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Sample records for oneidensis mr-1 fluxome

  1. Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions▿ †

    PubMed Central

    Tang, Yinjie J.; Hwang, Judy S.; Wemmer, David E.; Keasling, Jay D.

    2007-01-01

    The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values. PMID:17098921

  2. The Shewanella oneidensis MR-1 Fluxome under Various OxygenConditions

    SciTech Connect

    Tang, Yinjie J.; Hwang, Judy S.; Wemmer, David E.; Keasling, Jay D.

    2006-03-17

    The central metabolic fluxes of Shewanella oneidensis MR-1were examined under carbon-limited (aerobic) and oxygen-limited(micro-aerobic) chemostat conditions using 13C labeled lactate as thesole carbon source. The carbon labeling patterns of key amino acids inbiomass were probed using both GC-MS and 13C-NMR. Based on the genomeannotation, a metabolic pathway model was constructed to quantify thecentral metabolic flux distributions. The model showed that thetricarboxylic acid (TCA) cycle is the major carbon metabolism route underboth conditions. The Entner-Doudoroff and pentose phosphate pathways weremainly utilized for biomass synthesis (flux below 5 percent of thelactate uptake rate). The anapleurotic reactions (pyruvate to malate andoxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt wereactive. Under carbon-limited conditions, a substantial amount of carbonwas oxidized via the highly reversible serine metabolic pathway. Fluxesthrough the TCA cycle were less whereas acetate production was more underoxygen limitation than under carbon limitation. Although fluxdistributions under aerobic, micro-aerobic, and shake-flask cultureconditions were dramatically different, the relative flux ratios of thecentral metabolic reactions did not vary significantly. Hence, S.oneidensis metabolism appears to be quite robust to environmentalchanges. Our study also demonstrates the merit of coupling GC-MS with 13CNMR for metabolic flux analysis to reduce the use of 13C labeledsubstrates and to obtain more accurate flux values.

  3. Biotransformation and biomethylation of arsenic by Shewanella oneidensis MR-1.

    PubMed

    Wang, Juan; Wu, Mingyin; Lu, Gan; Si, Youbin

    2016-02-01

    The resistance of Shewanella oneidensis MR-1 to toxic arsenic was investigated by measuring the growth of the bacteria in the presence of As(III) and As(V) in different growth media. The bacteria were shown to biotransform arsenic through the partial methylation of inorganic arsenic into methylated metabolites. This biotransformation of inorganic arsenic by S. oneidensis MR-1 was affected by the methyl donor, the composition of the medium, and the presence of Fe(III). The relative content of methylated arsenic in the medium containing S-adenosyl methionine as the methyl donor was greater than that in the medium containing methylcobalamin. The biotransformation process driven by Fe-reducing bacteria, and occurred in combination with microbially mediated As-Fe reduction in the presence of Fe(III). The results demonstrate that S. oneidensis MR-1 methylates inorganic arsenic into less toxic organoarsenic compounds. This process has potential applications in the bioremediation of environmental arsenic, and the results provide new insights into the control of in situ arsenic pollution.

  4. Oxygen-dependent autoaggregation in Shewanella oneidensis MR-1

    SciTech Connect

    Mclean, Jeffrey S.; Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Bilskis, Christina L.; Zakrajsek, Brian A.; Hill, Eric A.; Saffarini, Daad; Romine, Margaret F.; Gorby, Yuri A.; Fredrickson, Jim K.; Beliaev, Alex S.

    2008-07-01

    In aerobic chemostat cultures maintained at 50% dissolved O2 tension (123.5 µM dissolved O2), Shewanella oneidensis strain MR-1 rapidly aggregated upon addition of 0.68 mM CaCl2 and retained this multi-cellular phenotype at high dilution rates. Confocal microscopy analysis of the extracellular matrix material contributing to the stability of the aggregate structures revealed the presence of extracellular DNA, protein, and glycoconjugates. Upon onset of O2-limited growth (dissolved O2 below detection) however, the Ca2+-supplemented chemostat cultures of strain MR-1 rapidly disaggregated and grew as motile dispersed cells. Global transcriptome analysis comparing aerobic aggregated to O2-limited unaggregated cells identified genes encoding cell-to-cell and cell-to-surface adhesion factors whose transcription increased upon exposure to increased O2 concentrations. The aerobic aggregated cells also revealed increased expression of putative anaerobic electron transfer and homologs of metal reduction genes, including mtrD (SO1782), mtrE (SO1781), and mtrF (SO1780). Our data indicate that mechanisms involved in autoaggregation of MR-1 are dependent on the function of pilD gene which encodes a putative prepilin peptidase. Mutants of S. oneidensis strain MR-1 deficient in PilD and associated pathways, including type IV and Msh pili biogenesis, displayed a moderate increase in sensitivity to H2O2. Taken together, our evidence indicates that aggregate formation in S. oneidensis MR-1 may serve as an alternative or an addition to biochemical detoxification to reduce the oxidative stress associated with production of reactive oxygen species during aerobic metabolism while facilitating the development of hypoxic conditions within the aggregate interior.

  5. Survival of Shewanella oneidensis MR-1 after UV radiation exposure.

    PubMed

    Qiu, Xiaoyun; Sundin, George W; Chai, Benli; Tiedje, James M

    2004-11-01

    We systematically investigated the physiological response as well as DNA damage repair and damage tolerance in Shewanella oneidensis MR-1 following UVC, UVB, UVA, and solar light exposure. MR-1 showed the highest UVC sensitivity among Shewanella strains examined, with D37 and D10 values of 5.6 and 16.5% of Escherichia coli K-12 values. Stationary cells did not show an increased UVA resistance compared to exponential-phase cells; instead, they were more sensitive at high UVA dose. UVA-irradiated MR-1 survived better on tryptic soy agar than Luria-Bertani plates regardless of the growth stage. A 20% survival rate of MR-1 was observed following doses of 3.3 J of UVC m(-2), 568 J of UVB m(-2), 25 kJ of UVA m(-2), and 558 J of solar UVB m(-2), respectively. Photoreactivation conferred an increased survival rate to MR-1 of as much as 177- to 365-fold, 11- to 23-fold, and 3- to 10-fold following UVC, UVB, and solar light irradiation, respectively. A significant UV mutability to rifampin resistance was detected in both UVC- and UVB-treated samples, with the mutation frequency in the range of 10(-5) to 10(-6). Unlike in E. coli, the expression levels of the nucleotide excision repair (NER) component genes uvrA, uvrB, and uvrD were not damage inducible in MR-1. Complementation of Pseudomonas aeruginosa UA11079 (uvrA deficient) with uvrA of MR-1 increased the UVC survival of this strain by more than 3 orders of magnitude. Loss of damage inducibility of the NER system appears to contribute to the high sensitivity of this bacterium to UVR as well as to other DNA-damaging agents.

