Functional diversity and electron donor dependence of microbial populations capable of U(VI) reduction in radionuclide-contaminated subsurface sediments

In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.Uranium contamination in subsurface environments is a widespread problem at mining and milling sites across North America, South America, and Eastern Europe (1). Uranium in the oxidized state, U(VI), is highly soluble and toxic and thus is a potential contaminant to local drinking-water supplies (46). Nitrate is often a cocontaminant with U(VI) as a result of the use of nitric acid in the processing of uranium and uranium-bearing waste (6, 45). Oxidized uranium can be immobilized in contaminated groundwater through the reduction of U(VI) to insoluble U(IV) by indirect (abiotic) and direct (enzymatic) processes catalyzed by microorganisms. Current remediation practices favor the stimulation of reductive uranium immobilization catalyzed by indigenous microbial communities along with natural attenuation and monitoring (5, 24, 40, 44, 65, 68, 69). Microbial uranium reduction activity in contaminated subsurface environments is often limited by carbon or electron donor availability (13, 24, 44, 69). Previous studies have indicated that U(VI) reduction does not proceed until nitrate is depleted (13, 16, 24, 44, 68, 69), as high nitrate concentrations inhibit the reduction of U(VI) by serving as a competing and more energetically favorable terminal electron acceptor for microorganisms (11, 16). The fate and transport of uranium in groundwater are also strongly linked through sorption and precipitation processes to the bioreduction of Fe minerals, including oxides, layer-silicate clay minerals, and sulfides (7, 23, 53).In order to appropriately design U(VI) bioremediation strategies, the potential function and phylogenetic structure of indigenous subsurface microbial communities must be further understood (24, 34, 46). Conflicting evidence has been presented on which microbial groups, Fe(III)- or sulfate-reducing bacteria (FeRB or SRB), effectively catalyze the reductive immobilization of U(VI) in the presence of amended electron donors (5, 44, 69). The addition of acetate to the subsurface at a uranium-contaminated site in Rifle, Colorado, initially stimulated FeRB within the family Geobacteraceae to reduce U(VI) (5, 65). However, with long-term acetate addition, SRB within the family Desulfobacteraceae, which are not capable of U(VI) reduction, increased in abundance and a concomitant reoxidation of U(IV) was observed (5, 65). At a uranium-contaminated site in Oak Ridge, Tennessee, in situ and laboratory-based experiments successfully employed ethanol amendments to stimulate denitrification followed by the reduction of U(VI) by indigenous microbial communities (13, 24, 44, 48, 50, 57, 68). In these studies, ethanol amendments stimulated both SRB and FeRB, with SRB likely catalyzing the reduction of U(VI). This suggests that the potential for bioremediation will be affected by the choice of electron donor amendment through effects on the functional diversity of U(VI)-reducing microbial populations. As uranium reduction is dependent on the depletion of nitrate, the microbial populations mediating nitrate reduction are also critical to the design of bioremediation strategies. Although nitrate-reducing bacteria (NRB) have been studied extensively in subsurface environments (2, 15, 19, 24, 56, 58, 70), the mechanisms controlling the in situ metabolism of NRB remain poorly understood.The dynamics of microbial populations capable of U(VI) reduction in subsurface sediments are poorly understood, and the differences in the microbial community dynamics during bioremediation have not been explored. Based on the results of previous studies (13, 44, 49, 57, 68, 69), we hypothesized that the activity of nitrate- and Fe(III)-reducing microbial populations, catalyzing the reductive immobilization of U(VI) in subsurface radionuclide-contaminated sediments, would be dependent on the choice of electron donor. The objectives of the present study were (i) to characterize structure-function relationships for microbial groups likely to catalyze or limit U(VI) reduction in radionuclide-contaminated sediments and (ii) to further develop a proxy for the metabolic activity of FeRB. Microbial activity was assessed by monitoring terminal electron-accepting processes (TEAPs), electron donor utilization, and Fe(III) mineral transformations in microcosms conducted with subsurface materials cocontaminated with high levels of U(VI) and nitrate. In parallel, microbial functional groups (i.e., NRB and FeRB) were enumerated and characterized using a combination of cultivation-dependent and -independent methods.     

Immobilization of Cr(VI) and Its Reduction to Cr(III) Phosphate by Granular Biofilms Comprising a Mixture of Microbes

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromate and remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubated aerobically reduced 0.2 mM Cr(VI) from a minimal medium at 0.15 mM day⁻¹ g⁻¹, with reduction of 0.17 mM day⁻¹ g⁻¹ under anaerobic conditions. There was negligible removal of Cr(VI) (i) without granular biofilms, (ii) with lyophilized granular biofilms, and (iii) with granules in the absence of an electron donor. Analyses by X-ray absorption near edge spectroscopy (XANES) of the granular biofilms revealed the conversion of soluble Cr(VI) to Cr(III). Extended X-ray absorption fine-structure (EXAFS) analysis of the Cr-laden granular biofilms demonstrated similarity to Cr(III) phosphate, indicating that Cr(III) was immobilized with phosphate on the biomass subsequent to microbial reduction. The sustained reduction of Cr(VI) by granular biofilms was confirmed in fed-batch experiments. Our study demonstrates the promise of granular-biofilm-based systems in treating Cr(VI)-containing effluents and wastewater.Chromium is a common industrial chemical used in tanning leather, plating chrome, and manufacturing steel. The two stable environmental forms are hexavalent chromium [Cr(VI)] and trivalent chromium [Cr(III)] (20). The former is highly soluble and toxic to microorganisms, plants, and animals, entailing mutagenic and carcinogenic effects (6, 22, 33), while the latter is considered to be less soluble and less toxic. Therefore, the reduction of Cr(VI) to Cr(III) constitutes a potential detoxification process that might be achieved chemically or biologically. Microbial reduction of Cr(VI) seemingly is ubiquitous; Cr(VI)-reducing bacteria have been isolated from both Cr(VI)-contaminated and -uncontaminated environments (6, 7, 23, 38, 39). Many archaeal/eubacterial genera, common to different environments, reduce a wide range of metals, including Cr(VI) (6, 16, 21). Some bacterial enzymes generate Cr(V) by mediating one-electron transfer to Cr(VI) (1, 4), while many other chromate reductases convert Cr(VI) to Cr(III) in a single step.Biological treatment of Cr(VI)-contaminated wastewater may be difficult because the metal''s toxicity potentially can kill the bacteria. Accordingly, to protect the cells, cell immobilization techniques were employed (31). Cells in a biofilm exhibit enhanced resistance and tolerance to toxic metals compared with free-living ones (15). Therefore, biofilm-based reduction of Cr(VI) and its subsequent immobilization might be a satisfactory method of bioremediation because (i) the biofilm-bound cells can tolerate higher concentrations of Cr(VI) than planktonic cells, and (ii) they allow easy separation of the treated liquid from the biomass. Ferris et al. (11) described microbial biofilms as natural metal-immobilizing matrices in aqueous environments. Bioflocs, the active biomass of activated sludge-process systems are transformed into dense granular biofilms in sequencing batch reactors (SBRs). As granular biofilms settle extremely well, the treated effluent is separated quickly from the granular biomass by sedimentation (9, 24). Previous work demonstrated that aerobic granular biofilms possess tremendous ability for biosorption, removing zinc, copper, nickel, cadmium, and uranium (19, 26, 31, 32, 40). However, no study has investigated the role of cellular metabolism of aerobically grown granular biofilms in metal removal experiments. Despite vast knowledge about biotransformation by pure cultures, very little is known about reduction and immobilization by mixed bacterial consortia (8, 12, 13, 16, 20, 31, 36). Our research explored, for the first time, the metabolically driven removal of Cr(VI) by microbial granules.The main aim of this study was to investigate Cr(VI) reduction and immobilization by mixed bacterial consortia, viz., aerobically grown granular biofilms. Such biofilm-based systems are promising for developing compact bioreactors for the rapid biodegradation of environmental contaminants (17, 24, 29). Accordingly, we investigated the microbial reduction of Cr(VI) by aerobically grown biofilms in batch and fed-batch experiments and analyzed the oxidation state and association of the chromium immobilized on the biofilms by X-ray absorption near edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS).     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

