Probing cytoplasmic peroxide metabolism in Shewanella oneidensis.

The facultative anaerobe Shewanella oneidensis respires an extensive set of electron acceptors and, as a consequence, can leak electrons to produce reactive oxygen species such as hydrogen peroxide (H2O2). However, the effects of respiration on cytoplasmic redox homeostasis are poorly characterized in comparison. In the present study, the H2O2 sensor HyPer-3 was deployed to interrogate cytoplasmic peroxide levels of both wild-type and gene deletion mutants lacking peroxide scavenging enzymes following exposure to H2O2. HyPer-3 signals were validated in the S. oneidensis wild-type strain and exhibited a dynamic range of 0-250 µM H2O2. As reported by the HyPer-3 sensor, the cytoplasm of H2O2-perturbed mutant strains lacking periplasmic glutathione peroxidase (PgpD) and double deletion mutants lacking catalase (KatB) and bifunctional catalase-peroxidases (KatG1 or KatG2) contained high H2O2 concentrations. The high cytoplasmic H2O2 concentrations correlated with impaired H2O2 removal rates displayed by the mutant strains. Results of the present study provide the first in vivo interrogation of the redox environment of the S. oneidensis cytoplasm with HyPer-3 sensors and indicate that proper redox conditions in minimal growth medium are maintained by the concerted action of both well-known (periplasmic PgpD, cytoplasmic KatB and KatG1) and previously overlooked (cytoplasmic KatG2) peroxidases and catalases.

Iodate Reduction by Shewanella oneidensis Requires Genes Encoding an Extracellular Dimethylsulfoxide Reductase.

Microbial iodate (IO3 -) reduction is a major component of the iodine biogeochemical reaction network in anaerobic marine basins and radioactive iodine-contaminated subsurface environments. Alternative iodine remediation technologies include microbial reduction of IO3 - to iodide (I-) and microbial methylation of I- to volatile gases. The metal reduction pathway is required for anaerobic IO3 - respiration by the gammaproteobacterium Shewanella oneidensis. However, the terminal IO3 - reductase and additional enzymes involved in the S. oneidensis IO3 - electron transport chain have not yet been identified. In this study, gene deletion mutants deficient in four extracellular electron conduits (EECs; ΔmtrA, ΔmtrA-ΔmtrDEF, ΔmtrA-ΔdmsEF, ΔmtrA-ΔSO4360) and DMSO reductase (ΔdmsB) of S. oneidensis were constructed and examined for anaerobic IO3 - reduction activity with either 20 mM lactate or formate as an electron donor. IO3 - reduction rate experiments were conducted under anaerobic conditions in defined minimal medium amended with 250 µM IO3 - as anaerobic electron acceptor. Only the ΔmtrA mutant displayed a severe deficiency in IO3 - reduction activity with lactate as the electron donor, which suggested that the EEC-associated decaheme cytochrome was required for lactate-dependent IO3 - reduction. The ΔmtrA-ΔdmsEF triple mutant displayed a severe deficiency in IO3 - reduction activity with formate as the electron donor, whereas ΔmtrA-ΔmtrDEF and ΔmtrA-ΔSO4360 retained moderate IO3 - reduction activity, which suggested that the EEC-associated dimethylsulfoxide (DMSO) reductase membrane-spanning protein DmsE, but not MtrA, was required for formate-dependent IO3 - reduction. Furthermore, gene deletion mutant ΔdmsB (deficient in the extracellular terminal DMSO reductase protein DmsB) and wild-type cells grown with tungsten replacing molybdenum (a required co-factor for DmsA catalytic activity) in defined growth medium were unable to reduce IO3 - with either lactate or formate as the electron donor, which indicated that the DmsAB complex functions as an extracellular IO3 - terminal reductase for both electron donors. Results of this study provide complementary genetic and phenotypic evidence that the extracellular DMSO reductase complex DmsAB of S. oneidensis displays broad substrate specificity and reduces IO3 - as an alternate terminal electron acceptor.

