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Neorickettsia sennetsu is an obligate intracellular bacterium of monocytes and macrophages and is the etiologic agent of human Sennetsu neorickettsiosis. Neorickettsia proteins expressed in mammalian host cells, including the surface proteins of Neorickettsia spp., have not been defined. In this paper, we isolated surface-exposed proteins from N. sennetsu by biotin surface labeling followed by streptavidin-affinity chromatography. Forty-two of the total of 936 (4.5%) N. sennetsu open reading frames (ORFs) were detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS), including six hypothetical proteins. Among the major proteins identified were the two major [beta]-barrel proteins: the 51-kDa antigen (P51) and Neorickettsia surface protein 3 (Nsp3). Immunofluorescence labeling not only confirmed surface exposure of these proteins but also showed rosary-like circumferential labeling with anti-P51 for the majority of bacteria and polar to diffuse punctate labeling with anti-Nsp3 for a minority of bacteria. We found that the isolated outer membrane of N. sennetsu had porin activity, as measured by a proteoliposome swelling assay. This activity allowed the diffusion of L-glutamine, the monosaccharides arabinose and glucose, and the tetrasaccharide stachyose, which could be inhibited with anti-P51 antibody. We purified native P51 and Nsp3 under nondenaturing conditions. When reconstituted into proteoliposomes, purified P51, but not Nsp3, exhibited prominent porin activity. This the first proteomic study of a Neorickettsia sp. showing new sets of proteins evolved as major surface proteins for Neorickettsia and the first identification of a porin for the genus Neorickettsia. doi: 10.1128/JB.00632-10

Methanogens represent some of the most oxygen-sensitive organisms in laboratory culture. Recent studies indicate that they have developed mechanisms to deal with brief oxygen exposure. MsvR is a transcriptional regulator that has a domain architecture unique to a select group of methanogens. Here, runoff in vitro transcription assays were used to demonstrate that MsvR regulates transcription of the divergently transcribed fpaA-rlp-rub operon in Methanothermobacter thermautotrophicus in addition to transcription from its own promoter. The protein products of the fpaA-rlp-rub operon have previously been implicated in oxidative stress responses in M. thermautotrophicus. Additionally, electrophoretic mobility shift assays (EMSAs) and DNase I footprinting were used to confirm a binding site inferred by bioinformatic analysis. Sequence mutations within these binding sites did not significantly alter EMSA shifting patterns on longer templates but did on shorter 50-bp fragments encompassing only the region containing the binding sites. Footprinting confirmed that the regions protected for the longer mutant templates are at different positions within the intergenic region compared to those seen in the intact intergenic region. Oxidized and reduced preparations of MsvR demonstrated different EMSA binding patterns and regions of protection on the intergenic sequence, suggesting that MsvR may play a role in detecting the redox state of the cell. doi: 10.1128/JB.00816-10

The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic C[O.sub.2] assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). In addition, a gene replacement mutation was constructed for a gene encoding a putative membrane-spanning subunit (ehbO). When grown in minimal medium plus acetate (McA), all ehb mutants had severe growth deficiencies except the [DELTA]ehbO::pac strain. The membrane-spanning ion translocator ([DELTA]ehbF) and the large hydrogenase subunit ([DELTA]ehbN) deletion strains displayed the severest growth defects. Deletion of the ehbN gene was of particular interest because this gene was not contiguous to the ehb operon. In-gel activity assays and Western blots confirmed that EhbN was part of the membrane-bound Ehb hydrogenase complex. The [DELTA]ehbN strain was also sensitive to growth inhibition by aryl acids, indicating that Ehb was coupled to the indolepyruvate oxidoreductase (Ior), further supporting the hypothesis that Ehb provides low potential reductants for the anabolic oxidoreductases in M. maripaludis. doi: 10.1128/JB.01446-09

