Complete genome sequence of Syntrophobacter fumaroxidans strain (MPOB(T)).

Syntrophobacter fumaroxidans strain MPOB(T) is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project.

Metabolic flexibility of sulfate-reducing bacteria.

Dissimilatory sulfate-reducing prokaryotes (SRB) are a very diverse group of anaerobic bacteria that are omnipresent in nature and play an imperative role in the global cycling of carbon and sulfur. In anoxic marine sediments sulfate reduction accounts for up to 50% of the entire organic mineralization in coastal and shelf ecosystems where sulfate diffuses several meters deep into the sediment. As a consequence, SRB would be expected in the sulfate-containing upper sediment layers, whereas methanogenic archaea would be expected to succeed in the deeper sulfate-depleted layers of the sediment. Where sediments are high in organic matter, sulfate is depleted at shallow sediment depths, and biogenic methane production will occur. In the absence of sulfate, many SRB ferment organic acids and alcohols, producing hydrogen, acetate, and carbon dioxide, and may even rely on hydrogen- and acetate-scavenging methanogens to convert organic compounds to methane. SRB can establish two different life styles, and these can be termed as sulfidogenic and acetogenic, hydrogenogenic metabolism. The advantage of having different metabolic capabilities is that it raises the chance of survival in environments when electron acceptors become depleted. In marine sediments, SRB and methanogens do not compete but rather complement each other in the degradation of organic matter. Also in freshwater ecosystems with sulfate concentrations of only 10-200 µM, sulfate is consumed efficiently within the top several cm of the sediments. Here, many of the δ-Proteobacteria present have the genetic machinery to perform dissimilatory sulfate reduction, yet they have an acetogenic, hydrogenogenic way of life. In this review we evaluate the physiology and metabolic mode of SRB in relation with their environment.

Global transcriptomics analysis of the Desulfovibrio vulgaris change from syntrophic growth with Methanosarcina barkeri to sulfidogenic metabolism.

Desulfovibrio vulgaris is a metabolically flexible micro-organism. It can use sulfate as an electron acceptor to catabolize a variety of substrates, or in the absence of sulfate can utilize organic acids and alcohols by forming a syntrophic association with a hydrogen-scavenging partner to relieve inhibition by hydrogen. These alternative metabolic types increase the chance of survival for D. vulgaris in environments where one of the potential external electron acceptors becomes depleted. In this work, whole-genome D. vulgaris microarrays were used to determine relative transcript levels as D. vulgaris shifted its metabolism from syntrophic in a lactate-oxidizing dual-culture with Methanosarcina barkeri to a sulfidogenic metabolism. Syntrophic dual-cultures were grown in two independent chemostats and perturbation was introduced after six volume changes with the addition of sulfate. The results showed that 132 genes were differentially expressed in D. vulgaris 2 h after addition of sulfate. Functional analyses suggested that genes involved in cell envelope and energy metabolism were the most regulated when comparing syntrophic and sulfidogenic metabolism. Upregulation was observed for genes encoding ATPase and the membrane-integrated energy-conserving hydrogenase (Ech) when cells shifted to a sulfidogenic metabolism. A five-gene cluster encoding several lipoproteins and membrane-bound proteins was downregulated when cells were shifted to a sulfidogenic metabolism. Interestingly, this gene cluster has orthologues found only in another syntrophic bacterium, Syntrophobacter fumaroxidans, and four recently sequenced Desulfovibrio strains. This study also identified several novel c-type cytochrome-encoding genes, which may be involved in the sulfidogenic metabolism.

Microbial dynamics in upflow anaerobic sludge blanket (UASB) bioreactor granules in response to short-term changes in substrate feed.

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 16S 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.

Separable functions of wingless in distal and ventral patterning of the Tribolium leg.

The gene wingless (wg) in Drosophila is an important factor in leg development. During embryonic development wg is involved in the allocation of the limb primordia. During imaginal disk development wg is involved in distal development and it has a separate role in ventral development. The expression pattern of wg is highly conserved in all arthropods (comprising data from insects, myriapods, crustaceans, and chelicerates), suggesting that its function in leg development is also conserved. However, recent work in other insects (e.g. the milkweed bug Oncopeltus fasciatus) argued against a role of wg in leg development. We have studied the role of wg in leg development of the flour beetle Tribolium castaneum. Using stage-specific staggered embryonic RNAi in wild-type and transgenic EGFP expressing enhancer trap lines we are able to demonstrate separable functions of Tribolium wg in distal and in ventral leg development. The distal role affects all podomeres distal to the coxa, whereas the ventral role is restricted to cells along the ventral midline of the legs. In addition, severe leg defects after injection into early embryonic stages are evidence that wg is also involved in proximal development and limb allocation in Tribolium. Our data suggest that the roles of wg in leg development are highly conserved in the holometabolous insects. Further studies will reveal the degree of conservation in other arthropod groups.