Inhibition of Nitrate Reduction by Chromium(VI) in Anaerobic Soil Microcosms

Chromium is often found as a cocontaminant at sites polluted with organic compounds. For nitrate-respiring microbes, Cr(VI) may be not only directly toxic but may also specifically interfere with N reduction. In soil microcosms amended with organic electron donors, Cr(VI), and nitrate, bacteria oxidized added carbon, but relatively low doses of Cr(VI) caused a lag and then lower rates of CO₂ accumulation. Cr(VI) strongly inhibited nitrate reduction; it occurred only after soluble Cr(VI) could not be detected. However, Cr(VI) additions did not eliminate Cr-sensitive populations; after a second dose of Cr(VI), bacterial activity was strongly inhibited. Differences in microbial community composition (assayed by PCR-denaturing gradient gel electrophoresis) driven by different organic substrates (glucose and protein) were smaller than when other electron acceptors had been used. However, the selection of bacterial phylotypes was modified by Cr(VI). Nine isolated clades of facultatively anaerobic Cr(VI)-resistant bacteria were closely related to cultivated members of the phylum Actinobacteria or Firmicutes. In Bacillus cereus GNCR-4, the nature of the electron donor (fermentable or nonfermentable) affected Cr(VI) resistance level and anaerobic nitrate metabolism. Our results indicate that carbon utilization and nitrate reduction in these soils were contingent upon the reduction of added Cr(VI). The amount of Cr(VI) required to inhibit nitrate reduction was 10-fold less than for aerobic catabolism of the same organic substrate. We speculate that the resistance level of a microbial process is directly related to the diversity of microbes capable of conducting it.Chromium(VI) is a toxic metal that can negatively affect bioremediation of organic compounds in sites where chromium and organic pollutants cooccur (36). Under oxygen-limited conditions, chromium(VI) can be reduced (biologically or chemically) to insoluble and relatively nontoxic Cr(III) (22). Despite the potential interactions between biotic and chemical components, the responses of anaerobic microbial activities to Cr(VI) have not been well studied (6, 7, 42, 43).Under anaerobic conditions, an important factor in the catabolism of organic carbon is the availability of electron acceptors. Nitrate is of special interest because it is often found as a copollutant in contaminated soils (18). Nitrate-reducing bacteria are facultative anaerobes commonly found in environmental samples and can couple the reduction of nitrate to the oxidation of diverse organic substrates (10, 13). The effect of Cr(VI) on natural denitrifying communities or pure cultures of denitrifying bacteria is not well characterized (8, 29). The environmental effects of Cr(VI) on denitrification are of particular interest because in addition to acute toxicity to the cell, Cr(VI) may compete with nitrate as an electron acceptor (15, 30). However, in other denitrifying bacteria (for example, Staphylococcus spp.), no competitive interactions were reported (45).The purpose of this study was to extend our work on the effects of Cr(VI) upon microbes in soil that mediate discrete chemoheterotrophic processes such as the use of O₂ (30) or Fe⁺³ (26) as terminal electron acceptors. We examined denitrification to determine whether the putative direct impact of Cr(VI) on the biochemistry of nitrate reduction would alter community dynamics from what had been observed with other terminal electron acceptors. In addition, we can add this data set to previous work to analyze the range of sensitivities to Cr(VI) that were found across a broad array of chemoheterotrophic processes.     

Significant Association between Sulfate-Reducing Bacteria and Uranium-Reducing Microbial Communities as Revealed by a Combined Massively Parallel Sequencing-Indicator Species Approach