Resistance of perfluorooctanoic acid to degradation by the microbially driven Fenton reaction.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) have received recent heightened attention as emerging contaminants. Due to widespread application in household products and aqueous film-forming foams, PFAS are globally distributed in the environment, and bioaccumulate in the blood and tissues of mammals including humans. The microbially driven Fenton reaction, a hybrid biotic/abiotic hydroxyl radical (HO•)-generating system, previously degraded a wide variety of persistant organic pollutants. In the present study, the microbially driven Fenton reaction was employed to attempt degradation of PFOA. Batch cultures of the facultatively anaerobic bacteria Shewanella oneidensis were amended with PFOA and Fe(III)-citrate. Under aerobic conditions, S. oneidensis reduced oxygen to hydrogen peroxide (H2O2), while under anaerobic conditions, S. oneidensis reduced Fe(III) to Fe(II). During aerobic-to-anaerobic transition periods, Fe(II) and H2O2 interacted chemically via the Fenton reaction to produce HO• radicals, which in turn interacted with PFOA. Batch reactors were cycled between aerobic and anaerobic phases for four cycles, residual PFOA was extracted via liquid-liquid extraction and analyzed by liquid chromatography combined with tandem mass spectrometry. Unlike degradation of other organic pollutants, PFOA concentrations remained unchanged, which indicated that PFOA was resistant to degradation by the microbially-driven Fenton reaction. Similar to abiotic (purely chemical) Fenton reaction systems, these results most likely reflect the inability of HO• radicals to oxidatively degrade PFOA.

Novel insights into the taxonomic diversity and molecular mechanisms of bacterial Mn(III) reduction.

Soluble ligand-bound Mn(III) can support anaerobic microbial respiration in diverse aquatic environments. Thus far, Mn(III) reduction has only been associated with certain Gammaproteobacteria. Here, we characterized microbial communities enriched from Mn-replete sediments of Lake Matano, Indonesia. Our results provide the first evidence for the biological reduction of soluble Mn(III) outside the Gammaproteobacteria. Metagenome assembly and binning revealed a novel betaproteobacterium, which we designate 'Candidatus Dechloromonas occultata.' This organism dominated the enrichment and expressed a porin-cytochrome c complex typically associated with iron-oxidizing Betaproteobacteria and a novel cytochrome c-rich protein cluster (Occ), including an undecaheme putatively involved in extracellular electron transfer. This occ gene cluster was also detected in diverse aquatic bacteria, including uncultivated Betaproteobacteria from the deep subsurface. These observations provide new insight into the taxonomic and functional diversity of microbially driven Mn(III) reduction in natural environments.

Metal Reduction and Protein Secretion Genes Required for Iodate Reduction by Shewanella oneidensis.

The metal-reducing gammaproteobacterium Shewanella oneidensis reduces iodate (IO3-) as an anaerobic terminal electron acceptor. Microbial IO3- electron transport pathways are postulated to terminate with nitrate (NO3-) reductase, which reduces IO3- as an alternative electron acceptor. Recent studies with S. oneidensis, however, have demonstrated that NO3- reductase is not involved in IO3- reduction. The main objective of the present study was to determine the metal reduction and protein secretion genes required for IO3- reduction by Shewanella oneidensis with lactate, formate, or H2 as the electron donor. With all electron donors, the type I and type V protein secretion mutants retained wild-type IO3- reduction activity, while the type II protein secretion mutant lacking the outer membrane secretin GspD was impaired in IO3- reduction. Deletion mutants lacking the cyclic AMP receptor protein (CRP), cytochrome maturation permease CcmB, and inner membrane-tethered c-type cytochrome CymA were impaired in IO3- reduction with all electron donors, while deletion mutants lacking c-type cytochrome MtrA and outer membrane ß-barrel protein MtrB of the outer membrane MtrAB module were impaired in IO3- reduction with only lactate as an electron donor. With all electron donors, mutants lacking the c-type cytochromes OmcA and MtrC of the metal-reducing extracellular electron conduit MtrCAB retained wild-type IO3- reduction activity. These findings indicate that IO3- reduction by S. oneidensis involves electron donor-dependent metal reduction and protein secretion pathway components, including the outer membrane MtrAB module and type II protein secretion of an unidentified IO3- reductase to the S. oneidensis outer membrane.IMPORTANCE Microbial iodate (IO3-) reduction is a major component in the biogeochemical cycling of iodine and the bioremediation of iodine-contaminated environments; however, the molecular mechanism of microbial IO3- reduction is poorly understood. Results of the present study indicate that outer membrane (type II) protein secretion and metal reduction genes encoding the outer membrane MtrAB module of the extracellular electron conduit MtrCAB are required for IO3- reduction by S. oneidensis On the other hand, the metal-reducing c-type cytochrome MtrC of the extracellular electron conduit is not required for IO3- reduction by S. oneidensis These findings indicate that the IO3- electron transport pathway terminates with an as yet unidentified IO3- reductase that associates with the outer membrane MtrAB module to deliver electrons extracellularly to IO3.

