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).     

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.     

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.     

Rhodobacter capsulatus Catalyzes Light-Dependent Fe(II) Oxidation under Anaerobic Conditions as a Potential Detoxification Mechanism

Diverse bacteria are known to oxidize millimolar concentrations of ferrous iron [Fe(II)] under anaerobic conditions, both phototrophically and chemotrophically. Yet whether they can do this under conditions that are relevant to natural systems is understood less well. In this study, we tested how light, Fe(II) speciation, pH, and salinity affected the rate of Fe(II) oxidation by Rhodobacter capsulatus SB1003. Although R. capsulatus cannot grow photoautotrophically on Fe(II), it oxidizes Fe(II) at rates comparable to those of bacteria that do grow photoautotrophically on Fe(II) as soon as it is exposed to light, provided it has a functional photosystem. Chelation of Fe(II) by diverse organic ligands promotes Fe(II) oxidation, and as the pH increases, so does the oxidation rate, except in the presence of nitrilotriacetate; nonchelated forms of Fe(II) are also more rapidly oxidized at higher pH. Salt concentrations typical of marine environments inhibit Fe(II) oxidation. When growing photoheterotrophically on humic substances, R. capsulatus is highly sensitive to low concentrations of Fe(II); it is inhibited in the presence of concentrations as low as 5 μM. The product of Fe(II) oxidation, ferric iron, does not hamper growth under these conditions. When other parameters, such as pH or the presence of chelators, are adjusted to promote Fe(II) oxidation, the growth inhibition effect of Fe(II) is alleviated. Together, these results suggest that Fe(II) is toxic to R. capsulatus growing under strictly anaerobic conditions and that Fe(II) oxidation alleviates this toxicity.Iron is one of the most (photo)redox-active metals involved in biochemical functions, and it can affect the cycling of many other key elements (e.g., C, S, N, and P), trace metals (33), metalloids, and organic compounds (6). It is well appreciated that microorganisms contribute greatly to iron cycling in nature through a diversity of processes, including both oxidation and reduction reactions (16). In the past decade, much attention has been paid to how such reactions can be used to support cellular growth (1, 7, 15, 17, 19, 37, 44-46) and/or iron acquisition (2, 42) under both aerobic and anaerobic conditions, and for some organisms, these processes are understood at the molecular level (10).Our lab has been particularly interested in one branch of the microbial Fe cycle: phototrophic Fe(II) oxidation under anaerobic conditions (9, 11, 12, 23-25). While most of the organisms we and others have studied can grow by coupling Fe(II) oxidation to CO₂ fixation (15, 23, 46), not all strains that oxidize Fe(II) can use it as an electron donor to support growth. An example of this is Rhodobacter capsulatus, which can benefit from Fe(II) oxidation only via an indirect pathway: it grows photoheterotrophically on low-molecular-weight organic compounds that form due to a photochemical reaction between biogenic Fe(III) and organic compounds that it cannot otherwise use (citrate and nitrilotriacetate [NTA]) (4). This observation led us to hypothesize that microbial Fe(II) oxidation might be more broadly useful to microorganisms by making refractory organic compounds, such as humic substances, more bioavailable through photochemical degradation (4).In this work, we set out to test this hypothesis using R. capsulatus. In addition, we sought to increase our understanding of Fe(II) oxidation by this organism by studying the effect of Fe(II) speciation and important environmental variables (e.g., light, pH, and [Cl⁻]) on the rate of Fe(II) oxidation. Along the way, we serendipitously discovered that low levels of Fe(II) are toxic to R. capsulatus when it is growing on humic substances under anaerobic conditions and that Fe(II) oxidation appears to alleviate this toxicity.     

Composition and Activity of an Autotrophic Fe(II)-Oxidizing,Nitrate-Reducing Enrichment Culture