Massively parallel sequencing has provided a more affordable and high-throughput method to study microbial communities, although it has mostly been used in an exploratory fashion. We combined pyrosequencing with a strict indicator species statistical analysis to test if bacteria specifically responded to ethanol injection that successfully promoted dissimilatory uranium(VI) reduction in the subsurface of a uranium contamination plume at the Oak Ridge Field Research Center in Tennessee. Remediation was achieved with a hydraulic flow control consisting of an inner loop, where ethanol was injected, and an outer loop for flow-field protection. This strategy reduced uranium concentrations in groundwater to levels below 0.126 μM and created geochemical gradients in electron donors from the inner-loop injection well toward the outer loop and downgradient flow path. Our analysis with 15 sediment samples from the entire test area found significant indicator species that showed a high degree of adaptation to the three different hydrochemical-created conditions. Castellaniella and Rhodanobacter characterized areas with low pH, heavy metals, and low bioactivity, while sulfate-, Fe(III)-, and U(VI)-reducing bacteria (Desulfovibrio, Anaeromyxobacter, and Desulfosporosinus) were indicators of areas where U(VI) reduction occurred. The abundance of these bacteria, as well as the Fe(III) and U(VI) reducer Geobacter, correlated with the hydraulic connectivity to the substrate injection site, suggesting that the selected populations were a direct response to electron donor addition by the groundwater flow path. A false-discovery-rate approach was implemented to discard false-positive results by chance, given the large amount of data compared.Massively parallel sequencing has increased our ability to study microbial communities to a greater depth and at decreased sequencing costs to an extent that replication and gradient interrogation are now reasonably attainable. However, this massive throughput has mostly been used in exploratory studies, given the challenges to analysis of the big data sets generated and the relative novelty of the technique. To date, no report of a study that has used this method to describe the microbial community over a large area influenced by complicated hydrogeochemical factors during bioremediation has been published. Here, we used pyrosequencing technology complemented with a hypothesis-based approach to identify bacteria associated with biostimulation of U(VI) reduction at Area 3 of the U.S. Department of Energy''s (DOE''s) Oak Ridge Field Research Center (FRC) at Oak Ridge, TN.The Oak Ridge FRC is one of the most-studied sites for uranium bioremediation (2, 8, 19-22, 27, 37, 45-48). Previously used as a uranium enrichment plant, the site remains contaminated with depleted uranium, nitrate, and acidity. To deal with uranium contamination, dissimilatory metal reduction has been studied as an alternative that reduces risk by converting toxic soluble metals and radionuclides to insoluble, less toxic forms (2, 3, 16, 21, 26, 45). For example, some microbes can use metals such as Cr(VI), Se(VI), and the radionuclides U(VI) and Tc(VII) as final electron acceptors, producing a reduced insoluble species, thus blocking dispersal and reducing bioavailability.The ability to reduce U(VI) to U(IV) has been found in several unrelated phylogenetic groups, i.e., Delta-, Beta-, and Gammaproteobacteria, Firmicutes, Deinococci, and Actinobacteria, among others (42). Most previous studies have focused on the Fe(III)-reducing bacteria (FRB), especially Geobacter, and the sulfate-reducing bacteria (SRB), especially Desulfovibrio. Uranium(VI) reduction for bioremediation purposes has been tested and confirmed in laboratory-scale experiments using serum bottles (13, 18, 48), microcosms (23, 32), sediment columns (14, 43), and in situ field studies (3, 21, 41, 45), with the last one demonstrating the feasibility of U(VI) remediation and the correlation of U(VI) reduction with FRB (3, 6, 18, 31, 41) or SRB (40), or both (8, 19, 49).During field studies at Area 3 of the Oak Ridge site, a hydraulic control system together with ethanol injection successfully promoted U(VI) reduction from 5 μM to levels below U.S. Environmental Protection Agency (EPA) maximum contaminant levels (MCLs) for drinking water (0.126 μM) over a 2-year period (46). Reduction of U(VI) to U(IV) was confirmed by X-ray absorption near edge structure (XANES) (22, 46). Previous microbial surveys of sediments and groundwater from Area 3 wells by the use of 16S rRNA gene clone libraries detected genera known to harbor U(VI)-reducing members, such as Geobacter, Desulfovibrio, Anaeromyxobacter, Desulfosporosinus, and Acidovorax, after U(VI) reduction was established (8, 19). In one study, microbial counts from sediments were correlated with the hydraulic path, suggesting differences in organic carbon availability throughout Area 3 (8). The study that tracked the groundwater microbial communities of four locations of Area 3 over a 1.5-year period during ethanol stimulation found that nitrate, uranium, sulfide, and ethanol were correlated with particular bacterial populations and that the engineering control of dissolved oxygen and delivered nutrients was also significant in explaining the microbial community variability (19). However, the analysis of communities has been focused on limited wells and the community of the entire test area has not been characterized.On the basis of the previous results, we further hypothesized that the hydrological control strategy employed for the remediation of the site constrained the geochemistry of the site by controlling the distribution of organic carbon substrates and other nutrients and that this in turn selected a characteristic microbial community that was distinguishable from its surrounding community. We used massively parallel sequencing of 16S rRNA genes from sediments of 15 wells to characterize the microbial communities along hydrological gradients from the microbiologically active and hydraulically protected inner-loop zone to less active and still contaminated areas outside the treatment area and downgradient. Our sediment-sampling strategy allows a more precise spatial characterization than the use of groundwater samples, where filtering large volumes of water is often required, and also because samples of the attached communities can differ from the planktonic ones, as expected in oligotrophic aquifers (15), such as this site. The deeper sequencing allowed a more extensive survey of the communities, higher confidence in the detection of less dominant but significant members, and a more statistically robust indicator species assessment. We were able to detect groups significantly associated with U(VI) reduction and to explain differences in community structure with hydrogeochemical conditions.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Microbial Community Changes in Response to Ethanol or Methanol Amendments for U(VI) Reduction

Microbial community responses to ethanol, methanol, and methanol plus humics amendments in relationship to U(VI) bioreduction were studied in laboratory microcosm experiments using sediments and ground water from a uranium-contaminated site in Oak Ridge, TN. The type of carbon source added, the duration of incubation, and the sampling site influenced the bacterial community structure upon incubation. Analysis of 16S rRNA gene clone libraries indicated that (i) bacterial communities found in ethanol- and methanol-amended samples with U(VI) reduction were similar due to the presence of Deltaproteobacteria and Betaproteobacteria (members of the families Burkholderiaceae, Comamonadaceae, Oxalobacteraceae, and Rhodocyclaceae); (ii) methanol-amended samples without U(VI) reduction exhibited the lowest diversity and the bacterial community contained 69.2 to 92.8% of the family Methylophilaceae; and (iii) the addition of humics resulted in an increase of phylogenetic diversity of Betaproteobacteria (Rodoferax, Polaromonas, Janthinobacterium, Methylophilales, and unclassified) and Firmicutes (Desulfosporosinus and Clostridium).The use of uranium in nuclear research, fuel production, and weapons manufacturing has resulted in environmental contamination at production, manufacturing, and storage sites throughout the United States. Although all of the common isotopes of uranium (²³⁸U [99.27%], ²³⁵U [0.72%], and ²³⁴U [0.005%]) are radioactive, it is the chemical toxicity of uranium that is usually of greatest concern when it is present as a contaminant.The U.S. Department of Energy (DOE) has ongoing efforts to identify and remediate contaminated areas under its control. Stimulating the in situ metabolism of microorganisms capable of reduction of U(VI) to U(IV), producing the insoluble mineral uraninite which precipitates and renders uranium immobile in ground water, has been proposed as an environmentally safe and a potentially cost-effective remediation method (37). Typically, an organic substrate is added to stimulate microbial growth and promote the development of anaerobic conditions, under which the reduction of U(VI) is favored (67). Various substrates (e.g., acetate, ethanol, glucose, and methanol) have been used either in the field or in microcosm studies, and most were capable of stimulating microbial U(VI) reduction (1, 8, 42, 43, 47, 60); however, the addition of methanol did not always result in U(VI) reduction (49). Many microorganisms are known to reduce U(VI) in pure culture, including a hyperthermophilic archaeon (28), a thermophilic bacterium Thermoterrabacterium ferrireducens (29), the mesophilic dissimilatory metal-reducing bacteria Geobacter and Shewanella (67) and Anaeromyxobacter dehalogenans (71), the sulfate-reducing bacterium Desulfovibrio sp. (61), and fermentative bacteria such as Clostridium spp. (20). These data suggest that U(VI) can be reduced by many microorganisms once suitable electron donors are available.The purpose of this study was to analyze the ability of various amendments to stimulate the reduction of U(VI) by the indigenous microbial communities found in subsurface sediments collected from a uranium-contaminated site. A previous publication from this project (42) gave a very limited analysis of the microbial community. Here we present a detailed phylogenetic analysis of the bacterial community structure and link community structure to capability of U(VI) reduction in sediments stimulated with ethanol and methanol. This study was designed to explore whether microbial communities that demonstrate U(VI) reduction after stimulation with different alcohols show a similar structure. Also, it was designed to detect differences between the methanol-stimulated communities that were capable of U(VI) reduction and those that were not capable of U(VI) reduction. Since humic substances have been reported to promote U(VI) reduction (10, 34), we also examined the effects of humics on the community structure and reduction of U(VI).     