The upflow anaerobic sludge blanket (UASB) reactor is a microcosm for the methanogenic degradation of organic matter in anaerobic environments, and depends on the auto-formation of dense 3D biofilms of 1-3 mm in diameter, referred to as granular sludge (biogranules). Past research has shown that UASB and other methanogenic reactors are extremely stable functionally, but the underlying basis of the functional stability is not well understood. In this study, microbial dynamics in the communities residing in UASB biogranules were analysed to determine responses to short-term perturbations (change in reactor feed). The reactor was fed with simulated brewery wastewater (SBWW) for 1.5 months (phase 1), acetate/sulfate for 2 months (phase 2), acetate alone for 3 months (phase 3) and then a return to SBWW for 2 months (phase 4). Analysis of 16S rRNA, methanogen-associated mcrA and sulfate reducer-associated dsrAB gene-based-clone libraries showed a relatively simple community composed mainly of the methanogenic archaea (Methanobacterium and Methanosaeta), members of the green non-sulfur (Chloroflexi) group of bacteria and Syntrophobacter, Spirochaeta, Acidobacteria and Cytophaga-related bacterial sequences. The mcrA clone libraries were dominated throughout by Methanobacterium- and Methanospirillum-related sequences. Although the reactor performance remained relatively stable throughout the experiment, community diversity levels generally decreased for all libraries in response to a change from SBWW to acetate alone feed. There was a large transitory increase noted in 1 6S diversity at the 2 month sampling on acetate alone, entirely related to an increase in bacterial diversity. Upon return to SBWW conditions in phase 4, all diversity measures returned to near phase 1 levels. Our results demonstrated that microbial communities, even highly structured ones such as in UASB biogranules, are very capable of responding to rapid and major changes in their environment. DOI 10.1099/mic.0.036715-0

The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to LL-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapLl group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Ammp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using LL-DAP and [alpha]-ketoglutarate as substrates was 24.3 [+ or -] 2.0 nmol [min.sup.-1] mg of [protein.sup.-1]. The gene encoding the DapL homolog in Methanocaidococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70[degrees]C and pH 8.0 to 9.0. The apparent [K.sub.m]s of MJ1391 for LL-DAP and [alpha]-ketoglutarate were 82.8 [+ or -] 10 [micro]M and 0.42 [+ or -] 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl- DAP as a substrate. Phyiogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales. doi: 10.1128/JB.00172-10

In eukaryotes, a complex of six highly related minichromosome maintenance (MCM) proteins is believed to function as the replicative helicase. Until recently, systems for exploring the molecular mechanisms underlying eukaryotic MCM function have been biochemically intractable. To overcome this, molecular studies of MCM function have been carried out using MCM homologues from the archaea. Archaeal MCM systems studied to date possess a single functional MCM, which forms a homohexameric complex that displays DNA binding, ATPase and helicase activities. We have identified an archaeal order that possesses multiple MCM homologues. BLAST searches of available Methanococcales genomes reveal that members of this order possess between two and eight MCM homologues. Phylogenetic analysis suggests that an ancient duplication in the Methanococcales gave rise to two major groups of MCMs. One group contains Methanococcus maripaludis S2 McmD and possesses a conserved C-terminal insert similar to one observed in eukaryotic MCM3, while the other group contains McmA, -B and -C. Analysis of the genome context of MCMs in the latter group indicates that these genes could have arisen from phagemediated events. When co-expressed in Escherichia coli, the four MCMs from M. maripaludis copurify, indicating the formation of heteromeric complexes in vitro. The presence of homologues from both groups in all Methanococcales indicates that there could be functionally important differences between these proteins and that Methanococcales MCMs may therefore provide an interesting additional model for eukaryotic MCM function. DOI 10.1099/mic.0.036707-0