  6. Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1

    PubMed Central

    El-Naggar, Mohamed Y.; Wanger, Greg; Leung, Kar Man; Yuzvinsky, Thomas D.; Southam, Gordon; Yang, Jun; Lau, Woon Ming; Nealson, Kenneth H.; Gorby, Yuri A.

    2010-01-01

    Bacterial nanowires are extracellular appendages that have been suggested as pathways for electron transport in phylogenetically diverse microorganisms, including dissimilatory metal-reducing bacteria and photosynthetic cyanobacteria. However, there has been no evidence presented to demonstrate electron transport along the length of bacterial nanowires. Here we report electron transport measurements along individually addressed bacterial nanowires derived from electron-acceptor–limited cultures of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. Transport along the bacterial nanowires was independently evaluated by two techniques: (i) nanofabricated electrodes patterned on top of individual nanowires, and (ii) conducting probe atomic force microscopy at various points along a single nanowire bridging a metallic electrode and the conductive atomic force microscopy tip. The S. oneidensis MR-1 nanowires were found to be electrically conductive along micrometer-length scales with electron transport rates up to 109/s at 100 mV of applied bias and a measured resistivity on the order of 1 Ω·cm. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA produce appendages that are morphologically consistent with bacterial nanowires, but were found to be nonconductive. The measurements reported here allow for bacterial nanowires to serve as a viable microbial strategy for extracellular electron transport. PMID:20937892

  7. Characterization of uraninite nanoparticles produced by Shewanella oneidensis MR-1

    SciTech Connect

    Burgos, William D.; McDonough, J.; Senko, John M.; Zhang, Gengxin; Dohnalkova, Alice; Kelly, Shelly D.; Gorby, Yuri A.; Kemner, Kenneth M.

    2008-10-15

    The reduction of uranium(VI) by Shewanella oneidensis MR-1 was studied to examine the effects of bioreduction kinetics and background electrolyte on the physical properties and reactivity to re-oxidation of the biogenic uraninite, UO2(s). Bioreduction experiments were conducted with uranyl acetate as the electron acceptor and sodium lactate as the electron donor under resting cell conditions in a 30 mM NaHCO3 buffer, and in a PIPES-buffered artificial groundwater (PBAGW). MR-1 was cultured in batch mode in a defined minimal medium with a specified air-to-medium volume ratio such that electron acceptor (O2) limiting conditions were reached just when cells were harvested for subsequent experiments. The rate of U(VI) bioreduction was manipulated by varying the cell density and the incubation temperature (1.0 _ 108 cell ml_1 at 20 _C or 2.0 _ 108 cell ml_1 at 37 _C) to generate U(IV) solids at ‘‘fast” and ‘‘slow” rates in the two different buffers. The presence of Ca in PBAGW buffer altered U(VI) speciation and solubility, and significantly decreased U(VI) bioreduction kinetics. High resolution transmission electron microscopy was used to measure uraninite particle size distributions produced under the four different conditions. The most common primary particle size was 2.9–3.0 nm regardless of U(VI) bioreduction rate or background electrolyte. Extended X-ray absorption fine-structure spectroscopy was also used to estimate uraninite particle size and was consistent with TEM results. The reactivity of the biogenic uraninite products with dissolved oxygen was tested, and neither U(VI) bioreduction rate nor background electrolyte had any statistical effect on oxidation rates. With MR-1, uraninite particle size was not controlled by the bioreduction rate of U(VI) or the background electrolyte. These results for MR-1, where U(VI) bioreduction rate had no discernible effect on uraninite particle size or oxidation rate, contrast with our recent research with

  8. High Pressure Reduction of Selenite by Shewanella oneidensis MR-1

    NASA Astrophysics Data System (ADS)

    Picard, A.; Daniel, I.; Testemale, D.; Letard, I.; Bleuet, P.; Cardon, H.; Oger, P.

    2007-12-01

    High-pressure biotopes comprise cold deep-sea environments, hydrothermal vents, and deep subsurface or deep-sea sediments. The latter are less studied, due to the technical difficulties to sample at great depths without contamination. Nevertheless, microbial sulfate reduction and methanogenesis have been found to be spatially distributed in deep deep-sea sediments (1), and sulfate reduction has been shown to be actually more efficient under high hydrostatic pressure (HHP) in some sediments (2). Sulfate-reducing bacteria obtained from the Japan Sea are characterized by an increased sulfide production under pressure (3,4). Unfortunately, investigations of microbial metabolic activity as a function of pressure are extremely scarce due to the experimental difficulty of such measurements at high hydrostatic pressure. We were able to measure the reduction of selenite Se(IV) by Shewanella oneidensis MR-1 as a function of pressure, to 150 MPa using two different high-pressure reactors that allow in situ X-ray spectroscopy measurements on a synchrotron source. A first series of measurements was carried out in a low-pressure Diamond Anvil Cell (DAC) of our own design (5) at ID22 beamline at ESRF (European Synchrotron Radiation Facility); a second one was performed in an autoclave (6) at the BM30B beamline at ESRF. Selenite reduction by strain MR-17 was monitored from ambient pressure to 150 MPa over 25 hours at 30 deg C by XANES spectroscopy (X-ray Analysis of Near Edge Structure). Spectra were recorded hourly in order to quantify the evolution of the oxidation state of selenium with time. Stationary-phase bacteria were inoculated at a high concentration into fresh growth medium containing 5 or 10 M of sodium selenite and 20 mM sodium lactate. Kinetic parameters of the Se (IV) reduction by Shewanella oneidensis strain MR-1 could be extracted from the data, as a function of pressure. They show 1) that the rate constant k of the reaction is decreased by a half at high pressure

  9. Energy-dependent stability of Shewanella oneidensis MR-1 biofilms.

    PubMed

    Saville, Renee M; Rakshe, Shauna; Haagensen, Janus A J; Shukla, Soni; Spormann, Alfred M

    2011-07-01

    Stability and resistance to dissolution are key features of microbial biofilms. How these macroscopic properties are determined by the physiological state of individual biofilm cells in their local physical-chemical and cellular environment is largely unknown. In order to obtain molecular and energetic insight into biofilm stability, we investigated whether maintenance of biofilm stability is an energy-dependent process and whether transcription and/or translation is required for biofilm dissolution. We found that in 12-hour-old Shewanella oneidensis MR-1 biofilms, a reduction in cellular ATP concentration, induced either by oxygen deprivation or by addition of the inhibitor of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), dinitrophenol (DNP), or CN(-), resulted in massive dissolution. In 60-hour-old biofilms, the extent of uncoupler-induced cell loss was strongly attenuated, indicating that the integrity of older biofilms is maintained by means other than those operating in younger biofilms. In experiments with 12-hour-old biofilms, the transcriptional and translational inhibitors rifampin, tetracycline, and erythromycin were found to be ineffective in preventing energy starvation-induced detachment, suggesting that neither transcription nor translation is required for this process. Biofilms of Vibrio cholerae were also induced to dissolve upon CCCP addition to an extent similar to that in S. oneidensis. However, Pseudomonas aeruginosa and P. putida biofilms remained insensitive to CCCP addition. Collectively, our data show that metabolic energy is directly or indirectly required for maintaining cell attachment, and this may represent a common but not ubiquitous mechanism for stability of microbial biofilms.