Whole-genome sequencing reveals that Shewanella haliotis Kim et al. 2007 can be considered a later heterotypic synonym of Shewanella algae Simidu et al. 1990.

Previously, experimental DNA-DNA hybridization (DDH) between Shewanellahaliotis JCM 14758T and Shewanellaalgae JCM 21037T had suggested that the two strains could be considered different species, despite minimal phenotypic differences. The recent isolation of Shewanella sp. MN-01, with 99 % 16S rRNA gene identity to S. algae and S. haliotis, revealed a potential taxonomic problem between these two species. In this study, we reassessed the nomenclature of S. haliotis and S. algae using available whole-genome sequences. The whole-genome sequence of S. haliotis JCM 14758T and ten S. algae strains showed ≥97.7 % average nucleotide identity and >78.9 % digital DDH, clearly above the recommended species thresholds. According to the rules of priority and in view of the results obtained, S. haliotis is to be considered a later heterotypic synonym of S. algae. Because the whole-genome sequence of Shewanella sp. strain MN-01 shares >99 % ANI with S. algae JCM 14758T, it can be confidently identified as S. algae.

Degradation of the recalcitrant oil spill components anthracene and pyrene by a microbially driven Fenton reaction.

Oil spill components include a range of toxic saturated, aromatic and polar hydrocarbons, including pyrene and anthracene. Such contaminants harm natural ecosystems, adversely affect human health and negatively impact tourism and the fishing industries. Current physical, chemical and biological remediation technologies are often unable to completely remove recalcitrant oil spill components, which accumulate at levels greater than regulatory limits set by the Environmental Protection Agency. In the present study, a microbially driven Fenton reaction, previously shown to produce hydroxyl (HO • ) radicals that degrade chlorinated solvents and associated solvent stabilizers, was also found to degrade source zone concentrations of the oil spill components, pyrene (10 µM) and anthracene (1 µM), at initial rates of 0.82 and 0.20 µM h -1 , respectively. The pyrene- and anthracene-degrading Fenton reaction was driven by the metal-reducing facultative anaerobe Shewanella oneidensis exposed to alternating aerobic and anaerobic conditions in the presence of Fe(III). Similar to the chlorinated solvent degradation system, the pyrene and anthracene degradation systems required neither the continual supply of exogenous H 2 O 2 nor UV-induced Fe(III) reduction to regenerate Fe(II). The microbially driven Fenton reaction provides the foundation for the development of alternate ex situ and in situ oil and gas spill remediation technologies.

Microbial manganese(III) reduction fuelled by anaerobic acetate oxidation.

Soluble manganese in the intermediate +III oxidation state (Mn3+ ) is a newly identified oxidant in anoxic environments, whereas acetate is a naturally abundant substrate that fuels microbial activity. Microbial populations coupling anaerobic acetate oxidation to Mn3+ reduction, however, have yet to be identified. We isolated a Shewanella strain capable of oxidizing acetate anaerobically with Mn3+ as the electron acceptor, and confirmed this phenotype in other strains. This metabolic connection between acetate and soluble Mn3+ represents a new biogeochemical link between carbon and manganese cycles. Genomic analyses uncovered four distinct genes that allow for pathway variations in the complete dehydrogenase-driven TCA cycle that could support anaerobic acetate oxidation coupled to metal reduction in Shewanella and other Gammaproteobacteria. An oxygen-tolerant TCA cycle supporting anaerobic manganese reduction is thus a new connection in the manganese-driven carbon cycle, and a new variable for models that use manganese as a proxy to infer oxygenation events on early Earth.

Simultaneous Transformation of Commingled Trichloroethylene, Tetrachloroethylene, and 1,4-Dioxane by a Microbially Driven Fenton Reaction in Batch Liquid Cultures.