The roles of three TATA binding protein (TBP) homologs (TBP1, TBP2, and TBP3) in the archaeon Methanosarcina acetivorans were investigated by using genetic and molecular approaches. Although tbp2 and tbp3 deletion mutants were readily obtained, a tbp1 mutant was not obtained, and the growth of a conditional tbp1 expression strain was tetracycline dependent, indicating that TBP1 is essential. Transcripts of tbp1 were 20-fold more abundant than transcripts of tbp2 and 100-to 200-fold more abundant than transcripts of tbp3, suggesting that TBP1 is the primary TBP utilized during growth. Accordingly, tbp1 is strictly conserved in the genomes of Methanosarcina species. [DELTA]tbp3 and [DELTA]tbp2 strains exhibited an extended lag phase compared with the wild type, although the lag phase for the [DELTA]tbp2 strain was less pronounced when this strain was transitioning from growth on methylotrophic substrates to growth on acetate. Acetate-adapted Atbp3 cells exhibited growth rates, final growth yields, and lag times that were significantly reduced compared with those of the wild type when the organisms were cultured with growth-limiting concentrations of acetate, and the acetate-adapted [DELTA]tbp2 strain exhibited a final growth yield that was reduced compared with that of the wild type when the organisms were cultured with growth-limiting acetate concentrations. DNA microarray analyses identified 92 and 77 genes with altered transcription in the [DELTA]tbp2 and [DELTA]tbp3 strains, respectively, which is consistent with a role for TBP2 and TBP3 in optimizing gene expression. Together, the results suggest that TBP2 and TBP3 are required for efficient growth under conditions similar to the conditions in the native environment of M. acetivorans. doi: 10.1128/JB.01165-09

Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastie pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei [DELTA]ech mutant which shows decreased ferredoxin-dependent membrane-bound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated [C.sub.1] compounds. doi: 10.1128/JB.01307-09

[tRNA.sup.His] guanylyltransferase (Thg1) post-transcriptionally adds a G (position-1) to the 5'-terminus of [tRNA.sup.His]. The Methanosarcina acetivorans Thg1 (MaThg1) gene contains an in-frame TAG (amber) codon. Although a UAG codon typically directs translation termination, its presence in Methanosarcina mRNA may lead to pyrrolysine (Pyl) incorporation achieved by Pyl-[tRNA.sup.Pyl], the product of pyrrolysyl-tRNA synthetase. Sequencing of the MaThg1 gene and transcript confirmed the amber codon. Translation of MaThg1 mRNA led to a full-length, Pyl-containing, active enzyme as determined by immunoblotting, mass spectrometry, and biochemical analysis. The nature of the inserted amino acid at the position specified by UAG is not critical, as Pyl or Trp insertion yields active MaThg1 variants in M. acetivorans and equal amounts of full-length protein. These data suggest that Pyl insertion is akin to natural suppression and unlike the active stop codon reassignment that is required for selenocysteine insertion. Only three Pyl-containing proteins have been characterized previously, a set of methylamine methyltransferases in which Pyl is assumed to have specifically evolved to be a key active-site constituent. In contrast, Pyl in MaThg1 is a dispensable residue that appears to confer no selective advantage. Phylogenetic analysis suggests that Thg1 is becoming dispensable in the archaea, and furthermore supports the hypothesis that Pyl appeared in MaThg1 as the result of neutral evolution. This indicates that even the most unusual amino acid can play an ordinary role in proteins. amber codon | Methanosarcina acetivorans | natural supression doi/10.1073/pnas.0912072106

Methanogenic oxidation of butyrate to acetate requires a tight cooperation between the syntrophically fermenting Syntrophomonas wolfei and the methanogen Methanospirillum hungatei, and a reversed electron transport system in S. wolfei was postulated to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH for [H.sub.2] generation. The metabolic activity of butyrate-oxidizing S. wolfei cells was measured via production of formazan and acetate from butyrate, with 2,3,5-triphenyltetrazolium chloride as electron acceptor. This activity was inhibited by trifluoperazine (TPZ), an antitubercular agent known to inhibit NADH:menaquinone oxidoreductase. In cell extracts of S. wolfei, the oxidation of NADH could be measured with quinones, viologens, and tetrazolium dyes as electron acceptors, and also this activity was inhibited by TPZ. The TPZ-sensitive NADH:acceptor oxidoreductase activity appeared to be membrane associated but could be dissociated from the membrane as a soluble protein and was semipurified by anion-exchange chromatography. Recovered proteins were identified by peptide mass fingerprinting, which indicated the presence of an NADH:acceptor oxidoreductase as part of a three-component [FeFe] hydrogenase complex and a selenocysteine-containing formate dehydrogenase. Furthermore, purification of butyryl-CoA dehydrogenase (Bcd) activity and peptide mass fingerprinting revealed two Bcd proteins different from the Bcd subunit of the Bcd/electron-transfer flavoprotein complex (Bcd/EtfAB) predicted from the genome sequence of S. wolfei. The results suggest that syntrophic oxidation of butyrate in S. wolfei involves a membrane-associated TPZ-sensitive NADH:acceptor oxidoreductase as part of a hydrogenase complex similar to the recently discovered 'bifurcating' hydrogenase in Thermotoga maritima and butyryl-CoA dehydrogenases that are different from Bcd of the Bcd/EtfAB complex. doi: 10.1128/JB.01605-08

Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukary. otic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis, a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslationai modifications.

Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:C[O.sub.2] oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:C[O.sub.2] interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the [[alpha].sub.2][[epsilon].sub.2] component from the [[alpha].sub.8][[beta].sub.8][[gamma].sub.8][[delta].sub.8][[epsilon].sub.8] CODH/acetyl-CoA decarbonylase/ synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and [H.sub.2]O/O[H.sup.-] bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the [epsilon]-subunit and FeS domain not present in nonmethanogenic Ni-CODHs. carbon dioxide | carbon monoxide | oxidoreductase | acetate | methanogenesis

The Cdc6-1 and -2 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind the minichromosome maintenance (MCM) helicase. It is shown here that Cdc6-2 protein dissociates the MCM complex. This observation supports the hypothesis that the Cdc6-2 protein functions as a helicase loader.

The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the M J0056 gene. Recombinant expression of the M J0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

Continuous culture, transcriptome arrays, and measurements of cellular amino acid pools and tRNA charging levels were used to determine the response of Methanococcus maripaludis to leucine limitation. For comparison, the responses to phosphate and [H.sub.2] limitations were measured as well. In addition, the effect of growth rate was determined. Leucine limitation resulted in a broad response, [tRNA.sup.Leu] charging decreased, but only small increases in mRNA were seen for amino acid biosynthesis genes. However, the cellular levels of free isoleucine and valine showed significant increases, indicating a coordinate regulation of branched-chain amino acids at a post-mRNA level. Leucine limitation also resulted in increased mRNA abundance for ribosomal protein genes, increased rRNA abundance, and decreased mRNA abundance for genes of methanogenesis. In contrast, phosphate limitation induced a specific response, a marked increase in mRNA levels for a phosphate transporter. Some mRNA levels responded to more than one factor; for example, transcripts for flagellum synthesis genes decreased under conditions of leucine limitation and increased under [H.sub.2] limitation. Increased growth rate resulted in increased mRNA levels for ribosomal protein genes, increased rRNA abundance, and increased mRNA for a gene encoding an S-layer protein.

A series of Methanosarcina barkeri mutants lacking the genes encoding the enzymes involved in the C1 oxidation/reduction pathway were constructed. Mutants lacking the methyl-tetrahydromethanopterin ([H.sub.4]MPT):coenzyme M (CoM) methyltransferase-encoding operon ([DELTA]mtr), the methylene-[H.sub.4]MPT reductase-encoding gene ([DELTA]mer), the methylene-[H.sub.4]MPT dehydrogenase-encoding gene ([DELTA]mtd), and the formyl-methanofuran:[H.sub.4]MPT formyl-transferase-encoding gene ([DELTA]flr) all failed to grow using either methanol or [H.sub.2]/C[O.sub.2] as a growth substrate, indicating that there is an absolute requirement for the C1 oxidation/reduction pathway for hydrogenotrophic and methylotrophic methanogenesis. The mutants also failed to grow on acetate, and we suggest that this was due to an inability to generate the reducing equivalents needed for biosynthetic reactions. Despite their lack of growth on methanol, the [DELTA]mtr and [DELTA]mer mutants were capable of producing methane from this substrate, whereas the [DELTA]mtd and [DELTA]ftr mutants were not. Thus, there is an Mtr/Mer bypass pathway that allows oxidation of methanol to the level of methylene-[H.sub.4]MPT in M. barkeri. The data further suggested that formaldehyde may be an intermediate in this bypass; however, no methanol dehydrogenase activity was found in [DELTA]mtr cell extracts, nor was there an obligate role for the formaldehyde-activating enzyme (Fae), which has been shown to catalyze the condensation of formaldehyde and [H.sub.4]MPT in vitro. Both the [DELTA]mer and [DELTA]mtr mutants were able to grow on a combination of methanol plus acetate, but they did so by metabolic pathways that are clearly distinct from each other and from previously characterized methanogenic pathways.

