No abstract
The hydrogenase (Hyd) isolated from the cytoplasmic membrane of Wolinellu succinogenes consists of three polypeptides (HydA, HydB and HydC) and contains cytochrome h (6.4 pmol/g protein), which was reduced upon the addition of H2. The enzyme catalyzed the reduction of 2,3-dimethyl-1, 4-naphthoquinone with H2, in contrast to an earlier preparation which was made up of HydA and HydB only and did not contain cytochrome b (Unden, G., Bocher, R., Knecht, J. & Kroger, A. (1982) FEBS Lett. 145, 230-234). This suggests that HydC is a cytochrome b which serves as a mediator in the electron transfer from H2 to the quinone.The hydrogenase genes were cloned, sequenced and identified by sequence comparison with the N-termini of the three subunits. The three genes were arranged in the order hydA, hydB, hydC, with the transcription start site in front of hydA, and were present only once on the genome. Separated by an intergene region of 69 nucleotides, hydC was followed by at least two more open reading frames of unknown function. The amino acid sequences derived from hydA, hydB and hydC were similar to those of the membrane Ni-hydrogenases of seven other bacteria. HydA and HydB also showed similarity to the small and the large subunits of periplasmic Ni-hydrogenases. HydC was predicted to contain four hydrophobic segments which might span the bacterial membrane. Two histidine residues located in hydrophobic segments are conserved in the corresponding sequences of the other membrane hydrogenases and might ligate the haem B.
Wolinella succinogenes contains a single formate dehydrogenase, but two gene loci (fdhl and fdhII) code for the subunits of the enzyme. The nucleotide sequence of fdhII is almost identical with that of fdhI in the region comprising fdhEABCD. The sequences of fdhI and fdhll differ in the promotor regions upstream of fdhE. Deletion mutants lacking either fdhI or fdhII synthesize functional formate dehydrogenases, as shown by growth with formate as electron donor and either fumarate or polysulfide as acceptor substrates, and by the presence of the FdhA subunit and of enzyme activity. In the wild-type strain, the fdhl genes appear to be expressed preferentially during growth with formate and fumarate. The six-times greater amount of the enzyme present upon growth with formate and polysulfide is due to the expression of both fdhI and fdhll. In a previous publication [4], the fdhA gene (of the fdhI cluster) was cloned from a genomic library of W. succinogenes using an antiserum raised against FdhA. The fdhA gene preceded the genes fdhB, fdhC and a fourth open reading frame (fdhD), the function of which is not known. The gene fdhA is predicted to encode a prepeptide of the catalytic subunit with an N-terminal signal peptide (32 residues) typical of certain periplasmic proteins or membraneous proteins facing the periplasm [15]. Consistently, the substrate site of the enzyme is oriented towards the periplasmic side of the membrane [6]. While the genes encoding the other electron-transport enzymes (fumarate reductase [16], hydrogenase [ 171 and polysulfide reductase [9]) were found only once in the genome of W succinogenes, there was evidence indicating that a functional formate dehydrogenase could be formed in the absence of the fdhl genes (41. Therefore, the second gene locus (fdhll) was sequenced, and the functions of fdhI and fdhlI were investigated using a AfdhI and a AfdhlI mutant. MATERIALS AND METHODSPhage, bacterial strains, plasmids and growth conditions. Propagation of phage AEMBL3 [18]
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