16S rRNA gene libraries from the lithoautotrophic Fe(II)-oxidizing, nitrate-reducing enrichment culture described by Straub et al. (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl. Environ. Microbiol. 62:1458-1460, 1996) were dominated by a phylotype related (95% 16S rRNA gene homology) to the autotrophic Fe(II) oxidizer Sideroxydans lithotrophicus. The libraries also contained phylotypes related to known heterotrophic nitrate reducers Comamonas badia, Parvibaculum lavamentivorans, and Rhodanobacter thiooxidans. The three heterotrophs were isolated and found to be capable of only partial (12 to 24%) Fe(II) oxidation, suggesting that the Sideroxydans species has primary responsibility for Fe(II) oxidation in the enrichment culture.A variety of microorganisms oxidize Fe(II) with nitrate under anaerobic, circumneutral pH conditions (29) and may contribute to an active microbially driven anoxic Fe redox cycle (1, 27-29, 31, 32). Straub et al. (28) obtained the first Fe(II)-oxidizing, nitrate-reducing (enrichment) culture capable of fully autotrophic growth by a reaction such as 5Fe²⁺ + NO₃⁻ + 12H₂O → 5Fe(OH)₃ + 0.5N₂ + 9H⁺. This process has since been demonstrated in detail with the hyperthermophilic archaeon Ferroglobus placidus (9) and with the mesophilic Proteobacteria Chromobacterium violacens strain 2002 (34) and Paracoccus ferrooxidans strain BDN-1 (16). Nitrate-dependent Fe(II) oxidation in the presence of fixed carbon has been documented for Dechlorosoma suillum strain PS (4), Geobacter metallireducens (7), Desulfitobacterium frappieri (23), and Acidovorax strain BoFeN1 (15). In addition to oxidizing insoluble Fe(II)-bearing minerals (33), the enrichment culture described by Straub et al. (28) is the only autotrophic Fe(II)-oxidizing, nitrate-reducing culture capable of near-complete oxidation of uncomplexed Fe(II) with reduction of nitrate to N₂. During Fe(II) oxidation, F. placidus reduces nitrate to nitrite, which may play a significant role in overall Fe(II) oxidation. Although both C. violacens and Paracoccus ferrooxidans reduce nitrate to N₂, C. violacens oxidizes only 20 to 30% of the initial Fe(II), and P. ferrooxidans uses FeEDTA²⁻ but not free (uncomplexed) Fe(II) in medium analogous to that used for cultivation of the enrichment culture described by Straub et al. (28). The enrichment culture described by Straub et al. (28) is thus the most robust culture capable of autotrophic growth coupled to nitrate-dependent Fe(II) oxidation available at present. The composition and activity of this culture was investigated with molecular and cultivation techniques. The culture examined is one provided by K. L. Straub to E. E. Roden in 1998 for use in studies of nitrate-dependent oxidation of solid-phase Fe(II) compounds (33) and has been maintained in our laboratory since that time.     

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.     

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).     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).     

Analysis of the Role of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola 1448a in Interaction with the Plant

In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the salicylic acid (SA)-signaling pathway.Type III secretion systems (T3SS) are complex and specialized machineries that inject effector proteins directly into the host cell cytosol (2). In Pseudomonas syringae, T3SS-mediated secretion is essential for disease in compatible hosts and for eliciting the hypersensitive response (HR) in incompatible hosts (1). P. syringae pathovars secrete a variable number of type III effectors that form their so-called secretomes and are expressed within the plant under the control of the alternative sigma factor HrpL (47). Understanding how the T3SS determines pathogenicity requires the functional characterization of the complete type III effector inventory. However, this characterization has been partially hindered by the fact that mutation of individual effectors, usually the most straightforward approach, rarely causes virulence attenuation (14). Thus, reports showing the contribution of the type III effector to virulence in P. syringae pathovars have resorted to ectopic expression in homolog-lacking related strains (40), plasmid-cured derivatives (21), double mutants (6, 28), or polymutants (3, 26). In relation to this, we have previously established the use of the competitive index (CI) in mixed infections (13, 42) as a more sensitive virulence assay for P. syringae pathovars than traditional assays (31). Using CIs, we demonstrated for the first time the individual contribution of AvrPto, an otherwise thoroughly characterized type III effector from Pseudomonas syringae pv. tomato (9, 17, 18, 27, 36, 39, 40, 46), to pathogen growth within its natural host (31). Therefore, analysis of effector mutants by use of the CI may provide the means to establish the quantitative contribution of the members of P. syringae T3SS secretomes to virulence. In addition, genetic analysis of the effects of combinations of effector mutations on virulence has already proven a useful approach to establishing the contribution of the members of the P. syringae pv. tomato DC3000 secretome to virulence by revealing a functional overlap (6, 26, 28). Thus, generation of knockouts in all individual effector genes of a given secretome, achieved in such a manner as to allow for easy combination of these strains into double or multiple mutant strains, is a desirable task, albeit a cumbersome one, considering the size of most secretomes.The secretome of the fully sequenced wild-type (wt) representative of the Pseudomonas syringae pv. phaseolicola 1448a strain (Pph1448a) has previously been analyzed, using a differential fluorescence induction screen (7) and bioinformatics (44), to identify effector genes. Our laboratory contributed to establishing this secretome through the development and application of a very sensitive assay for T3SS-mediated translocation based on CI assays (30). This assay represents an improvement over the sensitivity of the commonly used AvrRpt2 reporter assay. When fused to a T3SS-secreted protein, AvrRpt2^81-255 is translocated inside the host cell, eliciting a hypersensitive response (HR), dependent on the resistance protein RPS2 (32). By using CIs to measure the bacterial growth reduction associated with the AvrRpt2-RPS-mediated defense response, we detected translocation for two out of four Pph1448a effector candidates previously discarded by other assays, HopAJ1 and HopAK1 (30), and demonstrated translocation for two out of five previously untested candidates, HopAH2 and A0129. However, although in planta expression has been shown to take place in an HrpL-dependent manner for HopAJ1 and HopAK1 (7), it has not been established for HopAH2 and A0129. Effector nomenclature guidelines recommend that the abbreviation for the pathovars as well as the name of the strain should be included within the effector name (29). For simplicity, we include this indication only when effectors from other pathovars are mentioned. In summary, to date, 22 effectors in Pph1448a have been experimentally validated (7, 30, 44), one HrpL-regulated candidate has given inconsistent translocation results (AvrE1) (7), two translocated candidates have not been analyzed for expression in planta (HopAH2 and A0129) (30), one bioinformatically identified candidate has not been experimentally tested (AvrB4-2) (23), and six additional candidates have been proposed but experimentally ruled out (PSPPH3757, HopAN1, HopAJ2, HopW1-2, HopV1, and HopJ1) (7, 30).In this work, we analyzed the translocation and/or expression of these and other candidate effectors to close the type III effector inventory of Pph1448a. Our results indicate that the Pph1448a complete type III secretome is formed by 27 validated effectors, including a new one, HopAY1, which does not belong to any of the previously described effector families. The work includes the development of a simplified process for quick generation of single and double knockout mutants and its application to constructing a collection of single mutants for almost all members of the Pph1448a type III secretome. Additionally, we generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the four single and two double mutant strains as well as the double mutants expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to the virulence of Pph1448a. The tools and approach used in this work set the groundwork for dissecting the contribution of the entire Pph1448a type III secretome to virulence.     