  10. Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Chromate Insult

    SciTech Connect

    Thompson, Melissa R.; VerBerkmoes, Nathan C.; Chourey, Karuna; Brown, Steven D.; Hettich, Robert L.; Thompson, Dorothea K.

    2006-04-05

    Shewanella oneidensis MR-1 is a gram-negative, facultatively anaerobic bacterium originally isolated from a freshwater lake. S. oneidensis MR-1 has the ability to reduce toxic metal ions [e.g., Cr(VI) and U(VI)] found in industrial and governmental waste sites. Cells were grown and exposed to three different metal concentrations in order to probe the dosage response of S. oneidensis MR-1 to Cr(VI) in the form of chromate. Protein fractions were digested with trypsin and analyzed with a multidimensional HPLC-NanoESIMS/MS protocol. The goal of this work is to identify protein components of pathways/mechanisms responsible for both detoxification and reduction of chromate.

  11. Efficiencies of Bio-electrocatalytic Production of Hydrogen from Lactate Using Shewanella oneidensis MR-1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shewanella oneidensis MR-1 was grown in a chemostatic, continuously-fed bioelectrochemical cell under slightly aerated conditions. The start-up phase was controlled potentiostatically (0.4 V vs. SHE). When a stable performance was achieved, the reactor was switched to bio-electrocatalytic producti...

  12. Invariability of Central Metabolic Flux Distribution in Shewanella oneidensis MR-1 Under Environmental or Genetic Perturbations

    SciTech Connect

    Tang, Yinjie; Martin, Hector Garcia; Deutschbauer, Adam; Feng, Xueyang; Huang, Rick; Llora, Xavier; Arkin, Adam; Keasling, Jay D.

    2009-04-21

    An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time {approx} 3 hrs), salt stressed minimal medium (doubling time {approx} 6 hrs), and minimal medium with amino acid supplementation (doubling time {approx}1.5 hrs). {sup 13}C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, ten transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism.

  13. Biological accumulation of tellurium nanorod structures via reduction of tellurite by Shewanella oneidensis MR-1.

    PubMed

    Kim, Dong-Hun; Kanaly, Robert A; Hur, Hor-Gil

    2012-12-01

    The dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, reduced tellurite (Te(IV), TeO(3)(2-)) to elemental tellurium under anaerobic conditions resulting in the intracellular accumulation of needle shaped crystalline Te(0) nanorods. Fatty acid analyses showed that toxic Te(IV) increased the unsaturated fatty acid composition of the lipid components of the cell membrane, implying a deconstruction of the integrity of the cellular membrane structure. The current results suggest that dissimilatory metal reducing bacteria such as S. oneidensis MR-1 may play an important role in recycling toxic tellurium elements, and may be applied as a novel selective biological filter via the accumulation of industry-applicable rare materials, Te(0) nanorods, in the cell.

  14. Electron acceptor dependence of electron shuttle secretion and extracellular electron transfer by Shewanella oneidensis MR-1.

    PubMed

    Wu, Chao; Cheng, Yuan-Yuan; Li, Bing-Bing; Li, Wen-Wei; Li, Dao-Bo; Yu, Han-Qing

    2013-05-01

    Shewanella oneidensis MR-1 is an extensively studied dissimilatory metal-reducing bacterium with a great potential for bioremediation and electricity generation. It secretes flavins as electron shuttles which play an important role in extracellular electron transfer. However, the influence of various environmental factors on the secretion of flavins is largely unknown. Here, the effects of electron acceptors, including fumarate, ferrihydrite, Fe(III)-nitrilotriacetic acid (NTA), nitrate and trimethylamine oxide (TMAO), on the secretion of flavins were investigated. The level of riboflavin and riboflavin-5'-phosphate (FMN) secreted by S. oneidensis MR-1 varied considerably with different electron acceptors. While nitrate and ferrihydrite suppressed the secretion of flavins in relative to fumarate, Fe(III)-NTA and TMAO promoted such a secretion and greatly enhanced ferrihydrite reduction and electricity generation. This work clearly demonstrates that electron acceptors could considerably affect the secretion of flavins and consequent microbial EET. Such impacts of electron acceptors in the environment deserve more attention.

  15. Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1

    SciTech Connect

    Bendall, Matthew L.; Luong, Khai; Wetmore, Kelly M.; Blow, Matthew; Korlach, Jonas; Deutschbauer, Adam; Malmstrom, Rex

    2013-08-30

    We performed whole genome analyses of DNA methylation in Shewanella 17 oneidensis MR-1 to examine its possible role in regulating gene expression and 18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 19 revealed extensive methylation of adenine (N6mA) throughout the 20 genome. These methylated bases were located in five sequence motifs, 21 including three novel targets for Type I restriction/modification enzymes. The 22 sequence motifs targeted by putative methyltranferases were determined via 23 SMRT sequencing of gene knockout mutants. In addition, we found S. 24 oneidensis MR-1 cultures grown under various culture conditions displayed 25 different DNA methylation patterns. However, the small number of differentially 26 methylated sites could not be directly linked to the much larger number of 27 differentially expressed genes in these conditions, suggesting DNA methylation is 28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 29 of methylated GATC motifs in the origin of replication indicate DNA methylation 30 may regulate genome replication in a manner similar to that seen in Escherichia 31 coli. Furthermore, comparative analyses suggest that many 32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.

