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.     

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).     

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.     

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).     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

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.     

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.Cysteine thiols in proteins fulfill an important and diverse set of cellular functions. In particular, they participate in enzymatic catalysis; in metal coordination, such as in the generation of Fe-S-clusters; and in determining the spatial structure of proteins via disulfide bond formation (3, 22, 23, 38). Cysteines are strong nucleophiles amenable to posttranslational modifications by reactive oxygen species (ROS) and reactive nitrogen species, leading to disulfides; to sulfenic, sulfinic, or sulfonic acids; mixed disulfides with low-molecular-weight (LMW) thiols (S thiolations); and S nitrosylations (7, 16, 17, 27).The redox status of the cytoplasm is under physiological conditions in a reduced state. Thus, most cysteines are present as free thiols (6). Because aerobic organisms have to cope with oxidative stress caused by ROS, such as superoxide anions, hydrogen peroxide, or hydroxyl radicals, they need to employ effective mechanisms that maintain the reduced state. In gram-negative bacteria, the thiol-disulfide balance is accomplished by the glutathione (GSH) system, a thiol-based redox buffer. The GSH system consists of glutaredoxin (Grx), GSH (γ-glutamylcysteinyl glycine), GSH reductase, and GSH peroxidase (34). Reduction of disulfides occurs via sequential electron transfer from glutaredoxin and reduced GSH; oxidized GSH (GSSG) is reduced by the NADPH-dependent GSH reductase. GSH peroxidase enables the direct detoxification of ROS by GSH oxidation.However, many gram-positive bacteria lack genes for GSH biosynthesis. Actinomycetes instead use a thiol redox buffer based on mycothiol (50). Bacillus subtilis, Staphylococcus aureus, and other gram-positive bacteria rely on different thiol redox buffers based on cysteine, the novel 398-Da bacillithiol (BSH), or coenzyme A (CoA) (15, 52). To maintain the reduced state of the cytoplasm, most bacteria use enzymatic systems for disulfide bond reduction such as the thioredoxin (Trx) system, which is highly conserved in gram-negative bacteria (3, 10). The Trx system consists of thioredoxin (TrxA) and the NADPH-dependent thioredoxin reductase (TrxB).Any imbalance in the cellular redox state caused by ROS elicits expression of a repertoire of different proteins, commonly under the control of a redox-sensing regulator: for example, OxyR in Escherichia coli and PerR, OhrR, SarZ, and Spx in B. subtilis and S. aureus, respectively (11, 12, 41, 55, 58, 64-66). The subsequently induced proteins detoxify ROS and restore and protect the normal physiological redox state in the cell.Besides ROS and reactive nitrogen species, so-called “reactive electrophilic species” (RES) affect the thiol redox balance. RES include different chemical compounds such as aldehydes, quinones, and the azo compound diamide (2, 43, 45, 46, 53, 66). Quinones and aldehydes have electron-deficient centers that result in thiol-(S) alkylation of cysteine. Exposure of cells to diamide induces the oxidative as well as the electrophile stress response in B. subtilis (43, 45, 53). The toxicity of diamide is based on disulfide bond formation (40), which was recently visualized in B. subtilis and S. aureus by the fluorescence alkylation of oxidized thiols (FALKO) assay (32, 64). It was thought that the formation of nonnative inter- and intramolecular disulfide bonds results in damage of proteins.However, more recent findings demonstrate that diamide stress leads also to S thiolations: formation of disulfide bonds between proteins and LMW thiols (8, 13, 33). S thiolations prevent protein thiols from irreversible oxidation to sulfinic and sulfonic acids, but also affect enzyme activity (35, 47) and signal transduction (39, 42). In B. subtilis, we have identified a few cytoplasmic proteins that are S cysteinylated (33). In addition, the organic peroxide sensor OhrR was inactivated by an S bacillithiolation in B. subtilis (42).Cysteine, BSH, and CoA are also dominant LMW thiols in S. aureus (52). In this study, we have investigated in more detail the extents of S thiolations and inter- and intramolecular disulfide bond formation of B. subtilis and S. aureus in response to disulfide stress. The results showed that exposure to diamide leads to S thiolations in S. aureus. Using a nonreducing/reducing sodium dodecyl sulfate (SDS) diagonal electrophoresis approach, proteins with intermolecular disulfide bonds could be distinguished from proteins with intramolecular disulfide bonds (57). The results support that the majority of reversible thiol oxidations are based on S thiolations rather than disulfide bonds between proteins. Depletion of the free cysteine pool in B. subtilis after exposure to diamide supports this finding. To assess if GSH may have a bearing on the thiol redox buffer of B. subtilis, the gshF gene of Listeria monocytogenes (gshF_Lm) was expressed in B. subtilis, enabling GSH biosynthesis (29). Although GSH production does not enhance the resistance to oxidative stress in B. subtilis, it participates in the formation of S thiolations.     

