The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg2+ (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.
The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often post-translationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over a hundred proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed non-specifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically-localized. This approach provides an alternative strategy to study surface proteins in the archaea.
S-layer; surface proteins; Methanosarcina acetivorans; Methanosarcina mazei; biotinylation; mass spectrometry; glycoproteins; Concanavalin A
Currently, only one selectable marker is available for genetic studies in the archaeal genus Methanosarcina. Here we report the generation of selectable markers that encode resistance to pseudomonic acid (PAr) in Methanosarcina species by mutagenesis of the isoleucyl-tRNA synthetase gene (ileS) from Methanosarcina barkeri Fusaro. The M. barkeri ileS gene was obtained by screening of a genomic library for hybridization to a PCR fragment. The complete 3,787-bp DNA sequence surrounding and including the ileS gene was determined. As expected, M. barkeri IleS is phylogenetically related to other archaeal IleS proteins. The ileS gene was cloned into a Methanosarcina-Escherichia coli shuttle vector and mutagenized with hydroxylamine. Nine independent PAr clones were isolated after transformation of Methanosarcina acetivorans C2A with the mutagenized plasmids. Seven of these clones carry multiple changes from the wild-type sequence. Most mutations that confer PAr were shown to alter amino acid residues near the KMSKS consensus sequence of class I aminoacyl-tRNA synthetases. One particular mutation (G594E) was present in all but one of the PAr clones. The MIC of pseudomonic acid for M. acetivorans transformed with a plasmid carrying this single mutation is 70 μg/ml of medium (for the wild type, the MIC is 12 μg/ml). The highest MICs (560 μg/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PAr based on the mutant ileS alleles are described. Finally, the implications of the specific mutations we isolated with respect to binding of pseudomonic acid by IleS are discussed.
Methanosarcina is the only acetate-consuming genus of methanogenic archaea other than Methanosaeta and thus is important in methanogenic environments for the formation of the greenhouse gases methane and carbon dioxide. However, little is known about isotopic discrimination during acetoclastic CH4 production. Therefore, we studied two species of the Methanosarcinaceae family, Methanosarcina barkeri and Methanosarcina acetivorans, and a methanogenic rice field soil amended with acetate. The values of the isotope enrichment factor (ɛ) associated with consumption of total acetate (ɛac), consumption of acetate-methyl (ɛac-methyl) and production of CH4 (ɛCH4) were an ɛac of −30.5‰, an ɛac-methyl of −25.6‰, and an ɛCH4 of −27.4‰ for M. barkeri and an ɛac of −35.3‰, an ɛac-methyl of −24.8‰, and an ɛCH4 of −23.8‰ for M. acetivorans. Terminal restriction fragment length polymorphism of archaeal 16S rRNA genes indicated that acetoclastic methanogenic populations in rice field soil were dominated by Methanosarcina spp. Isotope fractionation determined during acetoclastic methanogenesis in rice field soil resulted in an ɛac of −18.7‰, an ɛac-methyl of −16.9‰, and an ɛCH4 of −20.8‰. However, in rice field soil as well as in the pure cultures, values of ɛac and ɛac-methyl decreased as acetate concentrations decreased, eventually approaching zero. Thus, isotope fractionation of acetate carbon was apparently affected by substrate concentration. The ɛ values determined in pure cultures were consistent with those in rice field soil if the concentration of acetate was taken into account.
The compatible solute Nɛ-acetyl-β-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of Nɛ-acetyl-β-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and β-lysine acetyltransferase (ablB), which are assumed to catalyze Nɛ-acetyl-β-lysine formation from α-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei Gö1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei Gö1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Δabl mutants of M. maripaludis no longer produced Nɛ-acetyl-β-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for Nɛ-acetyl-β-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.
In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793) was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458) was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.
Although some data link archaeal and eukaryotic translation, the overall mechanism of protein synthesis in archaea remains largely obscure. Both archaeal (aRF1) and eukaryotic (eRF1) single release factors recognize all three stop codons. The archaeal genus Methanosarcinaceae contains two aRF1 homologs, and also uses the UAG stop to encode the 22nd amino acid, pyrrolysine. Here we provide an analysis of the last stage of archaeal translation in pyrrolysine-utilizing species. We demonstrated that only one of two Methanosarcina barkeri aRF1 homologs possesses activity and recognizes all three stop codons. The second aRF1 homolog may have another unknown function. The mechanism of pyrrolysine incorporation in the Methanosarcinaceae is discussed.