  16. Shewanella oneidensis MR-1-Induced Fe(III) Reduction Facilitates Roxarsone Transformation

    PubMed Central

    Chen, Guowei; Ke, Zhengchen; Liang, Tengfang; Liu, Li; Wang, Gang

    2016-01-01

    Although microbial activity and associated iron (oxy)hydroxides are known in general to affect the environmental dynamics of 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), the mechanistic understanding of the underlying biophysico-chemical processes remains unclear due to limited experimental information. We studied how Shewanella oneidensis MR-1 –a widely distributed metal-reducing bacterium, in the presence of dissolved Fe(III), affects roxarsone transformations and biogeochemical cycling in a model aqueous system. The results showed that the MR-1 strain was able to anaerobically use roxarsone as a terminal electron acceptor and to convert it to a single product, 3-amino-4-hydroxybenzene arsonic acid (AHBAA). The presence of Fe(III) stimulated roxarsone transformation via MR-1-induced Fe(III) reduction, whereby the resulting Fe(II) acted as an efficient reductant for roxarsone transformation. In addition, the subsequent secondary Fe(III)/Fe(II) mineralization created conditions for adsorption of organoarsenic compounds to the yielded precipitates and thereby led to arsenic immobilization. The study provided direct evidence of Shewanella oneidensis MR-1-induced direct and Fe(II)-associated roxarsone transformation. Quantitative estimations revealed a candidate mechanism for the early-stage environmental dynamics of roxarsone in nature, which is essential for understanding the environmental dynamics of roxarsone and successful risk assessment. PMID:27100323

  17. Involvement of Shewanella oneidensis MR-1 LuxS in Biofilm Development and Sulfur Metabolism

    SciTech Connect

    Learman, Deric R.; Yi, Haakrho; Brown, Steven D.; Martin, Stanton L.; Geesey, Gill G.; Stevens, Ann M.; Hochella, Michael F.

    2009-01-05

    The role of LuxS in Shewanella oneidensis MR-1 has been examined by transcriptomic profiling, biochemical, and physiological experiments. The results indicate that a mutation in luxS alters biofilm development, not by altering quorum-sensing abilities but by disrupting the activated methyl cycle (AMC). The S. oneidensis wild type can produce a luminescence response in the AI-2 reporter strain Vibrio harveyi MM32. This luminescence response is abolished upon the deletion of luxS. The deletion of luxS also alters biofilm formations in static and flowthrough conditions. Genetic complementation restores the mutant biofilm defect, but the addition of synthetic AI-2 has no effect. These results suggest that AI-2 is not used as a quorum-sensing signal to regulate biofilm development in S. oneidensis. Growth on various sulfur sources was examined because of the involvement of LuxS in the AMC. A mutation in luxS produced a reduced ability to grow with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and to recycle homocysteine. These data suggest that LuxS is important to metabolizing methionine and the AMC in S. oneidensis.

  18. Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation.

    PubMed

    Brown, Ashley R; Correa, Elon; Xu, Yun; AlMasoud, Najla; Pimblott, Simon M; Goodacre, Royston; Lloyd, Jonathan R

    2015-01-01

    Biogeochemical processes mediated by Fe(III)-reducing bacteria such as Shewanella oneidensis have the potential to influence the post-closure evolution of a geological disposal facility for radioactive wastes and to affect the solubility of some radionuclides. Furthermore, their potential to reduce both Fe(III) and radionuclides can be harnessed for the bioremediation of radionuclide-contaminated land. As some such sites are likely to have significant radiation fluxes, there is a need to characterise the impact of radiation stress on such microorganisms. There have, however, been few global cell analyses on the impact of ionizing radiation on subsurface bacteria, so here we address the metabolic response of S. oneidensis MR-1 to acute doses of X-radiation. UV/Vis spectroscopy and CFU counts showed that although X-radiation decreased initial viability and extended the lag phase of batch cultures, final biomass yields remained unchanged. FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines. MALDI-TOF-MS detected an increase in total protein expression in cultures exposed to 12 Gy. At 95 Gy, a decrease in total protein levels was generally observed, although an increase in a putative cold shock protein was observed, which may be related to the radiation stress response of this organism. Multivariate statistical analyses applied to these FT-IR and MALDI-TOF-MS spectral data suggested that an irradiated phenotype developed throughout subsequent generations. This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response. Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear.

  19. Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation

    PubMed Central

    Brown, Ashley R.; Correa, Elon; Xu, Yun; AlMasoud, Najla; Pimblott, Simon M.; Goodacre, Royston; Lloyd, Jonathan R.

    2015-01-01

    Biogeochemical processes mediated by Fe(III)-reducing bacteria such as Shewanella oneidensis have the potential to influence the post-closure evolution of a geological disposal facility for radioactive wastes and to affect the solubility of some radionuclides. Furthermore, their potential to reduce both Fe(III) and radionuclides can be harnessed for the bioremediation of radionuclide-contaminated land. As some such sites are likely to have significant radiation fluxes, there is a need to characterise the impact of radiation stress on such microorganisms. There have, however, been few global cell analyses on the impact of ionizing radiation on subsurface bacteria, so here we address the metabolic response of S. oneidensis MR-1 to acute doses of X-radiation. UV/Vis spectroscopy and CFU counts showed that although X-radiation decreased initial viability and extended the lag phase of batch cultures, final biomass yields remained unchanged. FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines. MALDI-TOF-MS detected an increase in total protein expression in cultures exposed to 12 Gy. At 95 Gy, a decrease in total protein levels was generally observed, although an increase in a putative cold shock protein was observed, which may be related to the radiation stress response of this organism. Multivariate statistical analyses applied to these FT-IR and MALDI-TOF-MS spectral data suggested that an irradiated phenotype developed throughout subsequent generations. This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response. Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear. PMID

  20. Probing regulon of ArcA in Shewanella oneidensis MR-1 by integrated genomic analyses

    SciTech Connect

    Gao, Haichun; Wang, Xiaohu; Yang, Zamin Koo; Palzkill, Timothy; Zhou, Jizhong

    2008-01-01

    The Arc two-component system is a global regulator controlling many genes involved in aerobic/anaerobic respiration and fermentative metabolism in Escherichia coli. Shewanella oneidensis MR-1 contains a gene encoding a putative ArcA homolog with {approx} 81% amino acid sequence identity to the E. coli ArcA protein but not a full-length arcB gene. To understand the role of ArcA in S. oneidensis, an arcA deletion strain was constructed and subjected to both physiological characterization and microarray analysis. Compared to the wild-type MR-1, the mutant exhibited impaired aerobic growth and a defect in utilizing DMSO in the absence of O{sub 2}. Microarray analyses on cells grown aerobically and anaerobically on fumarate revealed that expression of 1009 genes was significantly affected (p < 0.05) by the mutation. In contrast to E. coli ArcA, the protein appears to be dispensable in regulation of the TCA cycle in S. oneidensis. To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map. By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in E. coli. These results indicate that the Arc system in S. oneidensis differs from that in E. coli substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.