Disruption of the Putative Cell Surface Polysaccharide Biosynthesis Gene SO3177 in Shewanella oneidensis MR-1 Enhances Adhesion to Electrodes and Current Generation in Microbial Fuel Cells

A microbial fuel cell (MFC) was inoculated with a random transposon insertion mutant library of Shewanella oneidensis MR-1 and operated with lactate as the sole fuel to select for mutants that preferentially grew in it. Agar plate cultivation of the resultant MFC enrichment culture detected an increased number of colonies exhibiting rough morphology. One such isolate, strain 4A, generated 50% more current in an MFC than wild-type MR-1. Determination of the transposon insertion site in strain 4A followed by deletion and complementation experiments revealed that the SO3177 gene, encoding a putative formyltransferase and situated in a cell surface polysaccharide biosynthesis gene cluster, was responsible for the increased current. Transmission electron microscopy showed that a layered structure at the cell surface, stainable with ruthenium red, was impaired in the SO3177 mutant (ΔSO3177), confirming that SO3177 is involved in the biosynthesis of cell surface polysaccharides. Compared to the wild type, ΔSO3177 cells preferentially attached to graphite felt anodes in MFCs, while physicochemical analyses revealed that the cell surface of ΔSO3177 was more hydrophobic. These results demonstrate that cell surface polysaccharides affect not only the cell adhesion to graphite anodes but also the current generation in MFCs.Dissimilatory metal-reducing bacteria (DMRB) conserve energy for growth by coupling the oxidation of organic compounds to the reduction of metal compounds (29). DMRB are of great interest not only for their importance in the biogeochemical cycling of metals (25) but also for their utility in biotechnological processes, such as microbial fuel cells (MFCs) (24, 40). In recent years, the ability of many DMRB, including members of the genera Shewanella (5, 12, 20, 31), Geobacter (2), Aeromonas (34), Desulfobulbus (19), and Phodoferax (9), to generate current in MFCs has been described.Among DMRB, Shewanella oneidensis MR-1 is one of the most extensively studied due to its metabolic versatility (28), annotated genome sequence (17), and genetic accessibility. In addition, since the first report in 1999 when this microorganism was shown to have the ability to transfer electrons to an anode without an exogenously added mediator (20), it has become a model organism for the study of microbial current generation in MFCs. Extensive studies have been performed to understand the mechanisms of extracellular electron transfer (EET) to solid materials, such as MFC anodes and metal oxides, in strain MR-1. Multiple mechanisms, including direct EET through the physical contact of bacterial cells via outer membrane (OM) cytochromes (42) and conductive nanowires (16) and mediated EET via self-produced electron shuttles such as quinones and flavins (27, 30, 39, 41), have been identified.Although OM cytochromes and electron shuttles have been identified to play central roles in EET, it is reasonable to speculate that this complex catabolic process is also influenced by other (extra)cellular components. To date, only limited studies have been done to investigate other cellular components involved in EET (7). A useful approach for identifying unknown cellular components (and genes) associated with a particular phenotype involves the construction and screening of a random mutant library for obtaining mutants with altered phenotypes. In the present study, we constructed a random transposon (Tn) insertion mutant library of S. oneidensis MR-1 and obtained mutants with altered colony morphologies (rough morphotypes) after the selection of mutants in an MFC. Analyses of one of such mutants suggest that cell surface capsular polysaccharides affect not only the adhesion of cells to graphite anodes but also the current generation in MFCs.     

Impact of Silver(I) on the Metabolism of Shewanella oneidensis

Anaerobic cultures of Shewanella oneidensis MR-1 reduced toxic Ag(I), forming nanoparticles of elemental Ag(0), as confirmed by X-ray diffraction analyses. The addition of 1 to 50 μM Ag(I) had a limited impact on growth, while 100 μM Ag(I) reduced both the doubling time and cell yields. At this higher Ag(I) concentration transmission electron microscopy showed the accumulation of elemental silver particles within the cell, while at lower concentrations the metal was exclusively reduced and precipitated outside the cell wall. Whole organism metabolite fingerprinting, using the method of Fourier transform infrared spectroscopy analysis of cells grown in a range of silver concentrations, confirmed that there were significant physiological changes at 100 μM silver. Principal component-discriminant function analysis scores and loading plots highlighted changes in certain functional groups, notably, lipids, amides I and II, and nucleic acids, as being discriminatory. Molecular analyses confirmed a dramatic drop in cellular yields of both the phospholipid fatty acids and their precursor molecules at high concentrations of silver, suggesting that the structural integrity of the cellular membrane was compromised at high silver concentrations, which was a result of intracellular accumulation of the toxic metal.Silver is an element that has been used widely in industrial processes as diverse as photographic processing, catalysis, mirror production, electroplating, alkaline battery production, and jewelry making (18). It has been known for some time that silver ions and silver-based compounds can be highly toxic to microorganisms, and with increasing concern about pathogenic “superbugs” with high resistance to conventional antibiotics, silver is attracting much interest as a potential biocide (11, 18, 36, 42). Silver has no known physiological functions and can exist in several oxidation states, although it is most commonly encountered in its elemental [Ag(0)] and monovalent [Ag(I)] forms. Although use of nanoscale elemental Ag(0) as a biocide has been increasing, for example, in wound dressings and as an antimicrobial coating on consumer products, little is known about its mode of toxicity. This is despite the surprising ability of actively growing Fe(III)-reducing bacteria such as Geobacter sulfurreducens to precipitate nanoscale Ag(0) particles within and around the cell surface via reduction of Ag(I) (18). Ionic Ag(I), in contrast, has been the focus of more studies on the mode of metal toxicity. Previous research showed that silver ions have antimicrobial activities against a wide diversity of bacteria (19). They have been shown to disrupt the respiratory chain of Escherichia coli (3) and inhibit the exchange of phosphate and its uptake (34). Ag(I) has also been linked to copper metabolism in E. coli, potentially competing with copper binding sites on the cell surface and subsequent copper transport into the cell (8). However, the toxicity of silver is not limited to prokaryotes, as long-term exposure in humans can cause argyria, impaired night vision, and abdominal pain (31, 32, 36). The detailed mechanism of toxicity in prokaryotes or eukaryotes remains to be identified, although it has been proposed that silver ions react with cellular proteins via SH groups (16), leading to the disruption of cellular metabolism.Microbial cells have evolved an extremely diverse range of mechanisms to survive high concentrations of toxic metals. The mechanisms invoked include biosorption, bioaccumulation, special efflux systems, alteration of solubility and toxicity via reduction or oxidation, extracellular complexation or precipitation of metals, and lack of specific metal transport systems (1). For example, for silver ions the bacterial cell wall can be an efficient permeability barrier to block the uptake of metal (21), with additional complexation in the periplasm by specific silver-binding proteins (35). Redox transformations also offer the potential to detoxify Ag(I) ions, e.g., through the reduction to insoluble elemental Ag(0) (30). In addition, the energy-dependent efflux of toxic Ag(I) is perhaps the best-studied resistance mechanism for silver, mediated via ATPases and chemiosmotic cation/protons antiporters (9).Shewanella spp., Gram-negative, dissimilatory metal-reducing bacteria, can use a wide variety of terminal electron acceptors for growth (23, 39), including high oxidation state metals such as Fe(III), Mn(IV), Cr(VI), U(VI), and Au(III) (5, 17, 26, 28, 41). Shewanella species also have the potential to reduce Ag(I), given their similar activities against Au(III), and the reduction of Ag(I) to form nanoscale deposits of Ag(0) within the cell has been documented for other Fe(III)-reducing bacteria (18). This metabolic versatility offers considerable potential for bioremediation applications, for example, via reduction of U(VI) to insoluble U(IV) (5, 17, 26, 28, 41), and the recovery of precious metals such as silver and gold via reductive precipitation. It also offers an interesting model organism to study the metabolism of toxic metals such as silver, including the physiological impact of ionic Ag(I) and nanoscale Ag(0).This paper describes interactions of Shewanella oneidensis MR-1 with various concentrations of Ag(I), including demonstrations of the reduction and deposition of silver nanoparticles under anaerobic conditions. A range of techniques, including X-ray diffraction (XRD) and analytical transmission electron microscopy (TEM), were used to investigate the nature and cellular localization of the precipitates, while Fourier transform infrared (FT-IR) spectroscopy metabolic profiling techniques were used to identify the impact of toxic metal accumulation on the cell. The disruption of membrane integrity was implied by these investigations and confirmed by fatty acid methyl ester (FAME) analysis, which showed a dramatic decrease in the quantities of membrane lipid components.     

Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.To escape toxic conditions or to acquire new sources of nutrients, prokaryotes often depend on some form of motility. Swimming motility, a common means by which many bacteria move from one place to another, usually depends on flagellar rotation to propel cells through liquid medium (24, 26, 34). These motility structures are also critical for the effective attachment of bacteria to surfaces.As in bacteria, rotating flagella are responsible for swimming motility in archaea, and recent studies suggest that archaea, like bacteria, also require flagella for efficient surface attachment (37, 58). However, in contrast to bacterial flagellar subunits, which are translocated via a specialized type III secretion apparatus, archaeal flagellin secretion and flagellum assembly resemble the processes used to translocate and assemble the subunits of bacterial type IV pili (34, 38, 54).Type IV pili are typically composed of major pilins, the primary structural components of the pilus, and several minor pilin-like proteins that play important roles in pilus assembly or function (15, 17, 46). Pilin precursor proteins are transported across the cytoplasmic membrane via the Sec translocation pathway (7, 20). Most Sec substrates contain either a class I or a class II signal peptide that is cleaved at a recognition site that lies subsequent to the hydrophobic portion of the signal peptide (18, 43). However, the precursors of type IV pilins contain class III signal peptides, which are processed at recognition sites that precede the hydrophobic domain by a prepilin-specific peptidase (SPase III) (38, 43, 45). Similarly, archaeal flagellin precursors contain a class III signal peptide that is processed by a prepilin-specific peptidase homolog (FlaK/PibD) (3, 8, 10, 11). Moreover, flagellar assembly involves homologs of components involved in the biosynthesis of bacterial type IV pili, including FlaI, an ATPase homologous to PilB, and FlaJ, a multispanning membrane protein that may provide a platform for flagellar assembly, similar to the proposed role for PilC in pilus assembly (38, 44, 53, 54). These genes, as well as a number of others that encode proteins often required for either flagellar assembly or function (flaCDEFG and flaH), are frequently coregulated with the flg genes (11, 26, 44, 54).Interestingly, most sequenced archaeal genomes also contain diverse sets of genes that encode type IV pilin-like proteins with little or no homology to archaeal flagellins (3, 39, 52). While often coregulated with pilB and pilC homologs, these genes are never found in clusters containing the motility-specific flaCDEFG and flaH homologs; however, the proteins they encode do contain class III signal peptides (52). Several of these proteins have been shown to be processed by an SPase III (4, 52). Moreover, in Sulfolobus solfataricus and Methanococcus maripaludis, some of these archaeal type IV pilin-like proteins were confirmed to form surface filaments that are distinct from the flagella (21, 22, 56). These findings strongly suggest that the genes encode subunits of pilus-like surface structures that are involved in functions other than swimming motility.In bacteria, type IV pili are multifunctional filamentous protein complexes that, in addition to facilitating twitching motility, mediate adherence to abiotic surfaces and make close intercellular associations possible (15, 17, 46). For instance, mating between Escherichia coli in liquid medium has been shown to require type IV pili (often referred to as thin sex pili), which bring cells into close proximity (29, 30, 57). Recent work has shown that the S. solfataricus pilus, Ups, is required not only for efficient adhesion to surfaces of these crenarchaeal cells but also for UV-induced aggregation (21, 22, 58). Frols et al. postulate that autoaggregation is required for DNA exchange under these highly mutagenic conditions (22). Halobacterium salinarum has also been shown to form Ca²⁺-induced aggregates (27, 28). Furthermore, conjugation has been observed in H. volcanii, which likely requires that cells be held in close proximity for a sustained period to allow time for the cells to construct the cytoplasmic bridges that facilitate DNA transfer between them (35).To determine the roles played by haloarchaeal flagella and other putative type IV pilus-like structures in swimming and surface motility, surface adhesion, autoaggregation, and conjugation, we constructed and characterized two mutant strains of H. volcanii, one lacking the genes that encode the flagellins and the other lacking pibD. Our analyses indicate that although this archaeon was previously thought to be nonmotile (14, 36), wild-type (wt) H. volcanii can swim in a flagellum-dependent manner. Consistent with the involvement of PibD in processing flagellins, the peptidase mutant is nonmotile. Unlike nonhalophilic archaea, however, the flagellum mutant can adhere to glass as effectively as the wild type. Conversely, the ΔpibD strain fails to adhere to glass surfaces, strongly suggesting that in H. volcanii surface adhesion involves nonflagellar, type IV pilus-like structures.     

Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp.Geobacter sulfurreducens is a member of the metal-reducing Geobacteraceae family and was originally isolated based on its ability to transfer electrons from internal oxidative reactions to extracellular electron acceptors such as insoluble Fe(III) or Mn(IV) oxides (5). G. sulfurreducens is also able to use an electrode as its sole electron acceptor for respiration, a phenotype which has many possible biotechnological applications (28, 29), and serves as a useful tool for direct measurement of electron transfer rates (2, 31). As G. sulfurreducens was the first Geobacteraceae genome sequence available (34) and the only member of this family with a robust genetic system (7), it serves as a model organism for extracellular electron transfer studies.The proteins facilitating electron transfer to insoluble Fe(III) oxides by individual Geobacter cells and how these cells interact in multicellular biofilms are not fully understood. Many genes implicated in Fe(III) and electrode reduction were identified based on proteomic and microarray analysis of cultures grown with fumarate versus Fe(III) citrate as a terminal electron acceptor (9, 15, 35). More recently, similar expression data from Fe(III) oxide and electrode-grown cultures have also become available (8, 12, 16). In most extracellular electron transfer studies, outer membrane proteins (such as c-type cytochromes) have been the focus (4, 23, 27, 32), leading to targeted knockout studies of at least 14 cytochromes to date.To reduce an insoluble electron acceptor, Geobacter spp. must achieve direct contact with the substrate (36). While contact with small Fe(III) oxide particles may be transient, growth on Fe(III)-coated surfaces or electron-accepting electrodes requires biofilm formation (31, 39). For example, when G. sulfurreducens produces an exponentially increasing rate of electron transfer at an electrode, this demonstrates that all newly divided cells remain embedded in the growing, conductive biofilm (2, 31). Thus, in addition to the need for an array of outer membrane cytochromes, there is also a need for control of both cell-cell contact and cell-surface contact.While a genetic system for G. sulfurreducens has been developed, conjugal transfer of a plasmid or a transposon has not been reported (7). The broad-host-range cloning vector pBBR1MCS-2 has previously been electroporated into G. sulfurreducens, but its mobilization capabilities were not utilized (7). Similarly, a number of suicide vectors have been identified for G. sulfurreducens, but none have been used to deliver transposons for mutagenesis. mariner-based transposon mutagenesis systems have been successful in a variety of Bacteria and Archaea, producing random insertions (20, 25, 40, 41, 43, 46, 48, 49). For example, genes involved in Shewanella oneidensis cytochrome maturation were discovered using the modified transposon mini-Himar RB1 (3).In this work, we describe a system for the conjugal transfer of the pBBR1MCS family of plasmids from Escherichia coli to G. sulfurreducens, which allowed transposon mutagenesis based on pMiniHimar RB1. Under strictly anaerobic conditions, a library of insertion mutants was constructed and screened to identify genes putatively involved in attachment and Fe(III) citrate reduction. Approximately 8,000 insertion mutants were isolated, with insertions distributed throughout the G. sulfurreducens chromosome. Subsequent characterization revealed mutants defective in metal reduction but unaffected in all aspects of electrode reduction, as well as mutants able to reduce metals but incapable of electrode reduction. These observations greatly expand the list of Geobacter mutants with defects in respiration or biofilm formation, and this library serves as a resource for further screening of extracellular electron transfer phenotypes.     

The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis

The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.Shewanella oneidensis strain MR-1 (MR-1) is a facultative anaerobe capable of respiring a variety of substrates, including various metals and metal oxides, a phenotype that is important for bioremediation and metal cycling in natural environments (22, 53). At near-neutral pH, Fe(III) and Mn(IV) often are present as insoluble oxide minerals. Dissimilatory metal-reducing bacteria such as MR-1 have developed pathways to transfer electrons from the interior of the cell to these external terminal electron acceptors. In some bacteria, these pathways also can transfer electrons to electrodes, which can be harnessed for renewable energy and remote biosensor applications (23, 26, 27). Beyond increasing our understanding of this unusual process, applying anaerobic microbial extracellular respiration to new technologies requires a thorough understanding of the molecular dynamics and cellular physiology of electron source utilization (substrate oxidation) and the reduction of insoluble terminal electron acceptor(s). There are four proposed mechanisms to explain how insoluble substrates are reduced by Shewanella: (i) direct contact, (ii) electron shuttling, (iii) chelation, and (iv) electrically conductive appendages (reviewed in reference 18). We will focus on the first three strategies here.Flavins recently have been discovered to accelerate the reduction of both iron oxide minerals (51) and electrodes (30) by MR-1. Riboflavin (vitamin B₂) is a precursor for the biosynthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (13). Riboflavin and FMN both can be observed to build up in the supernatant of anaerobic and aerobically grown cultures of MR-1 (30, 51). However, the mechanism by which flavins enhance the rate of iron oxide mineral or electrode reduction is unknown, although recent work is consistent with a critical role for these compounds in mediating solid Fe(III) reduction by MR-1 (42). Since soluble (chelated) Fe(III) is reduced faster than insoluble Fe(III) by MR-1 (6), one possible explanation for the enhancement of insoluble iron reduction by flavins is increased available soluble iron via chelation (1, 2, 30). Flavins also may be utilized as redox-active compounds to traffic electrons between extracellular reductases on the surface of the cell and insoluble substrates (30, 51), a process termed electron shuttling (18, 39, 41). The chelation of the terminal electron acceptor during electrode reduction is not relevant when the anode is composed of graphite. Therefore, electron shuttling likely is responsible for the flavin enhancement of current production on poised-potential electrodes (30). However, it is unclear if the chelation of metals by flavins influences insoluble metal reduction by S. oneidensis (30).The Mtr pathway is required for the reduction of metals and electrodes (5, 6, 9, 17). Five primary protein components have been identified in this pathway: OmcA, MtrC, MtrA, MtrB, and CymA (47). Current models of electron transfer in MR-1 assume that electrons from carbon source oxidation are passed via the menaquinone pool to the inner membrane-anchored c-type cytochrome CymA (19, 31). These electrons then are transferred to a periplasmic c-type cytochrome, MtrA, and eventually to outer membrane (OM)-anchored c-type cytochromes MtrC and OmcA, which interact with an integral OM scaffolding protein, MtrB (32, 33, 43). These OM cytochromes then can reduce various substrates, including iron oxides and electrodes (8, 9, 12, 36, 47). Since the Mtr system is required by MR-1 to reduce many different substrates (18), it also could be capable of reducing extracellular flavins. Indeed, electron transfer to carbon electrodes is impaired in strains lacking Mtr pathway components (9, 17), which may be explained simply by a decreased ability to reduce extracellular flavins. The observation that Mtr mutants produce less current on electrodes than the wild type could be due to (i) less current generated per cell (either direct reduction or flavin mediated), (ii) decreased attachment to the electrode surface, (iii) differences in external flavin concentrations, or (iv) a combination of these three possibilities. Determining the specific activity (current produced per unit of attached biomass) of Mtr mutants on electrodes under conditions where flavin levels were controlled would allow for differentiation between these possibilities. To date, this kind of analysis has not been reported.The results presented here extend our knowledge of how S. oneidensis catalyzes the reduction of insoluble substrates. Experiments using a model iron chelator and electron shuttle are consistent with electron shuttling being the primary mechanism by which flavins enhance insoluble iron oxide reduction rates. Moreover, we demonstrate that MR-1 reduces extracellular flavins at physiologically relevant rates and that the Mtr pathway accounts for at least 95% of this activity. The specific activities of various mutant strains lacking Mtr pathway components on poised-potential electrodes also are reported. Our data suggest that MtrC is responsible for most of the electron transfer to carbon electrodes, while OmcA is involved in attachment and has a lesser role in electron transfer. These observations could have broader implications regarding the role of OmcA in the reduction of soluble and insoluble substrates (8, 9, 36).     