Improper disposal of 1,4-dioxane and the chlorinated organic solvents trichloroethylene (TCE) and tetrachloroethylene (also known as perchloroethylene [PCE]) has resulted in widespread contamination of soil and groundwater. In the present study, a previously designed microbially driven Fenton reaction system was reconfigured to generate hydroxyl (HO˙) radicals for simultaneous transformation of source zone levels of single, binary, and ternary mixtures of TCE, PCE, and 1,4-dioxane. The reconfigured Fenton reaction system was driven by fed batch cultures of the Fe(III)-reducing facultative anaerobe Shewanella oneidensis amended with lactate, Fe(III), and contaminants and exposed to alternating anaerobic and aerobic conditions. To avoid contaminant loss due to volatility, the Fe(II)-generating, hydrogen peroxide-generating, and contaminant transformation phases of the microbially driven Fenton reaction system were separated. The reconfigured Fenton reaction system transformed TCE, PCE, and 1,4-dioxane either as single contaminants or as binary and ternary mixtures. In the presence of equimolar concentrations of PCE and TCE, the ratio of the experimentally derived rates of PCE and TCE transformation was nearly identical to the ratio of the corresponding HO˙ radical reaction rate constants. The reconfigured Fenton reaction system may be applied as an ex situ platform for simultaneous degradation of commingled TCE, PCE, and 1,4-dioxane and provides valuable information for future development of in situ remediation technologies. IMPORTANCE: A microbially driven Fenton reaction system [driven by the Fe(III)-reducing facultative anaerobe S. oneidensis] was reconfigured to transform source zone levels of TCE, PCE, and 1,4-dioxane as single contaminants or as binary and ternary mixtures. The microbially driven Fenton reaction may thus be applied as an ex situ platform for simultaneous degradation of at least three (and potentially more) commingled contaminants. Additional targets for ex situ and in situ degradation by the microbially driven Fenton reaction developed in the present study include multiple combinations of environmental contaminants susceptible to attack by Fenton reaction-generated HO˙ radicals, including commingled plumes of 1,4-dioxane, pentachlorophenol (PCP), PCE, TCE, 1,1,2-trichloroethane (TCA), and perfluoroalkylated substances (PFAS).

Direct conversion of cellulose and hemicellulose to fermentable sugars by a microbially-driven Fenton reaction.

The aim of this work was to develop a microbially-driven Fenton reaction that fragments cellulose and hemicellulose, degrades cellodextrins and xylodextrins, and produces short-chain oligosaccharides and monomeric sugars in a single bioreactor. The lignocellulose degradation system operates at neutral pH and does not require addition of conventional lignocellulose-degrading enzymes, thus avoiding problems associated with enzyme accessibility and specificity. The ability to produce useful bioproducts was demonstrated by production of the bioplastic polyhydroxybutyrate with the xylan degradation products as starting substrate.

Activation of an Otherwise Silent Xylose Metabolic Pathway in Shewanella oneidensis.

UNLABELLED: Shewanella oneidensis is unable to metabolize the sugar xylose as a carbon and energy source. In the present study, an otherwise silent xylose catabolic pathway was activated in S. oneidensis by following an adaptive evolution strategy. Genome-wide scans indicated that the S. oneidensis genome encoded two proteins similar to the xylose oxido-reductase pathway enzymes xylose reductase (SO_0900) and xylulokinase (SO_4230), and purified SO_0900 and SO_4230 displayed xylose reductase and xylulokinase activities, respectively. The S. oneidensis genome was missing, however, an Escherichia coli XylE-like xylose transporter. After 12 monthly transfers in minimal growth medium containing successively higher xylose concentrations, an S. oneidensis mutant (termed strain XM1) was isolated for the acquired ability to grow aerobically on xylose as a carbon and energy source. Whole-genome sequencing indicated that strain XM1 contained a mutation in an unknown membrane protein (SO_1396) resulting in a glutamine-to-histidine conversion at amino acid position 207. Homology modeling demonstrated that the Q207H mutation in SO_1396 was located at the homologous xylose docking site in XylE. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass. IMPORTANCE: The activation of an otherwise silent xylose metabolic system in Shewanella oneidensis is a powerful example of how accidental mutations allow microorganisms to adaptively evolve. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass.

Microbially driven Fenton reaction for degradation of the widespread environmental contaminant 1,4-dioxane.