  1. Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals a previously uncharacterized machinery for lactate utilization

    PubMed Central

    Pinchuk, Grigory E.; Rodionov, Dmitry A.; Yang, Chen; Li, Xiaoqing; Osterman, Andrei L.; Dervyn, Etienne; Geydebrekht, Oleg V.; Reed, Samantha B.; Romine, Margaret F.; Collart, Frank R.; Scott, James H.; Fredrickson, Jim K.; Beliaev, Alexander S.

    2009-01-01

    The ability to use lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal-reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial d- or l-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. By using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO_1522–SO_1518) containing lactate permease and candidate genes for both d- and l-lactate dehydrogenase enzymes. The predicted d-LDH gene (dld-II, SO_1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted l-LDH is encoded by 3 genes with previously unknown functions (lldEGF, SO_1520–SO_1518). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dld-II and lldEFG encode fully functional d-and l-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is a previously uncharacterized example of a multisubunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld-II in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism. PMID:19196979

  2. Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals previously uncharacterized machinery for lactate utilization

    SciTech Connect

    Pinchuk, Grigoriy E.; Rodionov, Dmitry A.; Yang, Chen; Li, Xiaoqing; Osterman, Andrei L.; Dervyn, Etienne; Geydebrekht, Oleg V.; Reed, Samantha B.; Romine, Margaret F.; Collart, Frank R.; Scott, J.; Fredrickson, Jim K.; Beliaev, Alex S.

    2009-02-24

    The ability to utilize lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial D- or L-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. Using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO1522-SO1518) containing lactate permease and candidate genes for both D- and L-lactate dehydrogenase enzymes. The predicted D-LDH gene (dldD, SO1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted L-LDH is encoded by three genes with previously unknown functions (lldEGF, SO1520-19-18). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dldD and lldEFG encode fully functional D-and L-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is the first described example of a multi-subunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.

  3. Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Acute Chromate Challenge

    SciTech Connect

    Thompson, Melissa R; Verberkmoes, Nathan C; Chourey, Karuna; Shah, Manesh B; Thompson, Dorothea K; Hettich, Robert {Bob} L

    2007-01-01

    Proteome alterations in the metal-reducing bacterium Shewanella oneidensis MR-1 in response to different acute dose challenges (0.3, 0.5, or 1 mM) of the toxic metal chromate [Cr(VI)] were characterized with multidimensional HPLC-MS/MS on a linear trapping quadrupole MS. A total of 2,406 functionally diverse proteins were identified, with a subset demonstrating dosage-dependent up- and down-regulated expression, such as proteins involved in detoxification and iron binding and transport.

  4. The octaheme SirA catalyses dissimilatory sulfite reduction in Shewanella oneidensis MR-1

    SciTech Connect

    Shirodkar, Sheetal; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad

    2011-01-01

    Shewanella oneidensis MR-1 is a metal reducer that uses a large number of electron acceptors that include thiosulfate, polysulfide, and sulfite. The enzyme required for thiosulfate and polysulfide respiration has been recently identified, but the mechanisms of sulfite reduction remained unexplored. Analysis of MR-1 cultures grown anaerobically with sulfite suggested that the dissimilatory sulfite reductase catalyzes six-electron reduction of sulfite to sulfide. Reduction of sulfite required menaquinones and c cytochromes but appeared to be independent of the intermediate electron carrier CymA. Furthermore, the terminal sulfite reductase, SirA, was identified as an octaheme c cytochrome with an atypical heme binding site that represents a new class of sulfite reductases. The sirA locus was identified in the genomes of several sequenced Shewanella genomes, and its presence appears to be linked to the ability of these organisms to reduce sulfite under anaerobic conditions.

  5. SO2426 is a positive regulator of siderophore expression in Shewanella oneidensis MR-1

    PubMed Central

    2011-01-01

    Background The Shewanella oneidensis MR-1 genome encodes a predicted orphan DNA-binding response regulator, SO2426. Previous studies with a SO2426-deficient MR-1 strain suggested a putative functional role for SO2426 in the regulation of iron acquisition genes, in particular, the siderophore (hydroxamate) biosynthesis operon so3030-3031-3032. To further investigate the functional role of SO2426 in iron homeostasis, we employed computational strategies to identify putative gene targets of SO2426 regulation and biochemical approaches to validate the participation of SO2426 in the control of siderophore biosynthesis in S. oneidensis MR-1. Results In silico prediction analyses revealed a single 14-bp consensus motif consisting of two tandem conserved pentamers (5'-CAAAA-3') in the upstream regulatory regions of 46 genes, which were shown previously to be significantly down-regulated in a so2426 deletion mutant. These genes included so3030 and so3032, members of an annotated siderophore biosynthetic operon in MR-1. Electrophoretic mobility shift assays demonstrated that the SO2426 protein binds to its motif in the operator region of so3030. A "short" form of SO2426, beginning with a methionine at position 11 (M11) of the originally annotated coding sequence for SO2426, was also functional in binding to its consensus motif, confirming previous 5' RACE results that suggested that amino acid M11 is the actual translation start codon for SO2426. Alignment of SO2426 orthologs from all sequenced Shewanella spp. showed a high degree of sequence conservation beginning at M11, in addition to conservation of a putative aspartyl phosphorylation residue and the helix-turn-helix (HTH) DNA-binding domain. Finally, the so2426 deletion mutant was unable to synthesize siderophores at wild-type rates upon exposure to the iron chelator 2,2'-dipyridyl. Conclusions Collectively, these data support the functional characterization of SO2426 as a positive regulator of siderophore-mediated iron

  6. Purification and characterization of the [NiFe]-hydrogenase of Shewanella oneidensis MR-1.

    PubMed

    Shi, Liang; Belchik, Sara M; Plymale, Andrew E; Heald, Steve; Dohnalkova, Alice C; Sybirna, Kateryna; Bottin, Hervé; Squier, Thomas C; Zachara, John M; Fredrickson, James K

    2011-08-15

    Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H(2)ase) that has been implicated in H(2) production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H(2)ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H(2)ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H(2)ase in trans restored the mutant's ability to produce H(2) at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H(2)ase coupled H(2) oxidation to reduction of Tc(VII)O(4)(-) and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H(2)ase-mediated reduction of Tc(VII)O(4)(-) but not methyl viologen. Under the conditions tested, all Tc(VII)O(4)(-) used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O(4)(-) was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ∼5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O(2)·nH(2)O, which was also the product of Tc(VII)O(4)(-) reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H(2)ase catalyzes Tc(VII)O(4)(-) reduction directly by coupling to H(2) oxidation.

