Nutrition and carbon metabolism of Methanococcus voltae.

Methanococcus voltae is a heterotrophic, H2-oxidizing methanogenic bacterium. In complex medium, this bacterium has a doubling time of 1.2 h at its temperature optimum of 38 degrees C. In defined medium, optimal growth is obtained with 0.75 mM isoleucine, 0.75 mM leucine, 2.5 mM acetate, 5 mM NH4Cl, 84 mM MgSO4, 0.4 M NaCl, 1 mM CaCl2, 10 microM Fe2O3, and 0.2 microM NiCl2. In addition, pantothenate, sodium selenate, and cobalt stimulate growth. Optimal growth is obtained between pH 6.0 and 7.0 with either H2 or formate as the electron donor. The volatile fatty acids 2-methylbutyrate and isovalerate can substitute for isoleucine and leucine, respectively. Cellular carbon is derived from acetate (31%), isoleucine (22%), leucine (25%), and carbon dioxide (23%). The amino acids and fatty acids are incorporated almost exclusively into protein. A comparison of the incorporation of U-14C-amino acids and 1-14C-fatty acids indicated that the fatty acids are degraded during incorporation into cell protein. The distribution of carbon from the amino acids suggests that acetyl coenzyme A is not a major intermediate in the degradation of these compounds. Thus, M. voltae may convert isoleucine and leucine to other amino acids by a unique mechanism. The lipid carbon is derived largely from acetate. Thus, the isoprenoid lipids are synthesized de novo from acetate rather than by degradation of leucine. The carbon in the nucleic acids is derived from carbon dioxide (45%), the C-1 of acetate (25%), the C-2 of acetate (22%), and isoleucine and leucine (7%). This labeling pattern is consistent with known biochemical pathways.     

Chemotaxis in the archaebacterium Methanococcus voltae.

The archaebacterium Methanococcus voltae, was shown to be chemotactic. Acetate, isoleucine, and leucine were identified as attractants; whereas histidine was not an attractant. A motile, generally nonchemotactic mutant was isolated.     

Formation and Regeneration of Methanococcus voltae Protoplasts

Methanococcus voltae cells were converted into protoplasts by suspension in anaerobic 0.1 M Tris-HCl buffer containing 0.4 M sucrose and 0.05 M NaCl as osmoprotectants. Protoplast formation was monitored microscopically by observing the conversion of the typical irregularly shaped (uneven peripheries) coccoid whole cells to rounded forms with smooth peripheries. Although the procedure resulted in about 50% lysis of the initial number of cells, the remainder were converted to the rounded form. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy of negatively stained cell preparations indicated that the treatment removed the wall layer from whole cells to yield protoplasts. Protoplast regeneration was evaluated by using optimized plating conditions and an anaerobic microplating technique. Between 50 and 63% of the initial number of protoplasts regenerated as colonies on agar medium (35°C, 7 days). The colony and cell morphologies of the regenerated protoplasts were indistinguishable from those of whole cells plated under identical conditions.     

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.     

Identification of the Methanococcus voltae S-layer structural gene.

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. voltae 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.     

Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae.

Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min(-1) mg of protein(-1) and had an apparent Km for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min(-1) mg of protein(-1). Hypoxanthine PRTase activity was present at 0.85 nmol min(-1) mg of protein(-1) with an apparent Km for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min(-1) mg of protein(-1). PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.     

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.     

Posttranslational processing of Methanococcus voltae preflagellin by preflagellin peptidases of M. voltae and other methanogens

Methanococcus voltae is a mesophilic archaeon with flagella composed of flagellins that are initially made with 11- or 12-amino-acid leader peptides that are cleaved prior to incorporation of the flagellin into the growing filament. Preflagellin peptidase activity was demonstrated in immunoblotting experiments with flagellin antibody to detect unprocessed and processed flagellin subunits. Escherichia coli membranes containing the expressed M. voltae preflagellin (as the substrate) were combined in vitro with methanogen membranes (as the enzyme source). Correct processing of the preflagellin to the mature flagellin was also shown directly by comparison of the N-terminal sequences of the two flagellin species. M. voltae preflagellin peptidase activity was optimal at 37 degrees C and pH 8.5 and in the presence of 0.4 M KCl with 0.25% (vol/vol) Triton X-100.     

Hydroxydiether Lipid Structures in Methanosarcina spp. and Methanococcus voltae

Hydroxylated diether lipids are the most abundant lipids in Methanosarcina acetivorans, Methanosarcina thermophila, and Methanosarcina barkeri MS and Fusaro, regardless of the substrate used for growth. Structural analysis of the lipid moiety freed of polar head groups revealed that the hydroxydiether lipids of all the Methanosarcina strains were hydroxylated at position 3 of sn-2 phytanyl chains. The finding that Methanosarcina strains synthesize the same hydroxydiether structure suggests that this is a taxonomic characteristic of the genus. Methanococcus voltae produced minor amounts of the 3-hydroxydiether characteristic of Methanosarcina spp. and also the 3′-hydroxydiether described previously for Methanosaeta concilii.     

Ultrastructure and biochemistry of the cell wall of Methanococcus voltae.