Microbial Iron Redox Cycling in a Circumneutral-pH Groundwater Seep

The potential for microbially mediated redox cycling of iron (Fe) in a circumneutral-pH groundwater seep in north central Alabama was studied. Incubation of freshly collected seep material under anoxic conditions with acetate-lactate or H₂ as an electron donor revealed the potential for rapid Fe(III) oxide reduction (ca. 700 to 2,000 μmol liter⁻¹ day⁻¹). Fe(III) reduction at lower but significant rates took place in unamended controls (ca. 300 μmol liter⁻¹ day⁻¹). Culture-based enumerations (most probable numbers [MPNs]) revealed significant numbers (10² to 10⁶ cells ml⁻¹) of organic carbon- and H₂-oxidizing dissimilatory Fe(III)-reducing microorganisms. Three isolates with the ability to reduce Fe(III) oxides by dissimilatory or fermentative metabolism were obtained (Geobacter sp. strain IST-3, Shewanella sp. strain IST-21, and Bacillus sp. strain IST-38). MPN analysis also revealed the presence of microaerophilic Fe(II)-oxidizing microorganisms (10³ to 10⁵ cells ml⁻¹). A 16S rRNA gene library from the iron seep was dominated by representatives of the Betaproteobacteria including Gallionella, Leptothrix, and Comamonas species. Aerobic Fe(II)-oxidizing Comamonas sp. strain IST-3 was isolated. The 16S rRNA gene sequence of this organism is 100% similar to the type strain of the betaproteobacterium Comamonas testosteroni ({"type":"entrez-nucleotide","attrs":{"text":"M11224","term_id":"151536","term_text":"M11224"}}M11224). Testing of the type strain showed no Fe(II) oxidation. Collectively our results suggest that active microbial Fe redox cycling occurred within this habitat and support previous conceptual models for how microbial Fe oxidation and reduction can be coupled in surface and subsurface sedimentary environments.Changes in iron (Fe) redox state are linked to carbon and energy flow as well as the behavior of various inorganic compounds in modern soils and sediments. Microorganisms play a pivotal role in the Fe redox cycle in such environments (29, 35, 39). A growing body of literature indicates that aerobic lithotrophic Fe(II)-oxidizing bacteria (FeOB) can contribute significantly to circumneutral-pH Fe(II) oxidation (4, 9, 15, 23, 25, 34) and that microbial catalysis can dominate Fe(II) oxidation in diffusion-limited reaction systems (32, 34). Microbial catalysis is strictly required for anaerobic nitrate-dependent Fe(II) oxidation (36), since an abiotic reaction between Fe(II) and nitrate does not take place under typical near-surface conditions (40).Circumneutral-pH Fe(II) oxidation produces Fe(III) oxide mineral phases which can function as electron acceptors for anaerobic respiration by dissimilatory Fe(III)-reducing bacteria (FeRB) (8, 37). This metabolism is widespread among prokaryotic taxa (19) and plays a key role in oxidation of natural organic compounds and in the bioremediation of organic and metal contaminants in the subsurface (18). The coupling of Fe(III) oxide reduction to oxidation of organic carbon or H₂ leads to release of Fe(II) into the aqueous phase. When the oxidative and reductive parts of the Fe redox cycle come together with ongoing input of energy, a self-sustaining microbial community based on Fe redox cycling may develop. Sustained microbial Fe redox cycling has been proposed in various redox interfacial environments like groundwater Fe seeps (8), plant roots (10), the sediment-water interface in circumneutral-pH (29, 33) and acidic (24) aquatic ecosystems, and hot springs and hydrothermal vents (16a, 24a).Here we present data that support the existence of a sustained microbial Fe redox cycle in a circumneutral-pH groundwater Fe seep in north central Alabama. Potential microbial involvement in Fe redox cycling was assessed by most probable number (MPN) enumerations, in vitro Fe(III) reduction experiments, and isolation of representative Fe(III)-reducing and Fe(II)-oxidizing microorganisms. A simple kinetic model was used to explore the impact that decay of dead chemolithotrophic biomass coupled to Fe(III) reduction could have on rates of Fe turnover.     