Translation termination; Archeon; Pyrrolysine-utilizing archea; aRF1; Polypeptide release factor
The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.
Methanosarcina acetivorans C2A encodes three putative hydrogenases, including one cofactor F420-linked (frh) and two methanophenazine-linked (vht) enzymes. Comparison of the amino acid sequences of these putative hydrogenases to those of Methanosarcina barkeri and Methanosarcina mazei shows that each predicted subunit contains all the known residues essential for hydrogenase function. The DNA sequences upstream of the genes in M. acetivorans were aligned with those in other Methanosarcina species to identify conserved transcription and translation signals. The M. acetivorans vht promoter region is well conserved among the sequenced Methanosarcina species, while the second vht-type homolog (here called vhx) and frh promoters have only limited similarity. To experimentally determine whether these promoters are functional in vivo, we constructed and characterized both M. acetivorans and M. barkeri strains carrying reporter gene fusions to each of the M. acetivorans and M. barkeri hydrogenase promoters. Generally, the M. acetivorans gene fusions are not expressed in either organism, suggesting that cis-acting mutations inactivated the M. acetivorans promoters. The M. barkeri hydrogenase gene fusions, on the other hand, are expressed in both organisms, indicating that M. acetivorans possesses the machinery to express hydrogenases, although it does not express its own hydrogenases. These data are consistent with specific inactivation of the M. acetivorans hydrogenase promoters and highlight the importance of testing hypotheses generated by using genomic data.
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.
We report here a comparative analysis of the genome sequence of Methanosarcina barkeri with those of Methanosarcina acetivorans and Methanosarcina mazei. The genome of M. barkeri is distinguished by having an organization that is well conserved with respect to the other Methanosarcina spp. in the region proximal to the origin of replication, with interspecies gene similarities as high as 95%. However, it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the distal semigenome. Of the 3,680 open reading frames (ORFs) in M. barkeri, 746 had homologs with better than 80% identity to both M. acetivorans and M. mazei, while 128 nonhypothetical ORFs were unique (nonorthologous) among these species, including a complete formate dehydrogenase operon, genes required for N-acetylmuramic acid synthesis, a 14-gene gas vesicle cluster, and a bacterial-like P450-specific ferredoxin reductase cluster not previously observed or characterized for this genus. A cryptic 36-kbp plasmid sequence that contains an orc1 gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143-nucleotide motif was detected in M. barkeri. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the relatively large M. acetivorans genome is the result of uniformly distributed multiple gene scale insertions and duplications, while the M. barkeri genome is characterized by localized inversions associated with the loss of gene content. In contrast, the short M. mazei genome most closely approximates the putative ancestral organizational state of these species.
The use of F420 as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H2 and CO2, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F420 was followed by high-resolution fluorescence spectroscopy. F420 concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein−1; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g−1 depending on growth conditions. The content of F420 in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F420 content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F420 approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F420 content. However, qCH4(F420), calculated by dividing the methane production rate by the coenzyme F420 concentration, almost paralleled qCH4(protein). These results suggest that F420 may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH4(F420) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.
In natural environments methane is usually produced by aceticlastic and hydrogenotrophic methanogenic archaea. However, some methanogens can use C1 compounds such as methanol as the substrate. To determine the contributions of individual substrates to methane production, the stable-isotope values of the substrates and the released methane are often used. Additional information can be obtained by using selective inhibitors (e.g., methyl fluoride, a selective inhibitor of acetoclastic methanogenesis). We studied stable carbon isotope fractionation during the conversion of methanol to methane in Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderi and generally found large fractionation factors (−83‰ to −72‰). We further tested whether methyl fluoride impairs methylotrophic methanogenesis. Our experiments showed that even though a slight inhibition occurred, the carbon isotope fractionation was not affected. Therefore, the production of isotopically light methane observed in the presence of methyl fluoride may be due to the strong fractionation by methylotrophic methanogens and not only by hydrogenotrophic methanogens as previously assumed.