NMR bioreactor development for live in-situ microbial functional analysis.

A live, in-situ metabolomics capability was developed for prokaryotic cultures under controlled growth conditions. Toward this goal, a radiofrequency-transparent bioreactor was developed and integrated with a commercial wide-bore nuclear magnetic resonance (NMR) imaging spectrometer and a commercial bioreactor controller. Water suppressed 1H NMR spectroscopy was used to monitor glucose and fructose utilization and byproduct excretion by Eubacterium aggregans (an anaerobic bacterial species relevant for biofuel production) under controlled batch and continuous culture conditions. The resulting metabolite profiles (short chain organic acids and ethanol) and trends are consistent with existing knowledge of its metabolism. However, our study also showed that E. aggregans produces lactate end product in significant concentrations-a result not previously reported. The advantages of live in-situ microbial metabolomics analysis and its complementariness with functional genomics/systems biology methods are discussed.

Protein oxidation: key to bacterial desiccation resistance?

For extremely ionizing radiation-resistant bacteria, survival has been attributed to protection of proteins from oxidative damage during irradiation, with the result that repair systems survive and function with far greater efficiency during recovery than in sensitive bacteria. Here we examined the relationship between survival of dry-climate soil bacteria and the level of cellular protein oxidation induced by desiccation. Bacteria were isolated from surface soils of the shrub-steppe of the US Department of Energy's Hanford Site in Washington State. A total of 63 isolates were used for phylogenetic analysis. The majority of isolates were closely related to members of the genus Deinococcus, with Chelatococcus, Methylobacterium and Bosea also among the genera identified. Desiccation-resistant isolates accumulated high intracellular manganese and low iron concentrations compared to sensitive bacteria. In vivo, proteins of desiccation-resistant bacteria were protected from oxidative modifications that introduce carbonyl groups in sensitive bacteria during drying. We present the case that survival of bacteria that inhabit dry-climate soils are highly dependent on mechanisms, which limit protein oxidation during dehydration.

Comparative transcriptome analysis of Desulfovibrio vulgaris grown in planktonic culture and mature biofilm on a steel surface.

Biofilm build-up of sulphate-reducing bacteria (SRB) on metal surfaces may lead to severe corrosion of iron. To understand the processes at molecular level, in this study, a whole-genome oligonucleotide microarray was used to examine differential expression patterns between planktonic populations and mature biofilm of Desulfovibrio vulgaris on a steel surface. Statistical analysis revealed that 472 genes were differentially expressed (1.5-fold or more with a q value less than 0.025) by comparing the biofilm cells with the planktonic cells. Among the differentially expressed genes were several that corresponded to genes identified in many aerobic bacterial biofilms (i.e., Pseudomonas species and Escherichia coli) such as genes encoding flagellin, a flagellar motor switch protein, chemotaxis proteins involved in cell motility, as well as genes involved in exopolysaccharide biosynthesis. In addition, the biofilm-bound cells of D. vulgaris exhibited decreased transcription of genes involved in protein synthesis, energy metabolism and sulfate reduction, as well as genes involved in general stress responses. These findings were all consistent with early suggestion that the average physiology of the biofilm cells were similar to cells reduced in growth. Most notably, up-regulation of large number of outer membrane proteins was observed in the D. vulgaris biofilm. Although their function is still unknown, the higher expression of these genes in the biofilm could implicate important roles in the formation and maintenance of multi-cellular consortium on a steel surface. The study provided insights into the metabolic networks associated with the formation and maintenance of a D. vulgaris biofilm on a steel surface.

Integrative analysis of transcriptomic and proteomic data: challenges, solutions and applications.