Methanotrophic bacteria constitute a ubiquitous group of microorganisms playing an important role in the biogeochemical carbon cycle and in control of global warming through natural reduction of methane emission. These bacteria share the unique ability of using methane as a sole carbon and energy source and have been found in a great variety of habitats. Phylogenetically, known methanotrophs constitute a rather limited group and have so far only been affiliated with the Proteobacteria. Here, we report the isolation and initial characterization of a nonproteobacterial obligately methanotrophic bacterium. The isolate, designated Kam1, was recovered from an acidic hot spring in Kamchatka, Russia, and is more thermoacidophilic than any other known methanotroph, with optimal growth at [approximately equal to] 55[degrees]C and pH 3.5. Kam1 is only distantly related to all previously known methanotrophs and belongs to the Verrucomicrobia lineage of evolution. Genes for methane monooxygenases, essential for initiation of methane oxidation, could not be detected by using standard primers in PCR amplification and Southern blot analysis, suggesting the presence of a different methane oxidation enzyme. Kam1 also lacks the well developed intracellular membrane systems typical for other methanotrophs. The isolate represents a previously unrecognized biological methane sink, and, due to its unusual phylogenetic affiliation, it will shed important light on the origin, evolution, and diversity of biological methane oxidation and on the adaptation of this process to extreme habitats. Furthermore, Kaml will add to our knowledge of the metabolic traits and biogeochemical roles of the widespread but poorly understood Verrucomicrobia phylum. extremophile | methanotroph

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 homoiogs 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.

Methanosarcina barkeri is a strictly anaerobic methanogenic archaeon, which can survive oxidative stress. The oxidative stress agent paraquat (PQ) suppressed growth of M. barkeri at concentrations of 50-200 [micro]M. Hydrogen peroxide ([H.sub.2][O.sub.2]) inhibited growth at concentrations of 0.4-1.6 mM. Catalase activity in cell-free extracts of M. barkeri increased about threefold during [H.sub.2][O.sub.2] stress (1.3 mM [H.sub.2][O.sub.2], 2-4 h exposure) and nearly twofold during superoxide stress (160 [micro]M PQ, 2 h exposure). PQ (160 [micro]M, 2-4 h exposure) and [H.sub.2][O.sub.2] (1.3 mM, 2 h exposure) also influenced superoxide dismutase activity in cell-free extracts of M. barkeri. Dot-blot analysis was performed on total RNA isolated from [H.sub.2][O.sub.2]- and PQ-exposed cultures, using labelled internal DNA fragments of the sod and kat genes. It was shown that [H.sub.2][O.sub.2] but not PQ strongly induced up-regulation of the kat gene. PQ and to a lesser degree [H.sub.2][O.sub.2] induced the expression of superoxide dismutase. The results indicate the regulation of the adaptive response of M. barkeri to different oxidative stresses.

Archaea have been shown to produce isoprenoids from mevalonate; however, genome analysis has failed to identify several genes in the mevalonate pathway on the basis of sequence similarity. A predicted archaeal kinase, coded for by the MJ0044 gene, was associated with other mevalonate pathway genes in the archaea and was predicted to be the 'missing' phosphomevalonate kinase. The MJ0044-derived protein was tested for phosphomevalonate kinase activity and was found not to catalyze this reaction. The MJ0044 gene product was found to phosphorylate isopentenyl phosphate, generating isopentenyl diphosphate. Unlike other known kinases associated with isoprene biosynthesis, Methanocaldococcus jannaschii isopentenyl phosphate kinase is predicted to be a member of the aspartokinase superfamily.