Tolerance of the Ralstonia eutropha Class I Polyhydroxyalkanoate Synthase for Translational Fusions to Its C Terminus Reveals a New Mode of Functional Display

Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized by many bacteria and some archaea in times of unbalanced nutrient availability (7, 14-16, 22). These polyesters are stored as water-insoluble inclusions inside the cells and serve as energy and carbon storage (11, 29, 30). PHA synthases catalyze the stereoselective conversion of (R)-3-hydroxyacyl-coenzyme A (CoA) to PHAs while CoA is released and intracellular PHA granules are formed (32). The PHA synthase remains covalently attached to the PHA granule surface and has been targeted by protein engineering, i.e., translational fusion to the dispensable and variable N terminus, to enable the display of various protein functions without affecting the synthase activity (8, 26). PHA granules displaying certain functionalities have been considered as biobeads for biotechnological and medical applications (11).PHA synthases can be divided into four classes. Class I and class II enzymes consist of only one subunit (PhaC) (28) and produce short-chain-length PHAs (class I) or medium-chain-length PHAs (class II), respectively (30, 33). Polyester synthases belonging to class III consist of two subunits, PhaC and PhaE, and produce short-chain-length PHAs (20, 21). Class IV PHA synthases are similar to enzymes belonging to class III. The synthases of this class comprise the two subunits PhaC and PhaR (23, 24).It was previously shown that the N terminus of PhaC is a highly variable region and not essential for PHA synthase activity (30, 35). In contrast, the C terminus is a rather conserved region among class I and class II PHA synthases and is essential for enzyme activity (31). Alignments of the amino acid sequences of different PHA synthases revealed that the C terminus of these enzymes is hydrophobic and was therefore suggested to interact with the hydrophobic core of PHA granules (30). The PhaC subunits of class III and class IV PHA synthases do not show a high hydrophobicity for their C- terminal regions. Previous studies showed that the PhaC subunit of the class IV PHA synthase from Bacillus megaterium tolerates fusions to its C terminus without a loss in activity as long as the hydrophobic second subunit, PhaR, is present as well (23).The aim of this study was to assess the effect of the conserved hydrophobic C terminus of PhaC on enzyme activity with regard to the possibility of translationally fusing protein functions for display at the PHA granule surface. This will be of interest for the display of proteins that require their free C terminus for activity.     

Analyses of Current-Generating Mechanisms of Shewanella loihica PV-4 and Shewanella oneidensis MR-1 in Microbial Fuel Cells

Although members of the genus Shewanella have common features (e.g., the presence of decaheme c-type cytochromes [c-cyts]), they are widely variable in genetic and physiological features. The present study compared the current-generating ability of S. loihica PV-4 in microbial fuel cells (MFCs) with that of well-characterized S. oneidensis MR-1 and examined the roles of c-cyts in extracellular electron transfer. We found that strains PV-4 and MR-1 exhibited notable differences in current-generating mechanisms. While the MR-1 MFCs maintained a constant current density over time, the PV-4 MFCs continued to increase in current density and finally surpassed the MR-1 MFCs. Coulombic efficiencies reached 26% in the PV-4 MFC but 16% in the MR-1 MFCs. Although both organisms produced quinone-like compounds, anode exchange experiments showed that anode-attached cells of PV-4 produced sevenfold more current than planktonic cells in the same chamber, while planktonic cells of MR-1 produced twice the current of the anode-attached cells. Examination of the genome sequence indicated that PV-4 has more c-cyt genes in the metal reductase-containing locus than MR-1. Mutational analysis revealed that PV-4 relied predominantly on a homologue of the decaheme c-cyt MtrC in MR-1 for current generation, even though it also possesses two homologues of the decaheme c-cyt OmcA in MR-1. These results suggest that current generation in a PV-4 MFC is in large part accomplished by anode-attached cells, in which the MtrC homologue constitutes the main path of electrons toward the anode.Some species of dissimilatory metal-reducing bacteria (DMRB) are able to reduce solid metal oxides as terminal electron acceptors and generate currents in microbial fuel cells (MFCs) (2, 11, 14, 30, 46). Although mixed cultures are often used in MFC experiments (13), studies seeking a mechanistic understanding of electron transfer to electrode surfaces typically target pure cultures of such DMRB, due to the complexity in microbial communities. Presently, two model DMRB, Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA (2, 3, 12, 18, 31), are used in most investigations.S. oneidensis MR-1 is a metabolically diverse DMRB that has been studied extensively for its potential use in bioremediation applications. For this reason, MR-1 was the first Shewanella species to have its genome completely sequenced and annotated (10). In addition, since the first report in 1999 when this microorganism was shown to have the ability to transfer electrons to the electrode without an exogenously added mediator (14), it has also become one of the model organisms for the study of electron transfer mechanisms in MFCs.Although the molecular mechanisms for extracellular electron transfer have not yet been elucidated fully, c-type cytochromes (c-cyts) appear to be the key cellular components involved in this process (38). In S. oneidensis MR-1, OmcA and MtrC are outer membrane (OM), decaheme c-cyts that are considered to be involved in the direct (directly attached) electron transfer to solid metal oxides and anodes of MFCs (9, 20, 22, 23, 47). Several pieces of evidence suggest that OmcA and MtrC form a complex and act in a cooperative manner (33, 37, 42), and these results correlate with the fact that the genes encoding these proteins constitute an operon-like cluster in the chromosome (1). It has also been shown that MtrC and OmcA have overlapping functions as terminal reductases of metal oxides (25, 38). OmcA and MtrC are also present on the surface of nanowires and may be involved in the long-range transfer of electrons (8). In addition to direct electron transfer, MR-1 has the ability to produce water-soluble electron-shuttle compounds (quinones and flavins) that are involved in the mediated electron transfer from cells to distant solid electron acceptors (metal oxides or MFC anodes) (21, 27, 44).Recently, the genome sequences of nearly 20 Shewanella strains have been completed and annotated, opening the door to study the diversity of their extracellular electron transfer mechanisms. A comparison of their genomes has shown that although they have some consensus OM c-cyt genes, variations exist in the number and order of these genes in their metal reductase-containing loci (6). One such species is S. loihica strain PV-4, which was recently isolated from an iron-rich microbial mat near a deep-sea hydrothermal vent located on the Loihi Seamount in Hawaii (7, 32). The phenotypic and phylogenetic characteristics of PV-4 were determined, with a subsequent study focusing on the metal reduction and iron biomineralization capabilities of this bacterium (32). Initial experiments performed in our laboratory revealed that PV-4 developed a c-cyt-dependent deep red color that was much more striking than that of strain MR-1 when grown anaerobically with iron oxide as the terminal electron acceptor (26). This allowed us to assume that PV-4 could have a high extracellular electron transfer ability. Accordingly, the present study evaluated the current-producing ability of strain PV-4 in MFCs and examined the roles of some c-cyts in extracellular electron transfer. Special attention was paid to the comparison of PV-4 with MR-1 to reveal differences in mechanisms for extracellular electron transfer. We report herein differences between these strains in the roles of OM c-cyts for extracellular electron transfer, the behaviors and metabolic patterns of MFC, and the resultant MFC performances.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Residue Histidine 669 Is Essential for the Catalytic Activity of Bacillus anthracis Lethal Factor