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).     

Thioredoxin 1 Participates in the Activity of the Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Type III Secretion System

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.In Escherichia coli, thioredoxin 1 (TrxA, encoded by trxA) is an evolutionary conserved 11-kDa cytosolic highly potent reductase that supports the activities of various oxidoreductases and ribonucleotide reductases (1, 29) and interacts with a number of additional cytoplasmic proteins through the formation of temporary covalent intermolecular disulphide bonds (32). Consequently, as trxA mutants of E. coli (51), Helicobacter pylori (13), and Rhodobacter sphaeroides (34) show increased sensitivity to hydrogen peroxide, TrxA has been defined as a significant oxidoprotectant. In addition, TrxA possess a protein chaperone function that is disconnected from cysteine interactions (30, 32).Salmonella enterica serovar Typhimurium is closely related to E. coli. During divergent evolution, the Salmonella genome acquired a number of virulence-associated genes (20). Many of these genes are clustered on genetic regions termed Salmonella pathogenicity islands (or SPIs). Of these, SPI1 and SPI2 code for separate type III secretion systems (T3SSs). T3SSs are supramolecular virulence-associated machineries that, in several pathogenic gram-negative bacterial species, enable injection of effector proteins from the bacteria into host cells (22, 57). The effector proteins, in turn, manipulate intrinsic host cell functions to facilitate the infection.The SPI1 T3SS of S. serovar Typhimurium is activated for expression in the intestine in response to increased osmolarity and decreased oxygen tension (22, 57). SPI1 effector proteins are primarily secreted into cells that constitute the epithelial layer and interfere with host cell Cdc42 and Rac-1 signaling and actin polymerization. This enables the bacteria to orchestrate their own actin-dependent uptake into nonphagocytic cells (57). SPI1 effector proteins also induce inflammatory signaling and release of interleukin-1β from infected cells (25, 26).Subsequent systemic progression of S. serovar Typhimurium from the intestinal tissue relies heavily on an ability to survive and replicate in phagocytic cells (18, 46, 53, 54). S. serovar Typhimurium uses an additional set of effector proteins secreted by the SPI2 T3SS for replication inside host cells and for coping with phagocyte innate responses to the infection (10, 11, 54). The functions of SPI2 effectors include diversion of vesicular trafficking, induction of apoptotic responses, and manipulation of ubiquitination of host proteins (28, 40, 45, 53). Hence, SPI2 effector proteins create a vacuolar environment that sustains intracellular replication of S. serovar Typhimurium (28).In addition to pathogenicity islands, the in vivo fitness of Salmonella spp. relies on selected functions shared with other enterobacteria. Thus, many virulence genes are integrated into “housekeeping” gene regulatory networks, coded for by a core genome, which steer bacterial stress responses (12, 17, 27, 55). Selected anabolic pathways also contribute to virulence of S. serovar Typhimurium (18, 27), evidently by providing biochemical building blocks for bacterial replication (36).In S. serovar Typhimurium, TrxA is a housekeeping protein that strongly contributes to virulence in cell culture and mouse infection models (8). However, the mechanism by which TrxA activity adds to virulence has not been defined. Here we show that the contribution of TrxA to virulence of S. serovar Typhimurium associates with its functional integration with the SPI2 T3SS under conditions that prevail in the intracellular vacuolar compartment of the host cell. These findings ascribe a novel role to TrxA in bridging environmental adaptations with virulence gene expression and illuminate a new aspect of the interaction between evolutionary conserved and horizontally acquired gene functions in bacteria.