A mutant of Methanosarcina barkeri (Fusaro) is able to grow on pyruvate as the sole carbon and energy source. During growth, pyruvate is converted to CH4 and CO2, and about 1.5 mol of ATP per mol of CH4 is formed (A.-K. Bock, A. Prieger-Kraft, and P. Schönheit, Arch. Microbiol. 161:33-46, 1994). The pyruvate-utilizing mutant of M. barkeri could also grow on pyruvate when methanogenesis was completely inhibited by bromoethanesulfonate (BES). The mutant grew on pyruvate (80 mM) in the presence of 2 mM BES with a doubling time of about 30 h up to cell densities of about 400 mg (dry weight) of cells per liter. During growth on pyruvate, the major fermentation products were acetate and CO2 (about 0.9 mol each per mol of pyruvate). Small amounts of acetoin, acetolactate, alanine, leucine, isoleucine, and valine were also detected. CH4 was not formed. The molar growth yield (Yacetate) was about 9 g of cells (dry weight) per mol of acetate, indicating an ATP yield of about 1 mol/mol of acetate formed. Growth on pyruvate in the presence of BES was limited; after six to eight generations, the doubling times increased and the final cell densities decreased. After 9 to 11 generations, growth stopped completely. In the presence of BES, suspensions of pyruvate-grown cells fermented pyruvate to acetate, CO2, and H2. CH4 was not formed. Conversion of pyruvate to acetate, in the complete absence of methanogenesis, was coupled to ATP synthesis. Dicyclohexylcarbodiimide, an inhibitor of H(+)-translocating ATP synthase, did not inhibit ATP formation. In the presence of dicyclohexylcarbodiimide, stoichiometries of up to 0.9 mol of ATP per mol of acetate were observed. The uncoupler arsenate completely inhibited ATP synthesis, while the rates of acetate, CO2, and H2 formation were stimulated up to fourfold. Cell extracts of M. barkeri grown on pyruvate under nonmethenogenic conditions contained pyruvate: ferredoxin oxidoreductase (0.5 U/mg), phosphate acetyltransferase (12 U/mg), and acetate kinase (12 U/mg). From these data it is concluded that ATP was synthesized by substrate level phosphorylation during growth of the M. barkeri mutant on pyruvate in the absence of methanogenesis. This is the first report of growth of a methanogen under nonmethanogenic conditions at the expense of a fermentative energy metabolism.
We used 13C-labeled methane to document the extent of trace
methane oxidation by Archaeoglobus fulgidus,
Archaeoglobus lithotrophicus, Archaeoglobus
thermoautotrophicum, Methanosarcina barkeri
and Methanosarcina acetivorans. The results indicate
trace methane oxidation during growth varied among different species
and among methanogen cultures grown on different substrates. The
extent of trace methane oxidation by Mb.
thermoautotrophicum (0.05 ± 0.04%, ± 2 standard
deviations of the methane produced during growth) was less than that
by M. barkeri (0.15 ± 0.04%), grown under
similar conditions with H2 and CO2.
Methanosarcina acetivorans oxidized more methane
during growth on trimethylamine (0.36 ± 0.05%) than during growth
on methanol (0.07 ± 0.03%). This may indicate that, in M.