The lethal factor (LF) of Bacillus anthracis is a Zn²⁺-dependent metalloprotease which plays an important role in anthrax virulence. This study was aimed at identifying the histidine residues that are essential to the catalytic activities of LF. The site-directed mutagenesis was employed to replace the 10 histidine residues in domains II, III, and IV of LF with alanine residues, respectively. The cytotoxicity of these mutants was tested, and the results revealed that the alanine substitution for His-669 completely abolished toxicity to the lethal toxin (LT)-sensitive RAW264.7 cells. The reason for the toxicity loss was further explored. The zinc content of this LF mutant was the same as that of the wild type. Also this LF mutant retained its protective antigan (PA)-binding activity. Finally, the catalytic cleavage activity of this mutant was demonstrated to be drastically reduced. Thus, we conclude that residue His-669 is crucial to the proteolytic activity of LF.Anthrax is a zoonotic disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis (24). Because infections are highly fatal, the organisms are easily produced, and the spores spread easily, B. anthracis has been used as a bioweapon in biological war and biological terrorism (38). If inhaled, the spores are phagocytosed by alveolar macrophages, where they germinate to produce vegetative bacteria (10, 24). The vegetative bacteria further release anthrax toxins, which inhibit the innate and adaptive immune responses of the hosts. This enables the capsulated bacteria to escape the lymph node defense barrier to reach the blood system, causing bacteremia and toxemia, which can rapidly kill the hosts (24, 26). The great threat posed by anthrax to the public is not only due to the highly lethal rate of inhaled anthrax, but also is due to the social panic caused by the lethality. Therefore, efficient ways to defend against anthrax infection and spreading are greatly needed. This mostly depends on a full understanding of the mechanisms of anthrax infection and toxicities.Anthrax toxins are the dominant virulence factors of Bacillus anthracis (6, 33, 37). They consist of three proteins: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and edema factor (EF; 89 kDa). The 83-kDa PA (PA₈₃) directly binds to cellular membrane receptors and was cleaved to an active fragment of 63-kDa PA (PA₆₃) by cellular proteases of the furin family or by serum proteases. The receptor-bound portion of PA₆₃ self-assembles into either ring-shaped heptamers, which bind to three molecules of LF and/or EF, resulting in (PA₆₃)₇(LF/EF)₃ (21), or octamers which bind up to four molecules of these moieties, resulting in (PA₆₃)₈(LF/EF)₄ complexes (16, 17). The catalytic partners (EF and/or LF) are subsequently transported across the membrane to the cell cytosol (24, 27). EF is a Ca²⁺- and calmodulin-dependent adenylate cyclase that, together with PA, forms edema toxin. EF causes a rapid increase in intracellular cyclic AMP (cAMP) levels in host cells and alters the elaborate balance of intracellular signaling pathways (20, 23). LF is a Zn²⁺-dependent protease that, together with PA, forms lethal toxin (LT). It is a dominant virulence factor and the major cause of death for the B. anthracis-infected animals (1, 29, 30). LF specifically cleaves the N-terminal domain of mitogen-activated protein kinase kinases (MAPKKs) (11, 35). Because the N-terminal domain of MAPKKs is essential for the interaction between MAPKKs and MAPKs, the cleavage of this domain impairs the activation of MAPKs (8, 11, 15) and leads to the inhibition of three major cellular signaling pathways—the ERK (extracellular signal-regulated kinase), p38, and JNK (c-Jun N-terminal kinase) pathways (29, 31)—and thus induces the lysis of the host cells in an unknown mechanism.The crystal structure of LF with the N-terminal domain of MEK2 has been reported (28). LF has 776 amino acids and comprises four different domains. Domain I (residues 1 to 254) is a PA-binding domain which delivers the remaining domains of the LF to the cell cytoplasm (3). The interface among domains II, III, and IV creates long, deep, 40-Å-long catalytic grooves into which the N terminus of MEK fits and forms an active site complex (28). Domain IV is central to catalytic activities of LF, containing two zinc-binding motifs (residues 686 to 690 and residues E735 to E739) and bound to a single Zn ion (18). However, which residues of LF are critical for efficient catalytic activities and execute the substrate cleavage remains unclear.Histidine is the only naturally occurring amino acid to contain an imidazole residue as a side chain. The catalytic activity of histidine mostly depends on the special features of the imidazole residue. The logarithm of the proton dissociation constant of imidazolyl in the histidine residue is about 6.5; thus, under the physiological condition, it tends to form hydrogen bonds and shares donor and acceptor properties that can take part in either nucleophilic or base catalysis. The speed of the imidazole residue to give or accept protons is very fast, with a half-life of less than 10 s. So in the process of natural selection, histidine was chosen as the catalytic structure, indicating that it plays an important role in the catalysis process of enzymes (9, 12, 14). There are 21 histidines in LF, with 9 of them in LF domain I and 12 of them in domains II, III, and IV. The histidine residues important to LF activities in domain I have been identified (2, 22). The other 12 histidine residues in the remaining three domains include His-277, His-280, and His-424 in domain II; His-309 in domain III; and His-588, His-645, His-654, His-669, His-686, His-690, His-745, and His-749 in domain IV (28). His-686 and His-690 in domain IV were demonstrated to form a zinc binding site constituting a thermolysin-like zinc metalloprotease motif, HEXXH (18). The activities of the remaining 10 histidine residues in domains II, III, and IV have not been explored yet. In this study, we replaced these 10 histidine residues separately with alanine residues by site-directed mutagenesis. By the cytotoxicity assay of all these mutants, the H669A mutant was found to lose cell toxicity completely. Further assay revealed that residue His-669 was involved in neither zinc stabilization nor PA binding but participated in the substrate proteolytic activity of LF.