The carcinogenic cyclic ether compound 1,4-dioxane is employed as a stabilizer of chlorinated industrial solvents and is a widespread environmental contaminant in surface water and groundwater. In the present study, a microbially driven Fenton reaction was designed to autocatalytically generate hydroxyl (HO•) radicals that degrade 1,4-dioxane. In comparison to conventional (purely abiotic) Fenton reactions, the microbially driven Fenton reaction operated at circumneutral pH and did not the require addition of exogenous H2O2 or UV irradiation to regenerate Fe(II) as Fenton reagents. The 1,4-dioxane degradation process was driven by pure cultures of the Fe(III)-reducing facultative anaerobe Shewanella oneidensis manipulated under controlled laboratory conditions. S. oneidensis batch cultures were provided with lactate, Fe(III), and 1,4-dioxane and were exposed to alternating aerobic and anaerobic conditions. The microbially driven Fenton reaction completely degraded 1,4-dioxane (10 mM initial concentration) in 53 h with an optimal aerobic-anaerobic cycling period of 3 h. Acetate and oxalate were detected as transient intermediates during the microbially driven Fenton degradation of 1,4-dioxane, an indication that conventional and microbially driven Fenton degradation processes follow similar reaction pathways. The microbially driven Fenton reaction provides the foundation for development of alternative in situ remediation technologies to degrade environmental contaminants susceptible to attack by HO• radicals generated by the Fenton reaction.

Flow dependent performance of microfluidic microbial fuel cells.

The integration of Microbial Fuel Cells (MFCs) in a microfluidic geometry can significantly enhance the power density of these cells, which would have more active bacteria per unit volume. Moreover, microfluidic MFCs can be operated in a continuous mode as opposed to the traditional batch-fed mode. Here we investigate the effect of fluid flow on the performance of microfluidic MFCs. The growth and the structure of the bacterial biofilm depend to a large extent on the shear stress of the flow. We report the existence of a range of flow rates for which MFCs can achieve maximum voltage output. When operated under these optimal conditions, the power density of our microfluidic MFC is about 15 times that of a similar-size batch MFC. Furthermore, this optimum suggests a correlation between the behaviour of bacteria and fluid flow.

Sulfur-mediated electron shuttling during bacterial iron reduction.

Microbial reduction of ferric iron [Fe(III)] is an important biogeochemical process in anoxic aquifers. Depending on groundwater pH, dissimilatory metal-reducing bacteria can also respire alternative electron acceptors to survive, including elemental sulfur (S(0)). To understand the interplay of Fe/S cycling under alkaline conditions, we combined thermodynamic geochemical modeling with bioreactor experiments using Shewanella oneidensis MR-1. Under these conditions, S. oneidensis can enzymatically reduce S(0) but not goethite (α-FeOOH). The HS(-) produced subsequently reduces goethite abiotically. Because of the prevalence of alkaline conditions in many aquifers, Fe(III) reduction may thus proceed via S(0)-mediated electron-shuttling pathways.

Identification of a molecular signature unique to metal-reducing Gammaproteobacteria.

Functional genes required for microbial (dissimilatory) metal reduction display high sequence divergence, which limits their utility as molecular biomarkers for tracking the presence and activity of metal-reducing bacteria in natural and engineered systems. In the present study, homologs of the outer membrane beta-barrel protein MtrB of metal-reducing Gammaproteobacteria were found to contain a unique N-terminal CXXC motif that was missing from MtrB homologs of nonmetal-reducing Gammaproteobacteria and metal- and nonmetal-reducing bacteria outside the Gammaproteobacteria. To determine whether the N-terminal CXXC motif of MtrB was required for dissimilatory metal reduction, each cysteine in the CXXC motif of the representative metal-reducing gammaproteobacterium Shewanella oneidensis was replaced with alanine, and the resulting site-directed mutants were tested for metal reduction activity. Anaerobic growth experiments demonstrated that the first, but not the second, conserved cysteine was required for metal reduction by S. oneidensis. The ability to predict metal reduction by Gammaproteobacteria with unknown metal reduction capability was confirmed with Vibrio parahaemolyticus, a pathogen whose genome encodes an MtrB homolog with an N-terminal CXXC motif. MtrB homologs with an N-terminal CXXC motif may thus represent a molecular signature unique to metal-reducing members of the Gammaproteobacteria.

Electron transport and protein secretion pathways involved in Mn(III) reduction by Shewanella oneidensis.

