[show abstract][hide abstract] ABSTRACT: Phototrophic microbial mats frequently exhibit sharp, light-dependent redox gradients that regulate microbial respiration on specific electron acceptors as a function of depth. In this work, a benthic phototrophic microbial mat from Hot Lake, a hypersaline, epsomitic lake located near Oroville in north-central Washington, was used to develop a microscale electrochemical method to study local electron transfer processes within the mat. To characterize the physicochemical variables influencing electron transfer, we initially quantified redox potential, pH, and dissolved oxygen gradients by depth in the mat under photic and aphotic conditions. We further demonstrated that power output of a mat fuel cell was light-dependent. To study local electron transfer processes, we deployed a microscale electrode (microelectrode) with tip size ~20 μm. To enrich a subset of microorganisms capable of interacting with the microelectrode, we anodically polarized the microelectrode at depth in the mat. Subsequently, to characterize the microelectrode-associated community and compare it to the neighboring mat community, we performed amplicon sequencing of the V1-V3 region of the 16S gene. Differences in Bray-Curtis beta diversity, illustrated by large changes in relative abundance at the phylum level, suggested successful enrichment of specific mat community members on the microelectrode surface. The microelectrode-associated community exhibited substantially reduced alpha diversity and elevated relative abundances of Prosthecochloris, Loktanella, Catellibacterium, other unclassified members of Rhodobacteraceae, Thiomicrospira, and Limnobacter, compared with the community at an equivalent depth in the mat. Our results suggest that local electron transfer to an anodically polarized microelectrode selected for a specific microbial population, with substantially more abundance and diversity of sulfur-oxidizing phylotypes compared with the neighboring mat community.
[show abstract][hide abstract] ABSTRACT: Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site and have been shown to significantly change in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single cell genomics techniques to shed light on the physiological niche of these microorganisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide-range of both intra- and extra-cellular carbohydrate-active enzymes, enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors (TBDRs), which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. CRISPR-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for survival by heterotrophic microorganisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area subsurface and biogeochemical shifts associated with Columbia River water intrusion.
[show abstract][hide abstract] ABSTRACT: The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decaheme cytochromes, MtrC and MtrA, brought together inside a transmembrane porin, MtrB, to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system containing a pool of internalized electron carriers was used to investigate how the topology of the MtrCAB complex relates to its ability to transport electrons across a lipid bilayer to externally located Fe(III) oxides. With MtrA facing the interior and MtrC exposed on the outer surface of the phospholipid bilayer, the established in vivo orientation, electron transfer from the interior electron carrier pool through MtrCAB to solid-phase Fe(III) oxides was demonstrated. The rates were 10(3) times higher than those reported for reduction of goethite, hematite, and lepidocrocite by S. oneidensis, and the order of the reaction rates was consistent with those observed in S. oneidensis cultures. In contrast, established rates for single turnover reactions between purified MtrC and Fe(III) oxides were 10(3) times lower. By providing a continuous flow of electrons, the proteoliposome experiments demonstrate that conduction through MtrCAB directly to Fe(III) oxides is sufficient to support in vivo, anaerobic, solid-phase iron respiration.
Proceedings of the National Academy of Sciences 03/2013; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: L-rhamnose (L-Rha) is a deoxy-hexose sugar commonly found in nature. L-Rha catabolic pathways were previously characterized in various bacteria including Escherichia coli. Nevertheless, homology searches failed to recognize all the genes for the complete L-Rha utilization pathways in diverse microbial species involved in biomass decomposition. Moreover, the regulatory mechanisms of L-Rha catabolism have remained unclear in most species. A comparative genomics approach was used to reconstruct the L-Rha catabolic pathways and transcriptional regulons in the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, and Thermotogae. The reconstructed pathways include multiple novel enzymes and transporters involved in the utilization of L-Rha and L-Rha-containing polymers. Large-scale regulon inference using bioinformatics revealed remarkable variations in transcriptional regulators for L-Rha utilization genes among bacteria. A novel bifunctional enzyme, L-rhamnulose-phosphate aldolase (RhaE) fused to L-lactaldehyde dehydrogenase (RhaW), which is not homologous to previously characterized L-Rha catabolic enzymes, was identified in diverse bacteria including Chloroflexi, Bacilli, and Alphaproteobacteria. By using in vitro biochemical assays we validated both enzymatic activities of the purified recombinant RhaEW proteins from Chloroflexus aurantiacus and Bacillus subtilis. Another novel enzyme of the L-Rha catabolism, L-lactaldehyde reductase (RhaZ), was identified in Gammaproteobacteria and experimentally validated by in vitro enzymatic assays using the recombinant protein from Salmonella typhimurium. C. aurantiacus induced transcription of the predicted L-Rha utilization genes when L-Rha was present in the growth medium and consumed L-Rha from the medium. This study provided comprehensive insights to L-Rha catabolism and its regulation in diverse Bacteria.
