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Stimulation of subsurface microorganisms to induce reductive immobilization of metals is a promising approach for bioremediation, yet the overall microbial community response is typically poorly understood. Here we used proteogenomics to test the hypothesis that excess input of acetate activates complex community functioning and syntrophic interactions among autotrophs and heterotrophs. A flow-through sediment column was incubated in a groundwater well of an acetate-amended aquifer and recovered during microbial sulfate reduction. De novo reconstruction of community sequences yielded near-complete genomes of Desulfobacter (Deltaproteobacteria), Sulfurovum- and Sulfurimonas-like Epsilonproteobacteria and Bacteroidetes. Partial genomes were obtained for Clostridiales (Firmicutes) and Desulfuromonadales-like Deltaproteobacteria. The majority of proteins identified by mass spectrometry corresponded to Desulfobacter-like species, and demonstrate the role of this organism in sulfate reduction (Dsr and APS), nitrogen fixation and acetate oxidation to CO2 during amendment. Results indicate less abundant Desulfuromonadales, and possibly Bacteroidetes, also actively contributed to CO2 production via the tricarboxylic acid (TCA) cycle. Proteomic data indicate that sulfide was partially re-oxidized by Epsilonproteobacteria through nitrate-dependent sulfide oxidation (using Nap, Nir, Nos, SQR and Sox), with CO2 fixed using the reverse TCA cycle. We infer that high acetate concentrations, aimed at stimulating anaerobic heterotrophy, led to the co-enrichment of, and carbon fixation in Epsilonproteobacteria. Results give an insight into ecosystem behavior following addition of simple organic carbon to the subsurface, and demonstrate a range of biological processes and community interactions were stimulated. [ABSTRACT FROM AUTHOR]

Dehalococcoides ethenogenes strain 195 (DE195) was grown in a sustainable syntrophic association with Desulfovibrio vulgaris Hildenborough (DVH) as a co-culture, as well as with DVH and the hydrogenotrophic methanogen Methanobacterium congolense (MC) as a tri-culture using lactate as the sole energy and carbon source. In the co- and tri-cultures, maximum dechlorination rates of DE195 were enhanced by approximately three times (11.0±0.01 μmol per day for the co-culture and 10.1±0.3 μmol per day for the tri-culture) compared with DE195 grown alone (3.8±0.1 μmol per day). Cell yield of DE195 was enhanced in the co-culture (9.0±0.5 × 107 cells per μmol Cl− released, compared with 6.8±0.9 × 107 cells per μmol Cl− released for the pure culture), whereas no further enhancement was observed in the tri-culture (7.3±1.8 × 107 cells per μmol Cl− released). The transcriptome of DE195 grown in the co-culture was analyzed using a whole-genome microarray targeting DE195, which detected 102 significantly up- or down-regulated genes compared with DE195 grown in isolation, whereas no significant transcriptomic difference was observed between co- and tri-cultures. Proteomic analysis showed that 120 proteins were differentially expressed in the co-culture compared with DE195 grown in isolation. Physiological, transcriptomic and proteomic results indicate that the robust growth of DE195 in co- and tri-cultures is because of the advantages associated with the capabilities of DVH to ferment lactate to provide H2 and acetate for growth, along with potential benefits from proton translocation, cobalamin-salvaging and amino acid biosynthesis, whereas MC in the tri-culture provided no significant additional benefits beyond those of DVH. [ABSTRACT FROM AUTHOR]

Metaproteomics is one of a suite of new approaches providing insights into the activities of microorganisms in natural environments. Proteins, the final products of gene expression, indicate cellular priorities, taking into account both transcriptional and posttranscriptional control mechanisms that control adaptive responses. Here, we report the proteomic composition of the < 1.2 μm fraction of a microbial community from Oregon coast summer surface waters, detected with two-dimensional liquid chromatography coupled with electrospray tandem mass spectrometry. Spectra corresponding to proteins involved in protein folding and biosynthesis, transport, and viral capsid structure were the most frequently detected. A total of 36% of all the detected proteins were best matches to the SAR11 clade, and other abundant coastal microbial clades were also well represented, including the Roseobacter clade (17%), oligotrophic marine gammaproteobacteria group (6%), OM43 clade (1%). Viral origins were attributed to 2.5% of proteins. In contrast to oligotrophic waters, phosphate transporters were not highly detected in this nutrient-rich system. However, transporters for amino acids, taurine, polyamines and glutamine synthetase were among the most highly detected proteins, supporting predictions that carbon and nitrogen are more limiting than phosphate in this environment. Intriguingly, one of the highly detected proteins was methanol dehydrogenase originating from the OM43 clade, providing further support for recent reports that the metabolism of one-carbon compounds by these streamlined methylotrophs might be an important feature of coastal ocean biogeochemistry. [ABSTRACT FROM AUTHOR]

Characterizing proteins recovered from natural microbial communities affords the opportunity to correlate protein expression and modification with environmental factors, including species composition and successional stage. Proteogenomic and biochemical studies of pellicle biofilms from subsurface acid mine drainage streams have shown abundant cytochromes from the dominant organism, Leptospirillum Group II. These cytochromes are proposed to be key proteins in aerobic Fe(II) oxidation, the dominant mode of cellular energy generation by the biofilms. In this study, we determined that posttranslational modification and expression of amino-acid sequence variants change as a function of biofilm maturation. For Cytochrome579 (Cyt579), the most abundant cytochrome in the biofilms, late developmental-stage biofilms differed from early-stage biofilms in N-terminal truncations and decreased redox potentials. Expression of sequence variants of two monoheme c-type cytochromes also depended on biofilm development. For Cyt572, an abundant membrane-bound cytochrome, the expression of multiple sequence variants was observed in both early and late developmental-stage biofilms; however, redox potentials of Cyt572 from these different sources did not vary significantly. These cytochrome analyses show a complex response of the Leptospirillum Group II electron transport chain to growth within a microbial community and illustrate the power of multiple proteomics techniques to define biochemistry in natural systems. [ABSTRACT FROM AUTHOR]