The ultrastructure and chemical composition of the cell wall of the marine archaebacterium Methanococcus voltae were studied by negative-staining and freeze-etch electron microscopy and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. M. voltae possesses a single regularly structured (RS) protein layer external to the plasma membrane. Freeze-etch preparations of cells indicated that the protein subunits are hexagonally arranged with a center-to-center spacing of approximately 10 nm. The extracted RS protein had a molecular weight of 76,000. It was present on envelopes prepared by shearing in a French press, osmotic lysis, or sonication, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NaCl was not required for attachment of the RS protein to the underlying plasma membrane. The hexagonal array could be demonstrated by platinum shadowing and freeze-etching of envelopes, but negative staining in the abscence of NaCl failed to stabilize the array. The RS protein could be solubilized by urea, guanidine hydrochloride, dithiothreitol, and several detergents, including Nonidet P-40, Triton X-100, and Tween 20. However, the most specific release of the wall protein from envelopes occurred after a heat treatment in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at 50 to 60 degrees C.     

Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii.

The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens. Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D. J. Hei and D. S. Clark, Appl. Environ. Microbiol. 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.     

Genetic transformation in the methanogen Methanococcus voltae PS.

Mutations causing requirements for histidine, purine, and vitamin B12 were obtained in strain PS of Methanococcus voltae (archaebacteria) upon irradiation with UV or gamma rays. The first two mutations were shown to revert at low frequencies and were used to demonstrate the occurrence of transformation with homologous, wild-type DNA. The transformation rates obtained for these presumably chromosomal markers were in the range of 2 to 100 transformants per microgram of DNA. Mutants resistant to 2-bromoethanesulfonate and to 5-methyl-DL-tryptophan were also isolated.     

Identification of a vanadate-sensitive, membrane-bound ATPase in the archaebacterium Methanococcus voltae.

Membrane-bound ATPase activity was detected in the methanogen Methanococcus voltae. The ATPase was inhibited by vanadate, a characteristic inhibitor of E1E2 ATPases. The enzyme activity was also inhibited by diethylstilbestrol. However, it was insensitive to N,N'-dicyclohexylcarbodiimide, ouabain, and oligomycin. The enzyme displayed a high preference for ATP as substrate, was dependent on Mg2+, and had a pH optimum of approximately 7.5. The enzyme was completely solubilized with 2% Triton X-100. The enzyme was insensitive to oxygen and was stabilized by ATP. There was no homology with the Escherichia coli F0F1 ATPase at the level of DNA and protein. The membrane-bound M. voltae ATPase showed properties similar to those of E1E2 ATPases.     

Physical and genetic map of the Methanococcus voltae chromosome

A physical map of the Methanococcus voltae chromosome was constructed on the basis of restriction mapping and cross-hybridization experiments, employing total and partial digests obtained with rarely cutting restriction enzymes. On the basis of the sum of the fragment sizes of digests with seven enzymes the chromosome length was calculated to be approximately 1900 kb. The derived map is circular. Hybridization of gene probes to mapped restriction fragments has led to a genetic map of genes for structural RNAs as well as proteins, including enzymes involved in the methanogenic pathway.     

The DNA polymerase gene from the methanogenic archaeon Methanococcus voltae.

Previous studies have identified intervening sequences that encode homing endonucleases within the genes encoding several archaeal DNA polymerases. We report the sequence of the gene encoding the DNA polymerase of Methanococcus voltae and describe evidence that it lacks analogous intervening sequences.     

Cloning and sequencing of a multigene family encoding the flagellins of Methanococcus voltae.

The flagellins of Methanococcus voltae are encoded by a multigene family of four related genes (flaA, flaB1, flaB2, and flaB3). All four genes map within the same region of the genome, with the last three arranged in a direct tandem. Northern (RNA) blot and primer extension analyses of total cellular RNA indicate that all four genes are transcribed. The flaB1, flaB2, and flaB3 flagellins are transcribed as part of a large polycistronic message which encodes at least one more protein which is not a flagellin. An intercistronic stem-loop followed by a poly(T) tract located between the flaB2 and flaB3 genes appears to increase the resistance of the flaB1/flaB2 portion of this polycistronic message to digestion by endogenous RNases. The flaA gene, located approximately 600 bp upstream from the tandem, is transcribed as a separate message at very low levels. The four open reading frames encode proteins of molecular weights 23,900, 22,400, 22,800, and 25,500, much less than the Mr values of 33,000 and 31,000 for the flagellins calculated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated flagellar filaments. Each flagellin contains multiple eukaryotic glycosylation signals (Arg-X-Ser/Thr), although they do not appear to be glycoproteins, and each has an 11- or 12-amino-acid leader peptide. The N termini of all four flagellins (amino acids 1 through 47 of the mature protein) are very hydrophobic, and this region shows a high degree of homology with the flagellins from Halobacterium halobium.     

Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus voltae.

An auxotrophic mutant of Methanococcus voltae was isolated that required coenzyme M (CoM) for growth. With the mutant as a recipient, conditions were developed that allowed the introduction of wild-type CoM+ DNA into the mutant methanogen via electroporation. This method also allowed the rescue of both a histidine and purine auxotroph as well as the introduction of DNA determining resistance to the CoM analog 2-bromoethanesulfonic acid. Electroporation of the CoM(+)-determining DNA was 50- to 80-fold more efficient than natural transformation.