Extracellular Iron Biomineralization by Photoautotrophic Iron-Oxidizing Bacteria

Iron oxidation at neutral pH by the phototrophic anaerobic iron-oxidizing bacterium Rhodobacter sp. strain SW2 leads to the formation of iron-rich minerals. These minerals consist mainly of nano-goethite (α-FeOOH), which precipitates exclusively outside cells, mostly on polymer fibers emerging from the cells. Scanning transmission X-ray microscopy analyses performed at the C K-edge suggest that these fibers are composed of a mixture of lipids and polysaccharides or of lipopolysaccharides. The iron and the organic carbon contents of these fibers are linearly correlated at the 25-nm scale, which in addition to their texture suggests that these fibers act as a template for mineral precipitation, followed by limited crystal growth. Moreover, we evidence a gradient of the iron oxidation state along the mineralized fibers at the submicrometer scale. Fe minerals on these fibers contain a higher proportion of Fe(III) at cell contact, and the proportion of Fe(II) increases at a distance from the cells. All together, these results demonstrate the primordial role of organic polymers in iron biomineralization and provide first evidence for the existence of a redox gradient around these nonencrusting, Fe-oxidizing bacteria.Fe(II) can serve as a source of electrons for phylogenetically diverse microorganisms that precipitate iron minerals as products of their metabolism (see, e.g., references 3, 5, 25, and 30). For example, mixotrophic or autotrophic bacteria can couple the oxidation of Fe(II) to the reduction of nitrate in anoxic and neutral-pH environments. With Fe(III) being highly insoluble at neutral pH, this metabolism leads to the formation of poorly to well-crystallized iron minerals (3, 18, 26, 27) that precipitate partly within the cell periplasm for some strains (22). Similar Fe minerals are also synthesized by autotrophic bacteria that perform anoxygenic photosynthesis, using Fe(II) as an electron donor and light as a source of energy for CO₂ fixation (8, 12, 30), according to the equation HCO₃⁻ + 4 Fe²⁺ + 10 H₂O ⇆ <CH₂O> + 4 Fe(OH)₃ + 7 H⁺.However, the biological mechanisms of iron oxidation in these bacteria and in particular the way they cope with the formation of minerals within their ultrastructures are still not fully understood. Indeed, iron minerals are potentially lethal since their precipitation may alter cellular ultrastructures but also catalyze the production of free radicals (2). Recent genetic studies of the phototrophic, iron-oxidizing bacteria Rhodobacter sp. strain SW2 (6) and Rhodopseudomonas palustris strain TIE-1 (16) have identified genes (fox and pio operons, respectively) encoding proteins specific for iron oxidation. Interestingly, Jiao and Newman (16) suggested that one of these proteins could have a periplasmic localization. However, in contrast to what has been observed in some other phototrophic iron oxidizers (25) and in some nitrate-reducing, iron-oxidizing bacteria (22), no iron-mineral precipitation occurs within the periplasm of the purple nonsulfur iron-oxidizing bacterium Rhodobacter sp. strain SW2 (3). Similarly to some other anaerobic neutrophilic (22, 25) and microaerobic iron-oxidizing bacteria (5, 10), this strain seems indeed to have the ability to localize iron biomineralization at a distance from the cells, leaving large areas of the cells free of precipitates (17, 25). While it has been shown that the Gallionella and Leptothrix genera, for example, produce extracellular polymers that facilitate the nucleation of iron minerals outside cells (see, e.g., references 5 and 9), only a little is known about the existence and function of such polymers in anaerobic, neutrophilic iron-oxidizing bacteria and particularly in the phototrophic strain SW2. In the present study, we investigate iron biomineralization by the photoautotrophic iron-oxidizing bacterium Rhodobacter sp. strain SW2. We use scanning transmission X-ray microscopy (STXM) to map and identify organic polymers produced by the cells as well as the redox state of iron at the 25-nanometer scale regularly during a 2 week-period. These results demonstrate the primordial role of organic polymers in iron biomineralization and provide the first evidence for the existence of a redox gradient around SW2 cells.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Siderophores Are Not Involved in Fe(III) Solubilization during Anaerobic Fe(III) Respiration by Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 respires a wide range of anaerobic electron acceptors, including sparingly soluble Fe(III) oxides. In the present study, S. oneidensis was found to produce Fe(III)-solubilizing organic ligands during anaerobic Fe(III) oxide respiration, a respiratory strategy postulated to destabilize Fe(III) and produce more readily reducible soluble organic Fe(III). In-frame gene deletion mutagenesis, siderophore detection assays, and voltammetric techniques were combined to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration were synthesized via siderophore biosynthesis systems and (ii) if the Fe(III)-siderophore reductase was required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. Genes predicted to encode the siderophore (hydroxamate) biosynthesis system (SO3030 to