Homologous recombination (HR) and lateral gene transfer are major processes in genome evolution. The combination of the two processes, HR between genes in different species, has been documented but is thought to be restricted to very similar sequences in relatively closely related organisms. Here we report two cases of interspecific HR in the gene encoding the core translational protein translation elongation factor 1[alpha] (EF-1[alpha]) between distantly related archaeal groups. Maximum-likelihood sliding window analyses indicate that a fragment of the EF-1[alpha] gene from the archaeal lineage represented by Methanopyrus kandleri was recombined into the orthologous gene in a common ancestor of the Thermococcales. A second recombination event appears to have occurred between the EF-1[alpha] gene of the genus Methanothermobacter and its ortholog in a common ancestor of the Methanosarcinales, a distantly related euryarchaeal lineage. These findings suggest that HR occurs across a much larger evolutionary distance than generally accepted and affects highly conserved essential 'informational' genes. Although difficult to detect by standard whole-gene phylogenetic analyses, interspecific HR in highly conserved genes may occur at an appreciable frequency, potentially confounding deep phylogenetic inference and hypothesis testing. hypothesis testing | lateral gene transfer | maximum likelihood | sliding window analysis

Methanosphaera stadtmanae has the most restricted energy metabolism of all methanogenic archaea. This human intestinal inhabitant can generate methane only by reduction of methanol with [H.sub.2] and is dependent on acetate as a carbon source. We report here the genome sequence of M. stadtmanae, which was found to be composed of 1,767,403 bp with an average G+C content of 28% and to harbor only 1,534 protein-encoding sequences (CDS). The genome lacks 37 CDS present in the genomes of all other methanogens. Among these are the CDS for synthesis of molybdopterin and for synthesis of the carbon monoxide dehydrogenase/acetylcoenzyme A synthase complex, which explains why M. stadtmanae cannot reduce C[O.sub.2] to methane or oxidize methanol to C[O.sub.2] and why this archaeon is dependent on acetate for biosynthesis of cell components. Four sets of mtaABC genes coding for methanol:coenzyme M methyltransferases were found in the genome of M. stadtmanae. These genes exhibit homology to mta genes previously identified in Methanosarcina species. The M. stadtmanae genome also contains at least 323 CDS not present in the genomes of all other archaea. Seventythree of these CDS exhibit high levels of homology to CDS in genomes of bacteria and eukaryotes. These 73 CDS include 12 CDS which are unusually long (>2,400 bp) with conspicuous repetitive sequence elements, 13 CDS which exhibit sequence similarity on the protein level to CDS encoding enzymes involved in the biosynthesis of cell surface antigens in bacteria, and 5 CDS which exhibit sequence similarity to the subunits of bacterial type I and III restriction-modification systems.

The archaeon Methanococcus voltae needs selenium for optimal growth. A gene group most likely involved in the demethylation of dimethylselenide was discovered, the expression of which is induced upon selenium deprivation. The operon comprises open reading frames for a corrinoid protein and two putative methyltransferases. It is shown that the addition of dimethylselenide to selenium-depleted growth medium relieves the lack of selenium, as indicated by the repression of a promoter of a transcription unit encoding selenium-free hydrogenases which is normally active only upon selenium deprivation. Knockout mutants of the corrinoid protein or one of the two methyltransferase genes did not show repression of the hydrogenase promoter in the presence of dimethylselenide. The mutation of the other methyltransferase gene had no effect. Growth rates of the two effective mutants were reduced compared to wild-type cells in selenium-limited medium in the presence of dimethylselenide.