  7. Role of electricity production in the anaerobic decolorization of dye mixture by exoelectrogenic bacterium Shewanella oneidensis MR-1.

    PubMed

    Cao, Dan-Ming; Xiao, Xiang; Wu, Yong-Min; Ma, Xiao-Bo; Wang, Ming-Na; Wu, Yan-You; Du, Dao-Lin

    2013-05-01

    This study investigated the anaerobic decolorization of the dye mixture containing methyl orange (MO) and naphthol green B (NGB) by Shewanella oneidensis MR-1. S. oneidensis MR-1 showed a strong ability to decolorize the dye mixture. MO was easier to get the electrons and inhibited the reduction of NGB, despite of its lower redox potential than NGB. The Mtr respiratory pathway played an important role in this process. Meantime, addition of extracellular electron shuttles accelerated the decolorization. Those results suggest that the decolorization capacity of S. oneidensis MR-1 is associated with the electricity production. The operating parameters, such as electron acceptors, temperature, and pH, were also investigated in this study. Thus, this work may facilitate a better understanding of the extensive nonspecific reduction capacity of exoelectrogens and is beneficial for promoting their application in bioremediation.

  8. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    PubMed Central

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  9. A Ferrous Iron Exporter Mediates Iron Resistance in Shewanella oneidensis MR-1

    PubMed Central

    Bennett, Brittany D.; Brutinel, Evan D.

    2015-01-01

    Shewanella oneidensis strain MR-1 is a dissimilatory metal-reducing bacterium frequently found in aquatic sediments. In the absence of oxygen, S. oneidensis can respire extracellular, insoluble oxidized metals, such as iron (hydr)oxides, making it intimately involved in environmental metal and nutrient cycling. The reduction of ferric iron (Fe3+) results in the production of ferrous iron (Fe2+) ions, which remain soluble under certain conditions and are toxic to cells at higher concentrations. We have identified an inner membrane protein in S. oneidensis, encoded by the gene SO_4475 and here called FeoE, which is important for survival during anaerobic iron respiration. FeoE, a member of the cation diffusion facilitator (CDF) protein family, functions to export excess Fe2+ from the MR-1 cytoplasm. Mutants lacking feoE exhibit an increased sensitivity to Fe2+. The export function of FeoE is specific for Fe2+, as an feoE mutant is equally sensitive to other metal ions known to be substrates of other CDF proteins (Cd2+, Co2+, Cu2+, Mn2+, Ni2+, or Zn2+). The substrate specificity of FeoE differs from that of FieF, the Escherichia coli homolog of FeoE, which has been reported to be a Cd2+/Zn2+ or Fe2+/Zn2+ exporter. A complemented feoE mutant has an increased growth rate in the presence of excess Fe2+ compared to that of the ΔfeoE mutant complemented with fieF. It is possible that FeoE has evolved to become an efficient and specific Fe2+ exporter in response to the high levels of iron often present in the types of environmental niches in which Shewanella species can be found. PMID:26341213

  10. Reconstruction of Extracellular Respiratory Pathways for Iron(III) Reduction in Shewanella Oneidensis Strain MR-1

    PubMed Central

    Coursolle, Dan; Gralnick, Jeffrey A.

    2012-01-01

    Shewanella oneidensis strain MR-1 is a facultative anaerobic bacterium capable of respiring a multitude of electron acceptors, many of which require the Mtr respiratory pathway. The core Mtr respiratory pathway includes a periplasmic c-type cytochrome (MtrA), an integral outer-membrane β-barrel protein (MtrB), and an outer-membrane-anchored c-type cytochrome (MtrC). Together, these components facilitate transfer of electrons from the c-type cytochrome CymA in the cytoplasmic membrane to electron acceptors at and beyond the outer-membrane. The genes encoding these core proteins have paralogs in the S. oneidensis genome (mtrB and mtrA each have four while mtrC has three) and some of the paralogs of mtrC and mtrA are able to form functional Mtr complexes. We demonstrate that of the additional three mtrB paralogs found in the S. oneidensis genome, only MtrE can replace MtrB to form a functional respiratory pathway to soluble iron(III) citrate. We also evaluate which mtrC/mtrA paralog pairs (a total of 12 combinations) are able to form functional complexes with endogenous levels of mtrB paralog expression. Finally, we reconstruct all possible functional Mtr complexes and test them in a S. oneidensis mutant strain where all paralogs have been eliminated from the genome. We find that each combination tested with the exception of MtrA/MtrE/OmcA is able to reduce iron(III) citrate at a level significantly above background. The results presented here have implications toward the evolution of anaerobic extracellular respiration in Shewanella and for future studies looking to increase the rates of substrate reduction for water treatment, bioremediation, or electricity production. PMID:22363330

  11. Expression of a tetraheme protein, Desulfovibrio vulgaris Miyazaki F cytochrome c(3), in Shewanella oneidensis MR-1

    NASA Technical Reports Server (NTRS)

    Ozawa, K.; Tsapin, A. I.; Nealson, K. H.; Cusanovich, M. A.; Akutsu, H.

    2000-01-01

    Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome c(3) gene from D. vulgaris Miyazaki F was used for transformation of S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine residue at the N terminus.

  12. Expression of a tetraheme protein, Desulfovibrio vulgaris Miyazaki F cytochrome c(3), in Shewanella oneidensis MR-1.

    PubMed

    Ozawa, K; Tsapin, A I; Nealson, K H; Cusanovich, M A; Akutsu, H

    2000-09-01

    Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome c(3) gene from D. vulgaris Miyazaki F was used for transformation of S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine residue at the N terminus.

  13. Global Transcriptome Analysis of Shewanella oneidensis MR-1 Exposed to Different Terminal Electron Acceptors

    SciTech Connect

    Beliaev, Alex S.; Klingeman, Dawn M.; Klappenbach, Joel; Wu, Liyou; Romine, Margaret F.; Tiedje, James M.; Nealson, Kenneth H.; Fredrickson, Jim K.; Zhou, Jizhong

    2005-10-01

    To gain insight into the complex structure of the energy-generating networks in the dissimilatory metal reducer Shewanella oneidensis MR-1, global mRNA patterns were examined in cells exposed to a wide range of metal and non-metal electron acceptors. Gene expression patterns were similar irrespective of which metal ion was used as electron acceptor, with 60% of the differentially expressed genes showing similar induction or repression relative to fumarate- respiring conditions. Several groups of genes exhibited elevated expression levels in the presence of metals, including those encoding putative multidrug efflux transporters, detoxification proteins, extracytoplasmic sigma factors and PAS-domain regulators. Only one of the 42 predicted c-type cytochromes in MR-1, SO3300, displayed significantly elevated transcript levels across all metal-reducing conditions. Genes encoding decaheme cytochromes MtrC and MtrA that were previously linked to the reduction of different forms of Fe(III) and Mn(IV), exhibited only slight decreases in relative mRNA abundances under metal-reducing conditions. In contrast, specific transcriptome responses were displayed to individual non-metal electron acceptors resulting in the identification of unique groups of nitrate-, thiosulfate- and TMAO-induced genes including previously uncharacterized multi-cytochrome gene clusters. Collectively, the gene expression results reflect the fundamental differences between metal and non-metal respiratory pathways of S. oneidensis MR-1, where the coordinate induction of detoxification and stress response genes play a key role in adaptation of this organism under metal-reducing conditions. Moreover, the relative paucity and/or the constitutive nature of genes involved in electron transfer to metals is likely due to the low-specificity and the opportunistic nature of the metal-reducing electron transport pathways.

