Desulfuromonas sp. 'CSMB_57', isolation and genomic insights from the most abundant bacterial taxon in eastern Australian coals.

One of the most abundant and ubiquitous taxa observed in eastern Australian coal seams is an uncultured Desulfuromonas species and part of the Coal Seam Microbiome dataset assigned as 'CSMB_57'. Despite this abundance and ubiquity, knowledge about this taxon is limited. The present study aimed to generate an enrichment culture of Desulfuromonas sp. 'CSMB_57' using culturing strategies that exploit its sulphur-reducing capabilities by utilizing a polysulfide solution in a liquid medium. Using dilution to extinction methods, a highly enriched culture was successfully generated. The full-length 16S rRNA sequence revealed that all closely related taxa were observed in subsurface environments suggesting that D. sp. 'CSMB_57' may be a subsurface specialist. Subsequently, the DNA from the enrichment culture was sequenced and the genome of D. sp. 'CSMB_57' was assembled. Genomic annotation revealed a high number of CRISPR arrays for viral defence, a large array of ABC transporters for amino acid and peptide uptake, as well as genes likely associated with syntrophy such as genes associated with type-IVa pilus, often used for direct interspecies electron transfer, and multiple hydrogenases capable of producing hydrogen. From the various genomic observations, a conceptual ecological model was developed that explores its possible syntrophic roles with hydrogenotrophic methanogens and acetogenic bacteria within coal-seam environments.

Desulfuromonas carbonis sp. nov., an Fe(III)-, S0- and Mn(IV)-reducing bacterium isolated from an active coalbed methane gas well.

A novel, mesophilic, obligately anaerobic, acetate-oxidizing, dissimilatory iron-, sulfur-, and manganese-reducing bacterium, designated strain ICBM(T), was obtained from an active, coalbed methane gas well in Indiana, USA. Strain ICBM(T) was a Gram-stain-negative, non-spore-forming, rod-shaped, non-motile bacterium that was rich in c-type cytochromes and formed red colonies in solid medium. Strain ICBM(T) conserved energy to support growth from the oxidation of acetate, propionate, pyruvate, malate, fumarate, succinate and dl-lactate, concomitant with dissimilatory iron reduction. Strain ICBM(T) fermented fumarate yielding succinate and acetate. Strain ICBM(T) was able to grow in the temperature range of 10 °C to 37 °C, NaCl concentration range of 0 to 1.2 M, and pH range of 6.5 to 8.0. The physiological characteristics of strain ICBM(T) indicated that it belongs to the Desulfuromonas cluster. The G+C content of its genomic DNA was 61.2 mol%. The predominant cellular fatty acids were C16 : 0 (39.3%), C16 : 1ω7c and/or iso-C15 : 0 2-OH (36.6%). The closest cultured phylogenetic relative of strain ICBM(T) was Desulfuromonas michiganensis BB1(T) with only 95% 16S rRNA gene sequence similarity. This confirmed that strain ICBM(T) is affiliated with the genus Desulfuromonas . On the basis of phenotypic and genotypic differences between strain ICBM(T) and other taxa of the genus Desulfuromonas , strain ICBM(T) represents a novel species for which the name Desulfuromonas carbonis sp. nov. is proposed (type strain ICBM(T) = DSM 29759(T) = JCM 30471(T)). Strain ICBM(T) is the first Fe(III)-, S(0)-, and Mn(IV)-reducing bacterium that was isolated from a coal bed.

Sulfur oxidation to sulfate coupled with electron transfer to electrodes by Desulfuromonas strain TZ1.

Microbial oxidation of elemental sulfur with an electrode serving as the electron acceptor is of interest because this may play an important role in the recovery of electrons from sulfidic wastes and for current production in marine benthic microbial fuel cells. Enrichments initiated with a marine sediment inoculum, with elemental sulfur as the electron donor and a positively poised (+300 mV versus Ag/AgCl) anode as the electron acceptor, yielded an anode biofilm with a diversity of micro-organisms, including Thiobacillus, Sulfurimonas, Pseudomonas, Clostridium and Desulfuromonas species. Further enrichment of the anode biofilm inoculum in medium with elemental sulfur as the electron donor and Fe(III) oxide as the electron acceptor, followed by isolation in solidified sulfur/Fe(III) medium yielded a strain of Desulfuromonas, designated strain TZ1. Strain TZ1 effectively oxidized elemental sulfur to sulfate with an anode serving as the sole electron acceptor, at rates faster than Desulfobulbus propionicus, the only other organism in pure culture previously shown to oxidize S° with current production. The abundance of Desulfuromonas species enriched on the anodes of marine benthic fuel cells has previously been interpreted as acetate oxidation driving current production, but the results presented here suggest that sulfur-driven current production is a likely alternative.

Identification of a novel fosfomycin-resistant UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from a soil metagenome.

A soil metagenomic library was constructed and screened for clones that conferred fosfomycin resistance. A novel protein with 46 % identity to UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Desulfuromonas acetoxidans DSM 684 (GenBank accession number: ZP_01311756) was identified. Multiple sequence alignment revealed that the novel protein was a natural MurA, in which an aspartic acid instead of a cysteine was located in the active site. An Asp120Cys mutant of Escherichia coli was constructed from the subclone through site-specific mutagenesis, and minimum inhibitory concentration of fosfomycin for the resistant subclone and its mutant were determined. These results showed that fosfomycin resistance was a result of the aspartic acid in the active site. Analysis of all existing MurA sequences revealed that MurAs with an active site aspartic acid that can confer fosfomycin resistance occur in ~14 % of bacteria.

Exploration of the 'cytochromome' of Desulfuromonas acetoxidans, a marine bacterium capable of powering microbial fuel cells.

Recent progress in bacterial genomic analysis has revealed a vast number of genes that encode c-type cytochromes that contain multiple heme cofactors. This high number of multiheme cytochromes in several bacteria has been correlated with their great respiratory flexibility, and in what concerns biotechnological applications, has been correlated with electricity production in Microbial Fuel Cells. Desulfuromonas acetoxidans, a member of the Geobactereaceae family, is one of these organisms for which the genome was recently made available, coding for 47 putative multiheme cytochromes. The growth of D. acetoxidans in different media allowed the identification of the cytochromes dominant in each condition. The triheme cytochrome c(7) is always present suggesting a key role in the bioenergetic metabolism of this organism, and a dodecaheme cytochrome of low homology with other proteins in the databases was also isolated. Different cytochromes are found for different growth conditions showing that their roles can be assigned to specific bioenergetic electron transfer routes.

Characterization of microbial community structure and population dynamics of tetrachloroethene-dechlorinating tidal mudflat communities.

Tetrachloroethene (PCE) and trichloroethene (TCE) are common groundwater contaminants that also impact tidal flats, especially near urban and industrial areas. However, very little is known about dechlorinating microbial communities in tidal flats. Titanium pyrosequencing, 16S rRNA gene clone libraries, and dechlorinator-targeted quantitative real-time PCR (qPCR) characterized reductive dechlorinating activities and populations in tidal flat sediments collected from South Korea's central west coast near Kangwha. In microcosms established with surface sediments, PCE dechlorination to TCE began within 10 days and 100% of the initial amount of PCE was converted to TCE after 37 days. cis-1,2-Dichloroethene (cis-DCE) was observed as dechlorination end product in microcosms containing sediments collected from deeper zones (i.e., 35-40 cm below ground surface). Pyrosequencing of bacterial 16S rRNA genes and 16S rRNA gene-targeted qPCR results revealed Desulfuromonas michiganensis-like populations predominanted in both TCE and cis-DCE producing microcosms. Other abundant groups included Desulfuromonas thiophila and Pelobacter acidigallici-like populations in the surface sediment microcosms, and Desulfovibrio dechloracetivorans and Fusibacter paucivorans-like populations in the deeper sediment microcosms. Dehalococcoides spp. populations were not detected in these sediments before and after incubation with PCE. The results suggest that tidal flats harbor novel, salt-tolerant dechlorinating populations and that titanium pyrosequencing provides more detailed insight into community structure dynamics of the dechlorinating microcosms than conventional 16S rRNA gene sequencing or fingerprinting methods.

Constraint-based modeling analysis of the metabolism of two Pelobacter species.

BACKGROUND: Pelobacter species are commonly found in a number of subsurface environments, and are unique members of the Geobacteraceae family. They are phylogenetically intertwined with both Geobacter and Desulfuromonas species. Pelobacter species likely play important roles in the fermentative degradation of unusual organic matters and syntrophic metabolism in the natural environments, and are of interest for applications in bioremediation and microbial fuel cells. RESULTS: In order to better understand the physiology of Pelobacter species, genome-scale metabolic models for Pelobacter carbinolicus and Pelobacter propionicus were developed. Model development was greatly aided by the availability of models of the closely related Geobacter sulfurreducens and G. metallireducens. The reconstructed P. carbinolicus model contains 741 genes and 708 reactions, whereas the reconstructed P. propionicus model contains 661 genes and 650 reactions. A total of 470 reactions are shared among the two Pelobacter models and the two Geobacter models. The different reactions between the Pelobacter and Geobacter models reflect some unique metabolic capabilities such as fermentative growth for both Pelobacter species. The reconstructed Pelobacter models were validated by simulating published growth conditions including fermentations, hydrogen production in syntrophic co-culture conditions, hydrogen utilization, and Fe(III) reduction. Simulation results matched well with experimental data and indicated the accuracy of the models. CONCLUSIONS: We have developed genome-scale metabolic models of P. carbinolicus and P. propionicus. These models of Pelobacter metabolism can now be incorporated into the growing repertoire of genome scale models of the Geobacteraceae family to aid in describing the growth and activity of these organisms in anoxic environments and in the study of their roles and interactions in the subsurface microbial community.

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer.

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.