SO3032), the Fe(III)-hydroxamate receptor (SO3033), and the Fe(III)-hydroxamate reductase (SO3034) were identified in the S. oneidensis genome, and corresponding in-frame gene deletion mutants were constructed. ΔSO3031 was unable to synthesize siderophores or produce soluble organic Fe(III) during aerobic respiration yet retained the ability to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. ΔSO3034 retained the ability to synthesize siderophores during aerobic respiration and to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. These findings indicate that the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are not synthesized via the hydroxamate biosynthesis system and that the Fe(III)-hydroxamate reductase is not essential for respiration of Fe(III)-citrate or Fe(III)-nitrilotriacetic acid (NTA) as an anaerobic electron acceptor.Bacterial electron transfer to sparingly soluble electron acceptors is a critical component of a wide variety of environmental and energy-generating processes, including biogeochemical cycling of metals, degradation of natural and contaminant organic matter, weathering of clays and minerals, biomineralization of Fe-bearing minerals, reductive precipitation of toxic metals and radionuclides, and electricity generation in microbial fuel cells (17, 33, 34). Anaerobic and facultatively anaerobic bacteria capable of respiring sparingly soluble (<10⁻²⁵ M at pH 7) Fe(III) oxides are ubiquitous in nature and may be found in marine, freshwater, and terrestrial environments, including metal- and radionuclide-contaminated subsurface aquifers (25, 34). Fe(III)-respiring prokaryotes are also deeply rooted and scattered throughout the domains Bacteria and Archaea (possibly indicating an ancient metabolic process) and include hyperthermophiles, psychrophiles, acidophiles, and extreme barophiles (34). Despite their potential environmental, energy-generating, and evolutionary significance, the molecular details of microbial Fe(III) respiration remain unclear.Fe(III)-respiring, neutrophilic bacteria are presented with a unique physiological challenge: they are required to respire anaerobically on electron acceptors found largely as sparingly soluble Fe(III) oxides presumably unable to contact periplasm- or inner membrane (IM)-localized electron transport systems. To overcome this problem, Fe(III)-respiring bacteria are postulated to employ novel respiratory strategies not found in other bacteria (e.g., aerobes, denitrifiers, sulfate-reducing bacteria, and methanogens) that respire soluble electron acceptors (17, 38). The novel respiratory strategies include (i) a direct-contact pathway in which terminal Fe(III) reductases are secreted to the cell outer membrane (OM), where they contact and deliver electrons directly to external Fe(III) oxides (18, 23, 40, 42, 48, 57, 64, 67), (ii) a two-step electron shuttling pathway in which bacterially reduced endogenous or exogenous electron shuttles deliver electrons to external Fe(III) oxides in a second (abiotic) electron transfer reaction (11, 26, 39, 45), and (iii) a two-step Fe(III) chelation (solubilization) pathway in which Fe(III) oxides are first nonreductively dissolved by endogenously synthesized organic ligands prior to reduction of the resulting soluble organic Fe(III) [Fe(III) bound to an organic molecule] complexes (36, 59).Candidate organic ligands for production of soluble organic Fe(III) during anaerobic Fe(III) oxide respiration include siderophores, the Fe(III)-chelating compounds synthesized and secreted by a wide variety of bacteria and fungi for solubilization and subsequent assimilation of otherwise inaccessible Fe(III) substrates (12, 44, 49, 63). Hydroxamate-type siderophores are produced via N⁶ hydroxylation and N⁶ acylation of l-ornithine and, in some cases, cyclization to macrocyclic ring structures (13). The macrocyclic siderophores bisucaberin and putrebactin, for example, are two structural analogs of the cyclic bis(hydroxamate) siderophore alcaligin, synthesized by Aliivibrio salmonicida and Shewanella putrefaciens strain 200, respectively (27, 32, 65). After transport across the cell envelope via a TonB-dependent pathway, Fe(III) is subsequently released from the Fe(III)-siderophore complex by ligand exchange reactions promoted by siderophore ligand hydrolysis and/or protonation or by Fe(III)-siderophore reduction and release of Fe(II) to acceptor ligands (9, 66).The main objectives of the present study were to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are synthesized by Fe(III)-siderophore biosynthesis systems and (ii) if Fe(III)-siderophore reductases are required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. The experimental strategy for this study included (i) identification of genes encoding the siderophore biosynthesis and Fe(III)-siderophore reductase systems in the S. oneidensis genome, (ii) generation of in-frame deletions in the corresponding siderophore biosynthesis and Fe(III)-siderophore reductase genes, (iii) tests of the resulting siderophore biosynthesis mutants for production of siderophores and soluble organic Fe(III) during aerobic and anaerobic Fe(III) oxide respiration, and (iv) tests of the resulting Fe(III)-siderophore reductase mutants for respiration of soluble organic Fe(III) as an anaerobic electron acceptor.