[show abstract][hide abstract] ABSTRACT: Phototrophic microbial mats are compact ecosystems composed of highly interactive organisms in which energy and element cycling take place over millimeter-to-centimeter-scale distances. Although microbial mats are common in hypersaline environments, they have not been extensively characterized in systems dominated by divalent ions. Hot Lake is a meromictic, epsomitic lake that occupies a small, endorheic basin in north-central Washington. The lake harbors a benthic, phototrophic mat that assembles each spring, disassembles each fall, and is subject to greater than tenfold variation in salinity (primarily Mg(2+) and SO(2-) 4) and irradiation over the annual cycle. We examined spatiotemporal variation in the mat community at five time points throughout the annual cycle with respect to prevailing physicochemical parameters by amplicon sequencing of the V4 region of the 16S rRNA gene coupled to near-full-length 16S RNA clone sequences. The composition of these microbial communities was relatively stable over the seasonal cycle and included dominant populations of Cyanobacteria, primarily a group IV cyanobacterium (Leptolyngbya), and Alphaproteobacteria (specifically, members of Rhodobacteraceae and Geminicoccus). Members of Gammaproteobacteria (e.g., Thioalkalivibrio and Halochromatium) and Deltaproteobacteria (e.g., Desulfofustis) that are likely to be involved in sulfur cycling peaked in summer and declined significantly by mid-fall, mirroring larger trends in mat community richness and evenness. Phylogenetic turnover analysis of abundant phylotypes employing environmental metadata suggests that seasonal shifts in light variability exert a dominant influence on the composition of Hot Lake microbial mat communities. The seasonal development and organization of these structured microbial mats provide opportunities for analysis of the temporal and physical dynamics that feed back to community function.
[show abstract][hide abstract] ABSTRACT: Shewanella species are isolated from the oxic/anoxic regions of seawater and aquatic sediments where redox conditions fluctuate in time and space. Colonization of these environments is by virtue of flexible respiratory chains, many of which are notable for the ability to reduce extracellular substrates including the Fe(III) and Mn(IV) contained in oxide and phyllosilicate minerals. Shewanella oneidensis MR-1 serves as a model organism to consider the biochemical basis of this flexibility. In the present paper, we summarize the various systems that serve to branch the respiratory chain of S. oneidensis MR-1 in order that electrons from quinol oxidation can be delivered the various terminal electron acceptors able to support aerobic and anaerobic growth. This serves to highlight several unanswered questions relating to the regulation of respiratory electron transport in Shewanella and the central role(s) of the tetrahaem-containing quinol dehydrogenase CymA in that process.
Biochemical Society Transactions 12/2012; 40(6):1217-21. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: The free energy profile for electron flow through the bacterial decahaem cytochrome MtrF has been computed using thermodynamic integration and classical molecular dynamics. The extensive calculations on two versions of the structure help to validate the method and results, because differences in the profiles can be related to differences in the charged amino acids local to specific haem groups. First estimates of reorganization free energies λ yield a range consistent with expectations for partially solvent-exposed cofactors, and reveal an activation energy range surmountable for electron flow. Future work will aim at increasing the accuracy of λ with polarizable forcefield dynamics and quantum chemical energy gap calculations, as well as quantum chemical computation of electronic coupling matrix elements.
Biochemical Society Transactions 12/2012; 40(6):1198-203. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decahaem cytochromes brought together inside a transmembrane porin to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system has been developed that contains Methyl Viologen as an internalized electron carrier and valinomycin as a membrane-associated cation exchanger. These proteoliposomes can be used as a model system to investigate MtrCAB function.
