutropha as well as in corresponding cbbRdeletion mutants. Mutations increasing the similarity of the −35 and/or −10 box to the consensus sequence stimulated PLactivity to various extents, whereas mutations deviating from the consensus decreased the activity. The length of the spacer region also proved to be critical. The conversion of the boxes, either individually or simultaneously, into the consensus sequences resulted in a highly active PL. All improved PL mutants, however, retained the activation under inducing or derepressing growth conditions, although the full-consensus promoter was nearly constitutive. They were also activated in the cbbR mutants. The activity of the overlapping, divergently oriented cbbRpromoter was less affected by the mutations. The half- and full-consensus PL mutants were comparably active inE. coli. Two major conclusions were drawn from the results: (i) the location and function of PL were verified, and (ii) indirect evidence was obtained for the involvement of another regulator(s), besides CbbR, in the transcriptional control of theR. eutropha cbb operons.

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (betadelta) and an NADH:acceptor oxidoreductase (alphagamma)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed.