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 genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (&A) and the first 23 amino acids of the large subunit of protein P, of glycine reductase ( g r d o belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11 742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklundii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.Eubacterium acidaminophilum utilizes glycine as both electron donor and electron acceptor, whereas sarcosine and betaine are exclusively used as electron acceptor [l]. Due to this fact, this organism is well suited to compare the biochemistry of the unusual reductive deaminations and the energy conservation associated with it. Glycine reductase, sarcosine reductase, and betaine reductase seem to be generally composed of three proteins: selenoprotein PA, protein Ps, and protein Pc, of which only P, is substrate-specific [2-101 (Fig. 1). In the first step the substrate is bound to protein P, that is specific for glycine, sarcosine, or betaine (M. Meyer and J. R. Andreesen, unpublished results). In all three reductases, the P,-activated substrate is proposed to be nucleophilically attacked by the selenol anion of protein PA to transform it to a carboxymethylated selenoether [3] and the respective amine [4]. By action of protein Pc, acetyl phosphate is formed in all cases leaving the selenoprotein PA in its oxidized state [3, 9, lo]. Finally the selenoprotein PA becomes reduced by the thioredoxin system to start a new catalytic cycle of reductive deamination [7, 81 (Fig. 1). In E. aciduminophilum the NADPH-derived electrons are transferred to protein PA by two proteins, a thioredoxin-reductase-like flavoprotein and an atypical thioredoxin [7,

Antigenic peptides have been found associated with heat shock proteins (HSP) including cytoplasmic HSP70 and heat shock cognate protein 70 as well as the endoplasmic reticulum‐resident glucose‐regulated protein 94. Recently, HSP70 transfection has been reported to increase MHC class I cell surface expression and antigen presentation on mouse melanoma B16 cells (Wells et al., Int. Immunol. 1998. 10: 609). To analyze the effect of HSP70 on MHC class I cell surface expression and lysability of target cells we transfected a human melanoma cell line with the rat Hsp70‐1 gene using the Tet‐On system for conditional overexpression of HSP70. Induction of HSP70 did not increase cell surface expression of HLA class I molecules in general or individual HLA‐A and B antigens in particular. Nonetheless, induction of HSP70 enhanced susceptibility of these cells to lysis by allospecific CTL. The same effect was observed using an HLA‐A2‐restricted tyrosinase‐specific CTL clone after pulsing the tyrosinase‐negative target cells with the specific peptide. Thus, HSP70 induction can increase killing by CTL without affecting MHC class I cell surface expression or antigen processing. This effect of HSP70 appears to be different from the commonly found protection exerted by HSP70 against stress like heat shock, and might be mediated by improving CTL‐induced apoptosis.

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