Biochemical Society Transactions 12/2012; 40(6):1257-60. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Originally discovered in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), key components of the Mtr (i.e. metal-reducing) pathway exist in all strains of metal-reducing Shewanella characterized. The protein components identified to date for the Mtr pathway of MR-1 include four multihaem c-Cyts (c-type cytochromes), CymA, MtrA, MtrC and OmcA, and a porin-like outer membrane protein MtrB. They are strategically positioned along the width of the MR-1 cell envelope to mediate electron transfer from the quinone/quinol pool in the inner membrane to Fe(III)-containing minerals external to the bacterial cells. A survey of microbial genomes has identified homologues of the Mtr pathway in other dissimilatory Fe(III)-reducing bacteria, including Aeromonas hydrophila, Ferrimonas balearica and Rhodoferax ferrireducens, and in the Fe(II)-oxidizing bacteria Dechloromonas aromatica RCB, Gallionella capsiferriformans ES-2 and Sideroxydans lithotrophicus ES-1. The apparent widespread distribution of Mtr pathways in both Fe(III)-reducing and Fe(II)-oxidizing bacteria suggests a bidirectional electron transfer role, and emphasizes the importance of this type of extracellular electron-transfer pathway in microbial redox transformation of iron. The organizational and electron-transfer characteristics of the Mtr pathways may be shared by other pathways used by micro-organisms for exchanging electrons with their extracellular environments.
Biochemical Society Transactions 12/2012; 40(6):1261-7. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many bacterial and archaeal species can couple growth to the respiratory reduction or oxidation of insoluble mineral oxides of transition metals. These solid substrates are abundant electron sinks and sources for life on Earth, but, since they are insoluble in water, they cannot enter the bacterial cells. So, to exploit these electron sinks and sources, specific respiratory electron-transfer mechanisms must overcome the physical limitations associated with electron transfer between a microbe and extracellular metal oxides. Recent microbiological, geochemical, biochemical, spectroscopic and structural work is beginning to shed light on the molecular mechanism and impacts of electron transfer at the microbe-mineral interface from a nanometre to kilometre scale. The research field is attracting attention in applied quarters from those with interests in nanowires, microbial fuel cells, bioremediation and microbial cell factories.
Biochemical Society Transactions 12/2012; 40(6):1163-6. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: The outer-membrane decahaem cytochrome MtrC is part of the transmembrane MtrCAB complex required for mineral respiration by Shewanella oneidensis. MtrC has significant sequence similarity to the paralogous decahaem cytochrome MtrF, which has been structurally solved through X-ray crystallography. This now allows for homology-based models of MtrC to be generated. The structure of these MtrC homology models contain ten bis-histidine-co-ordinated c-type haems arranged in a staggered cross through a four-domain structure. This model is consistent with current spectroscopic data and shows that the areas around haem 5 and haem 10, at the termini of an octahaem chain, are likely to have functions similar to those of the corresponding haems in MtrF. The electrostatic surfaces around haem 7, close to the β-barrels, are different in MtrF and MtrC, indicating that these haems may have different potentials and interact with substrates differently.
Biochemical Society Transactions 12/2012; 40(6):1181-5. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: A model-based analysis is conducted to investigate metabolism of Shewanella oneidensis MR-1 strain in aerobic batch culture, which exhibits an intriguing growth pattern by sequentially consuming substrate (i.e., lactate) and by-products (i.e., pyruvate and acetate). A general protocol is presented for developing a detailed network-based dynamic model for S. oneidensis based on the Lumped Hybrid Cybernetic Model (L-HCM) framework. The L-HCM, although developed from only limited data, is shown to accurately reproduce exacting dynamic metabolic shifts, and provide reasonable estimates of energy requirement for growth. Flux distributions in S. oneidensis predicted by the L-HCM compare very favorably with (13)C-metabolic flux analysis results reported in the literature. Predictive accuracy is enhanced by incorporating measurements of only a few intracellular fluxes, in addition to extracellular metabolites. The L-HCM developed here for S. oneidensis is consequently a promising tool for the analysis of intracellular flux distribution and metabolic engineering.
[show abstract][hide abstract] ABSTRACT: Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure has also been crystallographically resolved in complex with substrates, an Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.
[show abstract][hide abstract] ABSTRACT: A major goal of microbial community ecology is to understand the forces that structure community composition. Deterministic selection by specific environmental factors is sometimes important, but in other cases stochastic or ecologically neutral processes dominate. Lacking is a unified conceptual framework aiming to understand why deterministic processes dominate in some contexts but not others. Here we work toward such a framework. By testing predictions derived from general ecological theory we aim to uncover factors that govern the relative influences of deterministic and stochastic processes. We couple spatiotemporal data on subsurface microbial communities and environmental parameters with metrics and null models of within and between community phylogenetic composition. Testing for phylogenetic signal in organismal niches showed that more closely related taxa have more similar habitat associations. Community phylogenetic analyses further showed that ecologically similar taxa coexist to a greater degree than expected by chance. Environmental filtering thus deterministically governs subsurface microbial community composition. More importantly, the influence of deterministic environmental filtering relative to stochastic factors was maximized at both ends of an environmental variation gradient. A stronger role of stochastic factors was, however, supported through analyses of phylogenetic temporal turnover. Although phylogenetic turnover was on average faster than expected, most pairwise comparisons were not themselves significantly non-random. The relative influence of deterministic environmental filtering over community dynamics was elevated, however, in the most temporally and spatially variable environments. Our results point to general rules governing the relative influences of stochastic and deterministic processes across micro- and macro-organisms.
The ISME Journal 03/2012; 6(9):1653-64. · 8.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pyrosequencing analysis of 16S rRNA genes was used to study temporal dynamics of groundwater bacteria and archaea over 10 months within three well clusters separated by ~30 m and located 250 m from the Columbia River on the Hanford Site, WA. Each cluster contained three wells screened at different depths ranging from 10 to 17 m that differed in hydraulic conductivities. Representative samples were selected for analyses of prokaryotic 16S and eukaryotic 18S rRNA gene copy numbers. Temporal changes in community composition occurred in all nine wells over the 10-month sampling period. However, there were particularly strong effects near the top of the water table when the seasonal rise in the Columbia River caused river water intrusion at the top of the aquifer. The occurrence and disappearance of some microbial assemblages (such as Actinobacteria ACK-M1) were correlated with river water intrusion. This seasonal impact on microbial community structure was greater in the shallow saturated zone than deeper zone in the aquifer. Spatial and temporal patterns for several 16S rRNA gene operational taxonomic units associated with particular physiological functions (for example, methane oxidizers and metal reducers) suggests dynamic changes in fluxes of electron donors and acceptors over an annual cycle. In addition, temporal dynamics in eukaryotic 18S rRNA gene copies and the dominance of protozoa in 18S clone libraries suggest that bacterial community dynamics could be affected not only by the physical and chemical environment but also by top-down biological control.
The ISME Journal 03/2012; 6(9):1665-76. · 8.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: The microbial reduction of Fe(III) and U(VI) was investigated in shallow aquifer sediments collected from subsurface flood deposits near the Hanford Reach of the Columbia River in Washington State. Increases in 0.5 N HCl-extractable Fe(II) were observed in incubated sediments and (57)Fe Mössbauer spectroscopy revealed that Fe(III) associated with phyllosilicates and pyroxene was reduced to Fe(II). Aqueous uranium(VI) concentrations decreased in subsurface sediments incubated in sulfate-containing synthetic groundwater with the rate and extent being greater in sediment amended with organic carbon. X-ray absorption spectroscopy of bioreduced sediments indicated that 67-77% of the U signal was U(VI), probably as an adsorbed species associated with a new or modified reactive mineral phase. Phylotypes within the Deltaproteobacteria were more common in Hanford sediments incubated with U(VI) than without, and in U(VI)-free incubations, members of the Clostridiales were dominant with sulfate-reducing phylotypes more common in the sulfate-amended sediments. These results demonstrate the potential for anaerobic reduction of phyllosilicate Fe(III) and sulfate in Hanford unconfined aquifer sediments and biotransformations involving reduction and adsorption leading to decreased aqueous U concentrations.