  14. The Role of 4-Hydroxyphenylpyruvate Dioxygenase in Enhancement of Solid-Phase Electron Transfer by Shewanella oneidensis MR-1

    SciTech Connect

    Turick, Charles E.; Beliaev, Alex S.; Zakrajsek, Brian A.; Reardon, Catherine L.; Lowy, Daniel A.; Poppy, Tara E.; Maloney, Andrea; Ekechukwu, Amy A.

    2009-05-01

    ABSTRACT - While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane associated c-type cytochromes and electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of the tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione ([2-(2- chloro- 4- methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA, which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates at which MR-1 reduces hydrous ferric oxide were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E°') of S. oneidensis MR-1. Based on our findings, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in S. oneidensis MR-1.

  15. Identification of Mobile Elements and Pseudogenes in the Shewanella oneidensis MR-1 Genome

    SciTech Connect

    Romine, Margaret F.; Carlson, Timothy; Norbeck, Angela D.; McCue, Lee Ann; Lipton, Mary S.

    2008-05-01

    Shewanella oneidensis MR-1 is the first of 22 different Shewanella spp. whose genomes have been or are being sequenced and thus serves as the model organism for studying the functional repertoire of the Shewanella genus. The original MR-1 genome annotation revealed a large number of transposase genes and pseudogenes, indicating that many of the genome’s functions may be decaying. Comparative analyses of the sequenced Shewanella strains suggest that 209 genes in MR-1 have in-frame stop codons, frameshifts, or interruptions and/or are truncated and that 65 of the original pseudogene predictions were erroneous. Among the decaying functions are that of one of three chemotaxis clusters, type I pilus production, starch utilization, and nitrite respiration. Many of the mutations could be attributed to members of 41 different types of insertion sequence (IS) elements and three types of miniature inverted-repeat transposable elements identified here for the first time. The high copy numbers of individual mobile elements (up to 71) are expected to promote large-scale genome recombination events, as evidenced by the displacement of the algA promoter. The ability of MR-1 to acquire foreign genes via reactions catalyzed by both the integron integrase and the ISSod25-encoded integrases is suggested by the presence of attC sites and genes whose sequences are characteristic of other species downstream of each site. This large number of mobile elements and multiple potential sites for integrasemediated acquisition of foreign DNA indicate that the MR-1 genome is exceptionally dynamic, with many functions and regulatory control points in the process of decay or reinvention.

  16. Growth Inhibition and Stimulation of Shewanella oneidensis MR-1 by Surfactants and Calcium Polysulfide

    SciTech Connect

    Bailey, Kathryn L.; Tilton, Fred A.; Jansik, Danielle P.; Ergas, Sarina J.; Marshall, Matthew J.; Miracle, Ann L.; Wellman, Dawn M.

    2012-06-14

    Foam delivery technology (FDT) uses surfactant based foam to immobilize subsurface contaminants in situ. Where traditional approaches are impractical, FDT has the potential to overcome many of the technical challenges facing the remediation of contaminated deep vadose zone environments. However, little is known about the effects these reactive chemicals may have on microorganisms inhabiting the contaminated subsurface. In addition, there are currently no standard assays to assess microbial responses to subsurface remedial treatments while these agents are under development. The objective of this study was to develop a rapid laboratory assay to assess the potential growth inhibition and/or stimulation of microorganisms following exposure to candidate FDT components. Calcium polysulfide (CPS) and several surfactants (i.e. sodium laureth sulfate (SLES), sodium dodecyl sulfate (SDS), cocamidopropyl betaine (CAPB) and NINOL40-CO) have diverse chemistries and are candidate components of FDT. Shewanella oneidensis MR-1 cultures were exposed to a range of concentrations of these chemicals to determine the minimum bactericidal concentration (MBC) and the growth and viability potential of these components. Concentrations of SDS higher than 700 {micro}M were toxic to S. oneidensis MR-1 growth over the course of four days of exposure. The relative acute toxicity order for these compounds was SDS>>CPS>>NINOL40-CO>SLES-CAPB. Dose dependent growth decreases (20 to 100 mM) were observed in the CAPB and SLES treated cultures and both CPS and NINOL 40-CO were toxic at all concentrations tested (1.45 to 7.25 mM CPS). Both SLES (20 to 100 mM) and SDS at lower concentrations (20 to 500 {micro}M) were stimulatory to S. oneidensis MR-1 indicating a capacity to be used as a carbon source. These studies also identified potentially key component characteristics, such as precipitate formation and oxygen availability, which may prove valuable in assessing the response of subsurface

  17. Growth inhibition and stimulation of Shewanella oneidensis MR-1 by surfactants and calcium polysulfide.

    PubMed

    Bailey, Kathryn L; Tilton, Fred; Jansik, Danielle P; Ergas, Sarina J; Marshall, Matthew J; Miracle, Ann L; Wellman, Dawn M

