In methanogenic Archaea, the final step of methanogenesis generates methane and a heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB). Reduction of this heterodisulfide by heterodisulfide reductase to regenerate HS-CoM and HS-CoB is an exergonic process. Thauer et al. [Thauer, et al. 2008 Nat Rev Microbiol 6:579-591] recently suggested that in hydrogenotrophic methanogens the energy of heterodisulfide reduction powers the most endergonic reaction in the pathway, catalyzed by the formylmethanofuran dehydrogenase, via flavin-based electron bifurcation. Here we present evidence that these two steps in methanogenesis are physically linked. We identify a protein complex from the hydrogenotrophic methanogen, Methanococcus maripaludis, that contains heterodisulfide reductase, formylmethanofuran dehydrogenase, F 420 -nonreducing hydrogenase, and formate dehydrogenase. In addition to establishing a physical basis for the electronbifurcation model of energy conservation, the composition of the complex also suggests that either H 2 or formate (two alternative electron donors for methanogenesis) can donate electrons to the heterodisulfide-H 2 via F 420 -nonreducing hydrogenase or formate via formate dehydrogenase. Electron flow from formate to the heterodisulfide rather than the use of H 2 as an intermediate represents a previously unknown path of electron flow in methanogenesis. We further tested whether this path occurs by constructing a mutant lacking F 420 -nonreducing hydrogenase. The mutant displayed growth equal to wild-type with formate but markedly slower growth with hydrogen. The results support the model of electron bifurcation and suggest that formate, like H 2 , is closely integrated into the methanogenic pathway.energy conservation | Archaea | formate dehydrogenase | formylmethanofuran dehydrogenase | F 420 -nonreducing hydrogenase T he biochemical steps in methanogenesis from CO 2 are well known, but the interactions that lead to net energy conservation are not well understood. The steps in the pathway are diagrammed in Fig. 1 (1). The first step involves the reduction of CO 2 and covalent attachment to a unique cofactor, methanofuran (MFR), via the action of formylmethanofuran dehydrogenase (Fwd) to generate formyl-MFR. This represents an energy-consuming step in the pathway and is dependent on reduced ferredoxin, thought to be produced at the expense of a chemiosmotic membrane potential via the energy-conserving hydrogenase, Eha. Next, the formyl group is transferred to another carrier, tetrahydromethanopterin (H 4 MPT), and is then reduced to generate methyl-H 4 MPT. The methyl group is then transferred to yet another carrier, coenzyme M (HS-CoM), by methyl-H 4 MPT-CoM methyltransferase (Mtr) to generate methyl-S-CoM. At this point, Na + ions are translocated across the cell membrane. The final step involves reduction of the methyl group to CH 4 and capture of HS-CoM by coenzyme B (HS-CoB) to form a CoM-S-S-CoB heterodisulfide. To regenerate HS-CoM and HS-CoB, another enzyme is used, heterodisulfid...

BackgroundPorphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community.Results1156 P. gingivalis proteins were detected qualitatively during comparison of the three species model community with P. gingivalis incubated alone under the same conditions. Integration of spectral counting and summed signal intensity analyses of the dataset showed that 403 proteins were down-regulated and 89 proteins up-regulated. The proteomics results were inspected manually and an ontology analysis conducted using DAVID. Significant decreases were seen in proteins involved in cell shape and the formation of the cell envelope, as well as thiamine, cobalamin, and pyrimidine synthesis and DNA repair. An overall increase was seen in proteins involved in protein synthesis. HmuR, a TonB dependent outer membrane receptor, was up-regulated in the community and an hmuR deficient mutant was deficient in three species community formation, but was unimpaired in its ability to form mono- or dual-species biofilms.ConclusionCollectively, these results indicate that P. gingivalis can assemble into a heterotypic community with F. nucleatum and S. gordonii, and that a community lifestyle provides physiologic support for P. gingivalis. Proteins such as HmuR, that are up-regulated, can be necessary for community structure.