Acid mine drainage (AMD), an extreme environment characterized by low pH and high metal concentrations, can support dense acidophilic microbial biofilm communities that rely on chemoautotrophic production based on iron oxidation. Field determined production rates indicate that, despite the extreme conditions, these communities are sufficiently well adapted to their habitats to achieve primary production rates comparable to those of microbial communities occurring in some non-extreme environments. To enable laboratory studies of growth, production and ecology of AMD microbial communities, a culturing system was designed to reproduce natural biofilms, including organisms recalcitrant to cultivation. A comprehensive metabolic labeling-based quantitative proteomic analysis was used to verify that natural and laboratory communities were comparable at the functional level. Results confirmed that the composition and core metabolic activities of laboratory-grown communities were similar to a natural community, including the presence of active, low abundance bacteria and archaea that have not yet been isolated. However, laboratory growth rates were slow compared with natural communities, and this correlated with increased abundance of stress response proteins for the dominant bacteria in laboratory communities. Modification of cultivation conditions reduced the abundance of stress response proteins and increased laboratory community growth rates. The research presented here represents the first description of the application of a metabolic labeling-based quantitative proteomic analysis at the community level and resulted in a model microbial community system ideal for testing physiological and ecological hypotheses. [ABSTRACT FROM AUTHOR]

The human gut contains a dense, complex and diverse microbial community, comprising the gut microbiome. Metagenomics has recently revealed the composition of genes in the gut microbiome, but provides no direct information about which genes are expressed or functioning. Therefore, our goal was to develop a novel approach to directly identify microbial proteins in fecal samples to gain information about the genes expressed and about key microbial functions in the human gut. We used a non-targeted, shotgun mass spectrometry-based whole community proteomics, or metaproteomics, approach for the first deep proteome measurements of thousands of proteins in human fecal samples, thus demonstrating this approach on the most complex sample type to date. The resulting metaproteomes had a skewed distribution relative to the metagenome, with more proteins for translation, energy production and carbohydrate metabolism when compared to what was earlier predicted from metagenomics. Human proteins, including antimicrobial peptides, were also identified, providing a non-targeted glimpse of the host response to the microbiota. Several unknown proteins represented previously undescribed microbial pathways or host immune responses, revealing a novel complex interplay between the human host and its associated microbes.The ISME Journal (2009) 3, 179–189; doi:10.1038/ismej.2008.108; published online 30 October 2008 [ABSTRACT FROM AUTHOR]

Enhanced biological phosphorus removal (EBPR) selects for polyphosphate accumulating microorganisms to achieve phosphate removal from wastewater. We used high-resolution community proteomics to identify key metabolic pathways in ‘Candidatus Accumulibacter phosphatis’ (A. phosphatis)-mediated EBPR and to evaluate the contributions of co-existing strains within the dominant population. Overall, 702 proteins from the A. phosphatis population were identified. Results highlight the importance of denitrification, fatty acid cycling and the glyoxylate bypass in EBPR. Strong similarity in protein profiles under anaerobic and aerobic conditions was uncovered (only 3% of A. phosphatis-associated proteins exhibited statistically significant abundance differences). By comprehensive genome-wide alignment of 13 930 orthologous proteins, we uncovered substantial differences in protein abundance for enzyme variants involved in both core-metabolism and EBPR-specific pathways among the A. phosphatis population. These findings suggest an essential role for genetic diversity in maintaining the stable performance of EBPR systems and, hence, demonstrate the power of integrated cultivation-independent genomics and proteomics for the analysis of complex biotechnological systems.The ISME Journal (2008) 2, 853–864; doi:10.1038/ismej.2008.38; published online 1 May 2008 [ABSTRACT FROM AUTHOR]

Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5–1.2 and laden with toxic metals. To approach the mechanism of Fe(II) oxidation as a means of cellular energy acquisition, we isolated proteins from natural samples and found a conspicuous and novel cytochrome, Cyt572, which is unlike any known cytochrome. Both the character of its covalently bound prosthetic heme group and protein sequence are unusual. Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. The purified protein has a cytochrome c-type heme binding motif, CxxCH, but a unique spectral signature at 572 nm, and thus is called Cyt572. It readily oxidizes Fe2+ in the physiologically relevant acidic regime, from pH 0.95–3.4. Other physical characteristics are indicative of a membrane-bound multimeric protein. Circular dichroism spectroscopy indicates that the protein is largely beta-stranded, and 2D Blue-Native polyacrylamide gel electrophoresis and chemical crosslinking independently point to a multi-subunit structure for Cyt572. By analyzing environmental genomic information from biofilms in several distinctly different mine locations, we found multiple genetic variants of Cyt572. MS proteomics of extracts from these biofilms substantiated the prevalence of these variants in the ecosystem. Due to its abundance, cellular location and Fe2+ oxidation activity at very low pH, we propose that Cyt572 provides a critical function for fitness within the ecological niche of these acidophilic microbial communities.The ISME Journal (2008) 2, 542–550; doi:10.1038/ismej.2008.17; published online 28 February 2008 [ABSTRACT FROM AUTHOR]