Recent advances in high-throughput technologies enable quantitative monitoring of the abundance of various biological molecules and allow determination of their variation between biological states on a genomic scale. Two popular platforms are DNA microarrays that measure messenger RNA transcript levels, and gel-free proteomic analyses that quantify protein abundance. Obviously, no single approach can fully unravel the complexities of fundamental biology and it is equally clear that integrative analysis of multiple levels of gene expression would be valuable in this endeavor. However, most integrative transcriptomic and proteomic studies have thus far either failed to find a correlation or only observed a weak correlation. In addition to various biological factors, it is suggested that the poor correlation could be quite possibly due to the inadequacy of available statistical tools to compensate for biases in the data collection methodologies. To address this issue, attempts have recently been made to systematically investigate the correlation patterns between transcriptomic and proteomic datasets, and to develop sophisticated statistical tools to improve the chances of capturing a relationship. The goal of these efforts is to enhance understanding of the relationship between transcriptomes and proteomes so that integrative analyses may be utilized to reveal new biological insights that are not accessible through one-dimensional datasets. In this review, we outline some of the challenges associated with integrative analyses and present some preliminary statistical solutions. In addition, some new applications of integrated transcriptomic and proteomic analysis to the investigation of post-transcriptional regulation are also discussed.

Development and assessment of whole-genome oligonucleotide microarrays to analyze an anaerobic microbial community and its responses to oxidative stress.

The application of DNA microarray technology to investigate multiple-species microbial communities presents great challenges. In this study, we reported the design and quality assessment of four whole genome oligonucleotide microarrays for two syntroph bacteria, Desulfovibrio vulgaris and Syntrophobacter fumaroxidans, and two archaeal methanogens, Methanosarcina barkeri, and Methanospirillum hungatei, and their application to analyze global gene expression in a four-species microbial community in response to oxidative stress. In order to minimize the possibility of cross-hybridization, cross-genome comparison was performed to assure all probes unique to each genome so that the microarrays could provide species-level resolution. Microarray quality was validated by the good reproducibility of experimental measurements of multiple biological and analytical replicates. This study showed that S. fumaroxidans and M. hungatei responded to the oxidative stress with up-regulation of several genes known to be involved in reactive oxygen species (ROS) detoxification, such as catalase and rubrerythrin in S. fumaroxidans and thioredoxin and heat shock protein Hsp20 in M. hungatei. However, D. vulgaris seemed to be less sensitive to the oxidative stress as a member of a four-species community, since no gene involved in ROS detoxification was up-regulated. Our work demonstrated the successful application of microarrays to a multiple-species microbial community, and our preliminary results indicated that this approach could provide novel insights on the metabolism within microbial communities.

Evolution of the syntrophic interaction between Desulfovibrio vulgaris and Methanosarcina barkeri: Involvement of an ancient horizontal gene transfer.

The sulfate reducing bacteria Desulfovibrio vulgaris and the methanogenic archaea Methanosarcina barkeri can grow syntrophically on lactate. In this study, a set of three closely located genes, DVU2103, DVU2104, and DVU2108 of D. vulgaris, was found to be up-regulated 2- to 4-fold following the lifestyle shift from syntroph to sulfate reducer; moreover, none of the genes in this gene set were differentially regulated when comparing gene expression from various D. vulgaris pure culture experiments. Although exact function of this gene set is unknown, the results suggest that it may play roles related to the lifestyle change of D. vulgaris from syntroph to sulfate reducer. This hypothesis is further supported by phylogenomic analyses showing that homologies of this gene set were only narrowly present in several groups of bacteria, most of which are restricted to a syntrophic lifestyle, such as Pelobacter carbinolicus, Syntrophobacter fumaroxidans, Syntrophomonas wolfei, and Syntrophus aciditrophicus. Phylogenetic analysis showed that all three individual genes in the gene set tended to be clustered with their homologies from archaeal genera, and they were rooted on archaeal species in the phylogenetic trees, suggesting that they were horizontally transferred from archaeal methanogens. In addition, no significant bias in codon and amino acid usages was detected between these genes and the rest of the D. vulgaris genome, suggesting the gene transfer may have occurred early in the evolutionary history so that sufficient time has elapsed to allow an adaptation to the codon and amino acid usages of D. vulgaris. This report provides novel insights into the origin and evolution of bacterial genes linked to the lifestyle change of D. vulgaris from a syntrophic to a sulfate-reducing lifestyle.

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris.

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.

Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri.

We present a genome-scale metabolic model for the archaeal methanogen Methanosarcina barkeri. We characterize the metabolic network and compare it to reconstructions from the prokaryotic, eukaryotic and archaeal domains. Using the model in conjunction with constraint-based methods, we simulate the metabolic fluxes and resulting phenotypes induced by different environmental and genetic conditions. This represents the first large-scale simulation of either a methanogen or an archaeal species. Model predictions are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for wild type and mutants of the methanogenic pathway have a high level of agreement with experimental findings. We further examine the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction, and determine a stoichiometry for the nitrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation and further uncover the metabolic characteristics of methanogenesis.