SummaryThe ability of some microbial species to oxidize monomethylamine via glutamate-mediated pathways was proposed in the 1960s; however, genetic determinants of the pathways have never been described. In the present study we describe a gene cluster essential for operation of the N-methylglutamate pathway in the methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5. Four major polypeptides from protein fractions displaying high activities of N-methylglutamate synthetase, N-methylglutamate dehydrogenase and g-glutamylmethylamide synthetase were selected for mass spectrometry-based identification. The activities of enzymes were associated with the presence of peptides identified as ferredoxin-dependent glutamate synthase (GltB2), large subunit of putative heterotetrameric sarcosine oxidase (SoxA) and glutamine synthetase type III (GSIII) respectively. A gene cluster (8.3 kb) harbouring gltB2, soxA and gsIII-like genes was amplified from M. universalis FAM5, sequenced and assembled. Two partial and six complete open reading frames arranged in the order soxBDAG-gsIII-gltB132 were identified and subjected to mutational analysis, functional and metabolic profiling. We demonstrated that gltB-like and sox-like genes play a key role in methylamine utilization and encode N-methylglutamate synthetase and N-methylglutamate dehydrogenase respectively. Metabolic, enzymatic and mutational analyses showed that the gsIII-like gene encodes g-glutamylmethylamide synthetase; however, this enzyme is not essential for oxidation of methylamine.

Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 hours within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were over-expressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be under-expressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction.

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For the archaeon Methanococcus maripaludis, a fully sequenced and annotated model species of hydrogenotrophic methanogen, we report validation of quantitative protein level expression ratios on a proteome-wide basis. Using an approach based on quantitative multidimensional capillary HPLC and quadrupole ion trap mass spectrometry, coverage of gene expression approached that currently achievable with transcription microarrays. Comprehensive mass spectrometry-based proteomics and spotted cDNA arrays were used to compare global protein and mRNA levels in a wild-type (S2) and mutant strain (S40) of M. maripaludis. Using linear regression with 652 expression ratios generated by both the proteomic and microarray methods, a product moment correlation coefficient of 0.24 was observed. The correlation improved to 0.61 if only genes showing significant expression changes were included. A novel two-stage method of outlier detection was used for the protein measurements when Dixon's Q-test by itself failed to give satisfactory results. The log 2 transformations of the number of peptides or isotopic peptide pairs associated with each ORF, divided by the predicted molecular weight, were found to have moderately positive correlations with two bioinformatic predictors of gene expression based on codon bias. We detected peptides derived from 939 proteins or 55% of the genome coding capacity. Of these, 60 were overexpressed, and 34 were underexpressed in the mutant. Of the 1722 ORFs encoded in the genome, 1597 or 93% were probed by cDNA arrays. Of these, 50 were more highly expressed, and 45 showed lower expression levels in the mutant relative to the wild type. 15 ORFs were shown to be overexpressed by both methods, and two ORFs were shown to be overexpressed by proteomics and underexpressed by microarray. Molecular & Cellular Proteomics 5:, 868 -881.The hydrogenotrophic methanogens are a major group of Archaea that conserve energy from the use of molecular hydrogen or formate to reduce carbon dioxide to methane. Methanococcus maripaludis is a model species of hydrogenotrophic methanogen, having favorable laboratory growth behavior, excellent genetic tools (1), and a fully sequenced genome (2). In another publication (3) we describe the molecular biology of a mutant of M. maripaludis (S40) in which the operon encoding the Ehb hydrogenase was disrupted, and functional changes were observed that led to a model for the role of Ehb in energy metabolism. Here we present validation of the multidimensional capillary HPLC/tandem mass spectrometry approach (also known as MudPIT 1 (4, 5)) to quantitative and qualitative proteomics for M. maripaludis by comparison with mRNA expression. Transcriptome analysis was performed in which the ehb mutant and wild-type strains were compared globally for all known genes under the same culture conditions as for the proteomics. For a subset of genes the proteomic results were further validated by real time quantitative RT-PCR. The relative absence of evidence for posttranscriptional gene regulation and ...

