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During growth with formate as the sole energy source the autotrophic bacterium Alcaligenes eutrophus synthesizes a cytoplasmic formate dehydrogenase. The enzyme is a molybdo-iron-sulfur-flavo protein and the major NADH-producing system under these growth conditions, although it was estimated to constitute only 0.65% of the soluble cell protein. An electron microscopic analysis of the purified enzyme revealed that the particle is made up of four nonidentical submasses, corroborating previous structural data. The NH2-terminal amino acid sequences of the enzyme subunits exhibited significant similarities to those of only one other heteromeric formate dehydrogenase, the enzyme from the methane-utilizing bacterium Methylosinus trichosporium. Metal analyses yielded 21.5 g-atom iron, 2.18 g-atom nickel, 0.76 g-atom molybdenum, and 0.59 g-atom zinc per mol of enzyme. Initial electron paramagnetic resonance spectroscopic studies showed at least three distinct signals which appeared upon reduction of the enzyme with NADH or formate. The corresponding spin systems could be attributed to iron-sulfur centers of the enzyme. Comparative immunostaining and activity-staining experiments using cell extracts from various bacteria established immunological similarities between the soluble formate dehydrogenase of A. eutrophus and the soluble enzymes from all tested facultative autotrophs as well as from M. trichosporium.

We have established that the gene which we had previously identified as encoding the Methanococcus voltae P-type ATPase is, in fact, the structural gene for the M. voitae S-layer protein. This conclusion is based on a comparison of the N-terminal sequence of S-layer protein prepared by two independent methods with that derived from the nucleotide sequence of the cloned gene. This conclusion was further supported by immunocytochemical localization of the antigen directed against the antibodies used in the cloning experiments.High-resolution electron microscopy of thin-sectioned, freeze-etched, freeze-dried, and shadowed or negatively stained prokaryotes has shown that the outermost cell envelope in many species of bacteria and archaea consists of a crystalline cell surface layer, designated the S-layer. In most archaea the S-layer is the only cell envelope component outside the cytoplasmic membrane (1, 12). Among members of the methanogenic archaean order Methanococcales, the S-layer is composed of a regular hexagonal array of protein subunits that completely covers the cell surface (4, 5). Among the methanogens, primary sequence information is available only for the related species Methanothermus fervidus and Methanothermus sociabilis (2).Structural analysis. We have reported the nucleotide sequence of a gene that we believed was the structural gene for a membrane-associated Methanococcus voltae P-type ATPase (GenBank accession number M59200) (3). We now believe that the gene that we cloned encodes the M. voltae S-layer protein.Patel and coworkers developed a procedure for the conversion of M. voltae cells into protoplasts that led to the release of S-layer protein into the protoplasting buffer medium (8). We grew M. voltae PS (DSM 1537) in a medium containing vitamin and mineral supplements under a gas atmosphere of 20% H2 and 80% CO2 at 37°C (13). Cells were taken from the logarithmic growth phase of the culture, and the protoplasts were prepared according to the method described by Patel and coworkers (8) with the modification that 2% NaCl replaced 0.34 M NaCl in the original cell suspension. After removal of the protoplasts by centrifugation, we found that the supernatant was highly enriched in S-layer protein, which we resolved on a one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Fig.

is exemplified by a description of selected enzyme complexes located in the bacterial cytoplasm, in the cytoplasmic membrane or attached to it, in the periplasmic space, and attached to the cell wall or set free into the surrounding medium.