Global transcriptomic analysis of Desulfovibrio vulgaris on different electron donors.

Whole-genome microarrays of Desulfovibrio vulgaris were used to determine relative transcript levels in cells grown to exponential or stationary phase on a medium containing either lactate or formate as electron donor. The results showed that 158 and 477 genes were differentially expressed when comparing exponential to stationary phase in lactate- or formate-based media, respectively; and 505 and 355 genes were responsive to the electron donor used at exponential or stationary phase, respectively. Functional analyses suggested that the differentially regulated genes were involved in almost every aspect of cellular metabolism, with genes involved in protein synthesis, carbon, and energy metabolism being the most regulated. The results suggested that HynBA-1 might function as a primary periplasmic hydrogenase responsible for oxidation of H2 linked to the proton gradient in lactate-based medium, while several periplasmic hydrogenases including HynBA-1 and Hyd might carry out this role in formate-based medium. The results also indicated that the alcohol dehydrogenase and heterodisulfide reductase catalyzed pathway for proton gradient formation might be actively functioning for ATP synthesis in D. vulgaris. In addition, hierarchical clustering analysis using expression data across different electron donors and growth phases allowed the identification of the common electron donor independent changes in gene expression specifically associated with the exponential to stationary phase transition, and those specifically associated with the different electron donors independent of growth phase. The study provides the first global description and functional interpretation of transcriptomic response to growth phase and electron donor in D. vulgaris.

Analysis of methane monooxygenase genes in mono lake suggests that increased methane oxidation activity may correlate with a change in methanotroph community structure.

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.

Direct inhibition of methanogenesis by ferric iron.

Observed inhibition of methanogenesis under Fe(III)-reducing conditions is usually explained by competition of methanogens and Fe(III)-reducing bacteria for the common substrates acetate and hydrogen. However, substrate competition alone cannot explain the strong inhibition of methanogenesis during Fe(III)-reduction. We demonstrate direct inhibition of methanogenesis by amorphous Fe(OH)3 at concentrations between 0 and 10 mM in experiments with pure cultures of methanogens. The sensitivity toward Fe(III) was higher for Methanospirillum hungatei and Methanosarcina barkeri grown with H2/CO2 than for Methanosaeta concilii and Methanosarcina barkeri grown with acetate. Cultures of Methanosarcina barkeri grown with H2/CO2 and methanol demonstrated a capacity for Fe(III) reduction, which suggests that Fe(III)-reduction by methanogens may also contribute to Fe(III) inhibition of methanogenesis. Our results have important implications for kinetic modelling of microbial redox processes in anoxic soils and sediments.

Growth of sulfate-reducing bacteria and methanogenic archaea with methylated sulfur compounds: a commentary on the thermodynamic aspects.

Methylated sulfur compounds such as dimethylsulfoniopropionate, dimethylsulfide, methanethiol, and other methylated sulfur compounds can act as sources of carbon and energy for the growth under anoxic conditions of a number of sulfate-reducing bacteria and methanogenic archaea. We summarise the range of degradative reactions that do or might occur in such organisms, and present thermodynamic data for these processes. These data enable estimates of the feasibility of the reactions as growth-supporting systems, and of the possible maximum growth yields of the bacteria and archaea catalysing them. We compare our new estimates with the few data that are currently available from the literature, and show that some published growth-yield assessments need reevaluation.

Effect of sulfate and nitrate on acetate conversion by anaerobic microorganisms in a freshwater sediment.

Acetate is quantitatively the most important substrate for methane production in a freshwater sediment in The Netherlands. In the presence of alternative electron acceptors the conversion of acetate by methanogens was strongly inhibited. By modelling the results, obtained in experiments with and without (13)C-labelled acetate, we could show that the competition for acetate between methanogens and sulfate reducers is the main cause of inhibition of methanogenesis in the sediment. Although nitrate led to a complete inhibition of methanogenesis, acetate-utilising nitrate-reducing bacteria hardly competed with methanogens for the available acetate in the presence of nitrate. Most-probable-number enumerations showed that methanogens (2x10(8) cells cm(-3) sediment) and sulfate reducers (2x10(8) cells cm(-3) sediment) were the dominant acetate-utilising organisms in the sediment, while numbers of acetate-utilising nitrate reducers were very low (5x10(5) cells cm(-3) sediment). However, high numbers of sulfide-oxidising nitrate reducers were detected. Denitrification might result in the formation of toxic products. We speculate that the accumulation of low concentrations of NO (<0.2 mM) may result in an inhibition of methanogenesis.