Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

Abstract

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, ...

The Soluble NAD+-Reducing [NiFe]-Hydrogenase fromRalstonia eutrophaH16 Consists of Six Subunits and Can Be Specifically Activated by NADPH

“…Related hydrogenases are present in Rhodococcus opacus (24,56), in several species of cyanobacteria (65), and in Thiocapsa roseopersicina (53). The SH consists of four heterologous subunits forming two functional modules: a hydrogenase dimer encoded by the hoxH and hoxY genes and an NADH-dehydrogenase dimer encoded by hoxF and hoxU.…”

Identification and Mutagenesis by Allelic Exchange ofchoE, Encoding a Cholesterol Oxidase from the Intracellular PathogenRhodococcus equi

“…choE was preceded at the correct distance by a putative ribosome-binding sequence, GAGG, and its stop codon was followed by a palindromic sequence of 44 nucleotides that might act as a transcription terminator. Putative Ϫ35 and Ϫ10 sites (GCGACG and CA-GACC, respectively) were identified in the intergenic region upstream from choE on the basis of known Rhodococcus promoter sequences (1,13,23). The choE gene product, ChoE, is 552 amino acids long and has a predicted signal sequence of 45 residues (Fig.…”

The terminal structures of linear plasmids from Rhodococcus opacus

“…However, the macrorestriction maps of the two plasmids are different. The finding of a transposable element in M R l l by Grzeszik et al (1997a) offers a reasonable explanation, insofar as transposition events may have caused rearrangements and deletions in the plasmid.…”