BackgroundStreptococcus gordonii is one of several species that can initiate the formation of oral biofilms that develop into the complex multispecies microbial communities referred to as dental plaque. It is in the context of dental plaque that periodontal pathogens such as Porphyromonas gingivalis cause disease. We have previously reported a whole cell quantitative proteomics investigation of P. gingivalis in a model dental plaque community of S. gordonii, P. gingivalis, and Fusobacterium nucleatum. Here we report the adaptation of S. gordonii to the same model.Results1122 S. gordonii proteins were detected in S. gordonii control samples, 915 in communities with F. nucleatum, 849 with P. gingivalis, and 649 with all three organisms. Quantitative comparisons showed extensive proteome changes in association with F. nucleatum or P. gingivalis individually or both P. gingivalis and F. nucleatum together. The changes were species specific, though the P. gingivalis interaction may be dominant, indicated by large differences between the proteomes with F. nucleatum or P. gingivalis but limited changes between communities with P. gingivalis or both P. gingivalis and F. nucleatum. The results were inspected manually and an ontology analysis conducted using DAVID. Extensive changes were seen in nutrition pathways with increases in energy metabolism and changes in the resulting byproducts, while the acid and sugar repressed PTS (phosphoenolpyruvate dependent phosphotransferase system) sugar transport systems showed decreases. These results were seen across all the multispecies samples, though with different profiles according to the partner species. F. nucleatum association decreased proteins for the metabolic end products acetate and ethanol but increased lactate, the primary source of acidity from streptococcal cultures. P. gingivalis containing samples had a reduction in levels of proteins for ethanol and formate but increased proteins for both acetate and lactate production. The communities also showed increases in exopolysaccharide synthesis, amino acid biosynthesis, and oxidative stress protection and decreases in adhesion and transporter proteins.ConclusionThis study showed that S. gordonii demonstrates species specific responses during interactions with F. nucleatum or P. gingivalis. Extensive changes were seen in energy metabolism and byproduct production implicating nutrient transfer as an important community interaction.

Methanococcus maripaludis is a mesophilic archaeon that reduces CO 2 to methane with H 2 or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO 2 assimilation in methanococci.Methanogens are strictly anaerobic archaea that produce methane as the major product of their energy metabolism. They play an important role in the global carbon cycle, processing 1 to 2% of the carbon fixed per year and producing most of the earth's atmospheric methane (10, 21). Methanococcus maripaludis is a mesophile that reduces carbon dioxide to methane with H 2 or formate as electron donor (10). In addition, M. maripaludis assimilates acetate and some amino acids as carbon sources when they are present (24,25,33). Progress in genetics tools (30), relatively fast growth (11), suitability for chemostats (7), and a complete genomic sequence (9) make M. maripaludis an excellent model for the physiology of hydrogenotrophic methanogens.Hydrogenases catalyze the reaction H 2 3 2 H ϩ ϩ 2 e Ϫ . These enzymes are indispensable for the growth of hydrogenotrophic methanogens, which use H 2 as an electron donor. M. maripaludis contains six nickel-iron hydrogenases, including two coenzyme F 420 -reducing hydrogenases and two non-F 420 -reducing hydrogenases (9). Of each of these pairs of enzymes, one contains a selenocysteinyl residue and the other contains a cysteinyl residue at the active site (3). In addition, M. maripaludis contains genes for two separate multisubunit energyconserving hydrogenases, Eha and Ehb (9). These open reading frames (ORFs) include subunits that are homologous to the NADH-ubiquinone oxidoreductase or complex I of mitochondria (1). In the methanogens, the energy-converting [NiFe] hydrogenase (Ech) has been purified and characterized from Methanosarcina barkeri (12,16). M. barkeri is only distantly related to the methanococci. Although it can reduce CO 2 to CH 4 , it also utilizes acetate and methanol as substrates for methanogenesis. In this organism, the complex Ech enzyme contains six subunits, two predicted integral membrane-spanning proteins and...