acetivorans, either a methyltransferase related to growth on
trimethylamine plays a role in methane oxidation, or that methanol is
an intermediate of methane oxidation. Addition of possible electron
acceptors (O2, NO3–,
or H2 to the headspace did not substantially enhance or
diminish methane oxidation in M. acetivorans
cultures. Separate growth experiments with
FAD and NAD+ showed that inclusion of these electron
carriers also did not enhance methane oxidation. Our results suggest
trace methane oxidized during methanogenesis cannot be coupled to the
reduction of these electron acceptors in pure cultures, and that the
mechanism by which methane is oxidized in methanogens is independent
of H2 concentration. In contrast to the methanogens,
species of the sulfate-reducing genus Archaeoglobus
did not significantly oxidize methane during growth (oxidizing 0.003
± 0.01% of the methane provided to A. fulgidus,
0.002 ± 0.009% to A. lithotrophicus and 0.003
± 0.02% to A. profundus). Lack of observable
methane oxidation in the three Archaeoglobus species
examined may indicate that methyl-coenzyme M reductase, which is not
present in this genus, is required for the anaerobic oxidation of
methane, consistent with the “reverse methanogenesis”
anaerobic methane oxidation; Archaeoglobus; methanogen; reverse methanogenesis; stable isotope label
A liquid chromatography-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry approach was used to determine the differential abundance of proteins in acetate-grown cells compared to that of proteins in methanol-grown cells of the marine isolate Methanosarcina acetivorans metabolically labeled with 14N versus 15N. The 246 differentially abundant proteins in M. acetivorans were compared with the previously reported 240 differentially expressed genes of the freshwater isolate Methanosarcina mazei determined by transcriptional profiling of acetate-grown cells compared to methanol-grown cells. Profound differences were revealed for proteins involved in electron transport and energy conservation. Compared to methanol-grown cells, acetate-grown M. acetivorans synthesized greater amounts of subunits encoded in an eight-gene transcriptional unit homologous to operons encoding the ion-translocating Rnf electron transport complex previously characterized from the Bacteria domain. Combined with sequence and physiological analyses, these results suggest that M. acetivorans replaces the H2-evolving Ech hydrogenase complex of freshwater Methanosarcina species with the Rnf complex, which generates a transmembrane ion gradient for ATP synthesis. Compared to methanol-grown cells, acetate-grown M. acetivorans synthesized a greater abundance of proteins encoded in a seven-gene transcriptional unit annotated for the Mrp complex previously reported to function as a sodium/proton antiporter in the Bacteria domain. The differences reported here between M. acetivorans and M. mazei can be attributed to an adaptation of M. acetivorans to the marine environment.
The multi-copy single-stranded DNA (msDNA) is yielded by the action of reverse transcriptase of retro-element in a wide range of
pathogenic bacteria. Upon this phenomenon, it has been shown that msDNA is only produced by Eubacteria because many
Eubacteria species contained reverse transcriptase in their special retro-element. We have screened around 111 Archaea at KEGG
(Kyoto Encyclopedia of Genes and Genomes) database available at genome net server and observed three Methanosarcina species
(M.acetivorans, M.barkeri and M.mazei), which also contained reverse transcriptase in their genome sequences. This observation of
reverse transcriptase in Archaea raises questions regarding the origin of this enzyme. The evolutionary relationship between these
two domains of life (Eubacteria and Archaea) hinges upon the phenomenon of retrons. Interestingly, the evolutionary trees based
on the reverse transcriptases (RTs) and 16S ribosomal RNAs point out that all the Eubacteria RTs were descended from Archaea
RTs during their evolutionary times. In addition, we also have shown some significant structural features among the newly
identified msDNA-Yf79 in Yersinia frederiksenii with other of its related msDNAs (msDNA-St85, msDNA-Vc95, msDNA-Vp96,
msDNA-Ec78 and msDNA-Ec83) from pathogenic bacteria. Together the degree of sequence conservation among these msDNAs,
the evolutionary trees and the distribution of these ret (reverse transcriptase) genes suggest a possible evolutionary scenario. The
single common ancestor of the organisms of Eubacteria and Archaea subgroups probably achieved this ret gene during their
evolution through the vertical descent rather than the horizontal transformations followed by integration into this organism
genome by a mechanism related to phage recognition and/or transposition.
msDNA; reverse transcriptase; phylogenetic tree
The enzyme systems involved in the methyl group transfer from methanol and from tri- and dimethylamine to 2-mercaptoethanesulfonic acid (coenzyme M) were resolved from cell extracts of Methanosarcina barkeri Fusaro grown on methanol and trimethylamine, respectively. Resolution was accomplished by ammonium sulfate fractionation, anion-exchange chromatography, and fast protein liquid chromatography. The methyl group transfer reactions from tri- and dimethylamine, as well as the monomethylamine:coenzyme M methyltransferase reaction, were strictly dependent on catalytic amounts of ATP and on a protein present in the 65% ammonium sulfate supernatant. The latter could be replaced by methyltransferase-activating protein isolated from methanol-grown cells of the organism. In addition, the tri- and dimethylamine:coenzyme M methyltransferase reactions required the presence of a methylcobalamin:coenzyme M methyltransferase (MT2), which is different from the analogous enzyme from methanol-grown M. barkeri. In this work, it is shown that the various methylamine:coenzyme M methyltransfer steps proceed in a fashion which is mechanistically similar to the methanol:coenzyme M methyl transfer, yet with the participation of specific corrinoid enzymes and a specific MT2 isoenzyme.