The methanotrophic bacterium Methylosinus trichosporium OB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at Iow copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the smmo operon is initiated from a [[sigma].sup.N] promoter 5' of mmoX. In this study the genes encoding [[sigma].sup.N] (rpoN) and a typical [[sigma].sup.N] -dependent transcriptional activator (mmoR) were cloned and sequenced, mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 5' of the structural genes for the sMMO enzyme. Subsequent mutation of rpoN and mmoR by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of mmoX. An rpoN mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that [[sigma].sup.N] plays a role in both nitrogen and carbon metabolism in Ms. trichosporium OB3b. The data also indicate that mmoG is transcribed in a [[sigma].sup.N] and MmoR-independent manner. Marker-exchange mutagenesis of mmoG revealed that MmoG is necessary for smmo gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR in vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation of smmo gene expression.

The key enzyme in methane metabolism is methane monooxygenase (MMO), which catalyses the oxidation of methane to methanol. Some methanotrophs, including Methylococcus capsulatus (Bath), possess two distinct MMOs. The level of copper in the environment regulates the biosynthesis of the MMO enzymes in these methanotrophs. Under low-copper conditions, soluble MMO (sMMO) is expressed and regulation takes place at the level of transcription. The structural genes of sMMO were previously identified as mmoXYBZ, mmoD and mmoC. Putative transcriptional start sites, containing a [[sigma].sup.70] and a [[sigma].sup.N]-dependent motif, were identified in the 5' region of mmoX. The promoter region of mmoX was mapped using truncated 5' end regions fused to a promoterless green fluorescent protein gene. A 9.5 kb region, adjacent to the sMMO structural gene cluster, was analysed. Downstream (3') from the last gene of the operon, mmoC, four ORFs were found, mmoG, mmoQ, mmoS and minoR, mmoG shows significant identity to the large subunit of the bacterial chaperonin gene, groEL. In the opposite orientation, two genes, mmoQ and mmoS, showed significant identity to two-component sensor-regulator system genes. Next to mmoS, a gene encoding a putative [[sigma].sup.N]-dependent transcriptional activator, mmoR was identified. The mmoG and mmoR genes were mutated by marker-exchange mutagenesis and the effects of these mutations on the expression of sMMO was investigated. sMMO transcription was impaired in both mutants. These results indicate that mmoG and minor are essential for the expression of sMMO in Mc. capsulatus (Bath).

Research addresses the origin of methane precursors in an anoxic acidic bog peat and identification of microorganisms associated with methanogenesis. Results suggest that hydrogen is the substrate of the methanogens, hydrogen participates in the organic carbon degradation, and fatty acids inhibit methanogenesis. The methanogens belong to three bacterial families.

Results demonstrate that the early steps of riboflavin biosynthesis in Methanococcus jannaschii and M. thermophilia are very similar to those in yeasts and fungi. The biosynthesis is mediated by a pyrimidine nucleotide reductase.

The Methanococcus jannaschii MJ256 gene, termed cofD, encodes CofD enzyme that catalyzes the fourth step in the biosynthesis of coenzyme F(sub)420. On the basis of the reactions catalyzed by CofD, the enzyme is called lactyl(2) diphospho-(5')guanosine:Fo 2-phospho-L-lactate transferase. The enzyme requires magnesium for its activity.

Results demonstrate that zinc is required for methyltransferase enzyme activity and it tightly binds to the enzyme whereas thiolate binds to the metal center of the enzyme, together forming metal-thiolate bond. Data suggest a zinc-mediated thiol deprotonation in catalysis of methyl group transfer in Methanosarcina barkeri.

Results demonstrate that N-7-mercaptoheptanoylthreonine phosphate (CoBSH) mediates methane formation through class 1 mechanisms and that CoBSH is integrally involved in the C-S bond cleavage in methanogenesis catalyzed by methyl-coenzyme M reductase.

Research has been conducted on the coenzyme F (sub)420 in the methanoarchaea. The biochemical route for the phosphodiester bond formation in this coenzyme has been established and described.

Results reveal that in Methanosarcina barkeri oxaloacetate synthesizing activity is catalyzed by an alpha(sub)4beta(sub)4-type acetyl coenzyme A-independent pyruvate carboxylase. Data show that the enzyme consists of a biotinylated and an ATP-binding subunits.

Research describes the stability properties of elongation factor 2 from two phylogenetically related but thermally distinct methanogens as influenced by ribosomal and intracellular solutes. Results show that while GTPase activity is greatly stimulated by ribosomes, they moderately stabilize the elongation factor 2 proteins.