Soluble Mn(III) represents an important yet overlooked oxidant in marine and freshwater systems. The molecular mechanism of microbial Mn(III) reduction, however, has yet to be elucidated. Extracellular reduction of insoluble Mn(IV) and Fe(III) oxides by the metal-reducing γ-proteobacterium Shewanella oneidensis involves inner (CymA) and outer (OmcA) membrane-associated c-type cytochromes, the extracellular electron conduit MtrCAB, and GspD, the secretin of type II protein secretion. CymA, MtrCAB and GspD mutants were unable to reduce Mn(III) and Mn(IV) with lactate, H2, or formate as electron donor. The OmcA mutant reduced Mn(III) and Mn(IV) at near wild-type rates with lactate and formate as electron donor. With H2 as electron donor, however, the OmcA mutant was unable to reduce Mn(III) but reduced Mn(IV) at wild-type rates. Analogous Fe(III) reduction rate analyses indicated that other electron carriers compensated for the absence of OmcA, CymA, MtrCAB and GspD during Fe(III) reduction in an electron donor-dependent fashion. Results of the present study demonstrate that the S. oneidensis electron transport and protein secretion components involved in extracellular electron transfer to external Mn(IV) and Fe(III) oxides are also required for electron transfer to Mn(III) and that OmcA may function as a dedicated component of an H2 oxidation-linked Mn(III) reduction system.

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. DeltaSO3031 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. DeltaSO3034 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.

Adhesion of Shewanella oneidensis MR-1 to iron (Oxy)(Hydr)oxides: microcolony formation and isotherm.

The adhesion of dissimilatory metal reducing bacteria (DMRB) to iron (oxy)(hydr)oxides may play an important role in their respiration on ferric iron-containing minerals, but few quantitative surface cell density measurements have been made thus far. We used confocal microscopy to examine the adhesion of a common DMRB species, Shewanella oneidensis MR-1, onto iron (oxy)(hydr)oxide particulate-coated glass slides across a broad range of bulk (i.e., solution phase) cell densities from 10(5) cells/mL to 2 x 10(9) cells/mL. At bulk cell densities less than 1 x 10(7) cells/mL, cells adhered to the slide surface formed an evenly distributed, homogeneous monolayer, while at the bulk cell densities higher than 2 x 10(8) cells/mL the adhered cells formed distinct microcolonies. As a result of this complex adhesion behavior, simple Langmuir or Freundlich adsorption isotherms do not capture the relationship between the surface cell density and the bulk cell density over the entire range of bulk cell densities. Thus a new, two-step isotherm was developed that incorporated both isolated attached cells at low cell densities as well as microcolonies at higher cell densities.

Shewanella oneidensis MR-1 mutants selected for their inability to produce soluble organic-Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor.

Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic-Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic-Fe(III) complexes to include Shewanella oneidensis MR-1. Soluble organic-Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O(2), nitrate or trimethylamine-N-oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic-Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)-citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic-Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild-type and Sol mutant strains, and all four Sol mutants retained wild-type siderophore production capability. Results of this study indicate that the production of soluble organic-Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.

Outer membrane-associated serine protease involved in adhesion of Shewanella oneidensis to Fe(III) oxides.

The facultative anaerobe Shewanella oneidensis MR-1 respires a variety of anaerobic electron acceptors, including insoluble Fe(III) oxides. S. oneidensis employs a number of novel strategies for respiration of insoluble Fe(III) oxides, including localization of respiratory proteins to the cell outer membrane (OM). The molecular mechanism by which S. oneidensis adheres to and respires Fe(III) oxides, however, remains poorly understood. In the present study, whole cell fractionation and MALDI-TOF-MS/MS techniques were combined to identify a serine protease (SO3800) associated with the S. oneidensis OM. SO3800 contained predicted structural motifs similar to cell surface-associated serine proteases that function as bacterial adhesins in other gram-negative bacteria. The gene encoding SO3800 was deleted from the S. oneidensis genome, and the resulting mutant strain (DeltaSO3800) was tested for its ability to adhere to and respire Fe(III) oxides. DeltaSO3800 was severely impaired in its ability to adhere to Fe(III) oxides, yet retained wild-type Fe(III) respiratory capability. Laser Doppler velocimetry and cryoetch high-resolution SEM experiments indicated that DeltaSO3800 displayed a lower cell surface charge and higher amount of surface-associated exopolysaccharides. Results of this study indicate that S. oneidensis may respire insoluble Fe(III) oxides at a distance, negating the requirement for attachment prior to electron transfer.