The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkeri Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labelling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.
Members of all four families of methanogenic bacteria were analyzed for polyamine concentrations. High-performance liquid chromatography analysis of dansylated cell extracts revealed typical polyamine patterns for each family. Members of Methanobacteriaceae (family I) were characterized by very low polyamine concentrations; members of Methanococcaceae (family II) were characterized by putrescine and high spermidine concentrations; members of Methanomicrobiaceae (family III) were characterized by the presence of putrescine, spermidine, and sym-homospermidine; and members of Methanosarcinaceae (family IV) contained only high concentrations of sym-homospermidine in addition to putrescine. The highest polyamine concentration was found in Methanosarcina barkeri Jülich, with 0.35% putrescine in the dry cell material. The polyamine distribution found coincides with the dendrogram based on comparative cataloguing of 16S rRNA and offers a new, rapid chemotaxonomic method for characterizing methanogenic bacteria. Variation of the growth substrates (H2-CO2, methanol, acetate, and trimethylamine) for M. barkeri resulted in quantitative but not qualitative differences in polyamine composition.
Methanosarcina barkeri 227 and Methanosarcina mazei S-6 grew with acetate as the substrate; we found little effect of H2 on the rate of aceticlastic growth in the presence of various H2 pressures between 2 and 810 Pa. We used physical (H2 addition or flushing the headspace to remove H2) and biological (H2-producing or -utilizing bacteria in cocultures) methods for controlling H2 pressure in Methanosarcina cultures growing on acetate. Added H2 (ca. 100 Pa) was removed rapidly (a few hours) by M. barkeri and slowly (within a day) by M. mazei. When the H2 produced by the aceticlastic methanogens was removed by coculturing with an H2-using Desulfovibrio sp., the H2 pressure was about 2.2 Pa. Under these conditions the stoichiometry of aceticlastic methanogenesis did not change. H2-grown inocula of M. barkeri grew with acetate as the sole catabolic substrate if the inoculum culture was transferred during logarithmic growth to acetate-containing medium or if the transfer was accomplished within 1 or 2 days after exhaustion of H2. H2-grown cultures incubated for 4 or more days after exhaustion of H2 were able to grow with H2 but not with acetate as the sole catabolic substrate. Addition of small quantities of H2 to acetate-containing medium permitted these cultures to initiate growth on acetate.
The ability of hydrolytic products of coenzyme F420 to substitute for F420 in the hydrogenase and nicotinamide adenine dinucleotide phosphate-liniked hydrogenase systems of Methanobacterium strain M.o.H. was kinetically determined. The nicotinamide adenine dinucleotide phosphate-linked hydrogenase system was employed to quantitate the levels of F420 in a number of methanogenic bacteria as well as in some nonmethanogens. Methanobacterium ruminantium and Methanosarcina barkeri contained low levels of F420, whereas other methanogens tested contained high levels (100 to 400 mg/kg of cells). F420 from six of the seven methanogens was tested by thin-layer electrophoresis and was found to be electrophoretically identical to that purified from Methanobacterium strain M.o.H. The only exception was M. barkeri, which contained a more electronegative derivative of F420. Acetobacterium woodii, Escherichia coli, and yeast extract contained no compounds able to substitute for F420 in the nicotinamide adenine dinucleotide phosphate-linked hydrogenase system.
Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo.
tRNAPyl; pyrrolysine; mischarging; Methanosarcina barkeri
Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.
A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of Escherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen, Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins. In vitro radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously in E. coli. Introduction of the 4 genes into E. coli resulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of E. coli.