Results demonstrate that there is an obligatory nutritional relationship between methanotrophs and heterotrophs as well as metabolic interactions aiding the transformation of the complex linear alkylbenzenesulfonate molecule. Data further suggest that such interactions of the microbial populations favored adaptation to various environmental conditions as a community.

Results demonstrate that in high-spirochete communities a dramatic shift in the relative abundance of fermentative bacteria is seen upon glucose shock whereas in low-spirochete communities perturbation effects are smaller. Data point out that community stability and functional stability are inversely correlated.

Research demonstrates that methanogenic bioreactor communities composed of different populations of bacteria exhibit functional stability based on their substrate processing type. Parallel substrate processing leads to more stability than serial substrate processing.

Production and consumption of nitric oxide by three different groups of methanotrophs involved production of small amounts of nitrous oxide as well. However, Methylobacter strain T20 produced large amounts of nitric oxide originating from reduction of nitrate to nitrite, which then decomposing to nitric oxide.

Research reveals that the halophilic methanogen, Methanohalophilus portucalensis exhibits an highly specific and high-affinity transport system for glycine betaine. Data show that the osmoprotectant is preferentially taken up from exterior instead of being synthesized.

Research reveals that methanogenesis from tri-, di-, and monomethylamine is mediated by three different methyltransferases in the archaeal bacteria. Results indicate that genes encoding the enzymes exhibit in-frame amber codons.

Results show that the elongation factor 2 protein from the Antartic methanogens require additional cytoplasmic factors for complete adaptation in vivo. Data also indicate that structural changes in the protein occur in response to cold adaptation.

A toluene-degrading methanogenic consortium enriched from creosote-contaminated aquifer material has been characterized with a molecular approach. Six eubacterial sequences and two archaeal sequences were found to be the most abundant ones based on RFLP analysis Relative abundance of the putatitve species was estimated by use of fluorescent in situ hybridization (FISH). It is likely, based on results, that a eubacterial spcies related to the genus Desultfotomaculum initiates the processing of toluene. Other species detected are likely related to genera Methanosaeta and Methanospirillum.

The hydroxylation of methylcubane catalyzed by methane monooxygenase (MMO) from Methylosinus trichosporium OB3b has been studied. The reaction produces in cubylmethanol and methylcubanols with methyl hydroxylation dominant over cubyl hydroxylation. This finding suggests that a radical intermediate is formed during the reaction indicating that the MMO is radical-based. The results obtained differ from those for M. capsulatus MMO even if the two enzymes have similar structures and reaction cycle intermediates.

Acetate-fed upflow anaerobic sludge blanket reactors containing Methanosaeta concilii GP6, immobilized Methanosarcina mazeii S-6 or a combination of both species are used in studying the immobilization patterns of two different species of methanogens utilizing acetate and their interactions. Results indicated that the reactor with M. mazeei S-6 had the fastest immobilization time. The reactor also had the highest effluent concentration of acetate while the reactor with a combination of both species had the lowest effluent concentration.

The role of salt on the in vivo and in vitro diazotrophy of various cations and anions of the methanogenic archaeon Methanosarcina barkeri 227 strain has been studied. The osmolyte accumulation in both diazotrophic and ammonium-grown cells have also been investigated to find out if there was a shift towards nonnitrogenous osmolytes under diazotrophic regimes. Results indicate that inhibition of growth was not greater with N2 than with NH4+. Cells grown with N2 and with NH4+ produced equal amounts of alpha-glutamate at low salt concentrations.

A new DNA polymerase family is identified in archaebacteria. The DNA polymerase II of Pyrococcus furiosus contains an alpha-like DNA polymerase. The P. furiosus genes coding for this enzyme have homologs in the genome of Methanococcus jannaschii, a methonogenic archaebacteria. These genes were sequenced, cloned and expressed in a heterologous expression system using Escherichia coli. The gene products exhibit DNA polymerase activity as well as 3' to 5' nuclease activity.