    2012-06-01

    Foam delivery technology (FDT) uses surfactant based foam to immobilize subsurface contaminants in situ. Where traditional approaches are impractical, FDT has the potential to overcome many of the technical challenges facing the remediation of contaminated deep vadose zone environments. However, little is known about the effects these reactive chemicals may have on microorganisms inhabiting the contaminated subsurface. In addition, there are currently no standard assays to assess microbial responses to subsurface remedial treatments while these agents are under development. The objective of this study was to develop a rapid laboratory assay to assess the potential growth inhibition and/or stimulation of microorganisms following exposure to candidate FDT components. Calcium polysulfide (CPS) and several surfactants (i.e. sodium laureth sulfate (SLES), sodium dodecyl sulfate (SDS), cocamidopropyl betaine (CAPB) and NINOL40-CO) have diverse chemistries and are candidate components of FDT. Shewanella oneidensis MR-1 cultures were exposed to a range of concentrations of these chemicals to determine the minimum bactericidal concentration (MBC) and the growth and viability potential of these components. Concentrations of SDS higher than 700 μM were toxic to S. oneidensis MR-1 growth over the course of four days of exposure. The relative acute toxicity order for these compounds was SDS > CPS > NINOL 40-CO>SLES≥CAPB. Dose dependent growth decreases (20-100mM) were observed in the CAPB and SLES treated cultures and both CPS and NINOL 40-CO were toxic at all concentrations tested (1.45-7.25 mM CPS). Both SLES (20-100mM) and SDS at lower concentrations (20-500 μM) were stimulatory to S. oneidensis MR-1 indicating a capacity to be used as a carbon source. These studies also identified potentially key component characteristics, such as precipitate formation and oxygen availability, which may prove valuable in assessing the response of subsurface microorganisms. This benchtop

  18. Decolorization and detoxification of a sulfonated triphenylmethane dye aniline blue by Shewanella oneidensis MR-1 under anaerobic conditions.

    PubMed

    Wu, Yongmin; Xiao, Xiang; Xu, Cancan; Cao, Danming; Du, Daolin

    2013-08-01

    In this work, the extracellular decolorization of aniline blue, a sulfonated triphenylmethane dye, by Shewanella oneidensis MR-1 was confirmed. S. oneidensis MR-1 showed a high capacity for decolorizing aniline blue even at a concentration of up to 1,000 mg/l under anaerobic conditions. Maximum decolorization efficiency appeared at pH 7.0 and 30 °C. Lactate was a better candidate of electron donor for the decolorization of aniline blue. The addition of nitrate, hydrous ferric oxide, or trimethylamine N-oxide all could cause a significant decline of decolorization efficiency. The Mtr respiratory pathway was found to be involved into the decolorization of aniline blue by S. oneidensis MR-1. The toxicity evaluation through phytotoxicity and genotoxicity showed that S. oneidensis MR-1 could decrease the toxicity of aniline blue during the decolorization process. Thus, this work may facilitate a better understanding on the degradation mechanisms of the triphenylmethane dyes by Shewanella and is beneficial to their application in bioremediation.

  19. THE ROLE OF 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE IN ENHANCEMENT OF SOLID-PHASE ELECTRON TRANSFER BY SHEWANELLA ONEIDENSIS MR-1

    SciTech Connect

    Turick, C; Amy Ekechukwu, A

    2007-06-01

    While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane-associated c-type cytochromes and redox active electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. In this study, we determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione (2-(2-chloro-4-methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates, with which MR-1 reduces hydrous ferric oxide, were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E{sup o}{prime}) of S. oneidensis MR-1. Based on this work, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in Shewanella oneidensis.

  20. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms.

    PubMed

    Gorby, Yuri A; Yanina, Svetlana; McLean, Jeffrey S; Rosso, Kevin M; Moyles, Dianne; Dohnalkova, Alice; Beveridge, Terry J; Chang, In Seop; Kim, Byung Hong; Kim, Kyung Shik; Culley, David E; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Hill, Eric A; Shi, Liang; Elias, Dwayne A; Kennedy, David W; Pinchuk, Grigoriy; Watanabe, Kazuya; Ishii, Shun'ichi; Logan, Bruce; Nealson, Kenneth H; Fredrickson, Jim K

    2006-07-25

    Shewanella oneidensis MR-1 produced electrically conductive pilus-like appendages called bacterial nanowires in direct response to electron-acceptor limitation. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA, and those that lacked a functional Type II secretion pathway displayed nanowires that were poorly conductive. These mutants were also deficient in their ability to reduce hydrous ferric oxide and in their ability to generate current in a microbial fuel cell. Nanowires produced by the oxygenic phototrophic cyanobacterium Synechocystis PCC6803 and the thermophilic, fermentative bacterium Pelotomaculum thermopropionicum reveal that electrically conductive appendages are not exclusive to dissimilatory metal-reducing bacteria and may, in fact, represent a common bacterial strategy for efficient electron transfer and energy distribution.

  1. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms

    PubMed Central

    Gorby, Yuri A.; Yanina, Svetlana; McLean, Jeffrey S.; Rosso, Kevin M.; Moyles, Dianne; Dohnalkova, Alice; Beveridge, Terry J.; Chang, In Seop; Kim, Byung Hong; Kim, Kyung Shik; Culley, David E.; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad A.; Hill, Eric A.; Shi, Liang; Elias, Dwayne A.; Kennedy, David W.; Pinchuk, Grigoriy; Watanabe, Kazuya; Ishii, Shun’ichi; Logan, Bruce; Nealson, Kenneth H.; Fredrickson, Jim K.

    2006-01-01

    Shewanella oneidensis MR-1 produced electrically conductive pilus-like appendages called bacterial nanowires in direct response to electron-acceptor limitation. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA, and those that lacked a functional Type II secretion pathway displayed nanowires that were poorly conductive. These mutants were also deficient in their ability to reduce hydrous ferric oxide and in their ability to generate current in a microbial fuel cell. Nanowires produced by the oxygenic phototrophic cyanobacterium Synechocystis PCC6803 and the thermophilic, fermentative bacterium Pelotomaculum thermopropionicum reveal that electrically conductive appendages are not exclusive to dissimilatory metal-reducing bacteria and may, in fact, represent a common bacterial strategy for efficient electron transfer and energy distribution. PMID:16849424

  2. Shewanella oneidensis MR-1 sensory box protein involved in aerobic and anoxic growth.

    PubMed

    Sundararajan, A; Kurowski, J; Yan, T; Klingeman, D M; Joachimiak, M P; Zhou, J; Naranjo, B; Gralnick, J A; Fields, M W

    2011-07-01

    Although little is known of potential function for conserved signaling proteins, it is hypothesized that such proteins play important roles to coordinate cellular responses to environmental stimuli. In order to elucidate the function of a putative sensory box protein (PAS domains) in Shewanella oneidensis MR-1, the physiological role of SO3389 was characterized. The predicted open reading frame (ORF) encodes a putative sensory box protein that has PAS, GGDEF, and EAL domains, and an in-frame deletion mutant was constructed (ΔSO3389) with approximately 95% of the ORF deleted. Under aerated conditions, wild-type and mutant cultures had similar growth rates, but the mutant culture had a lower growth rate under static, aerobic conditions. Oxygen consumption rates were lower for mutant cultures (1.5-fold), and wild-type cultures also maintained lower dissolved oxygen concentrations under aerated growth conditions. When transferred to anoxic conditions, the mutant did not grow with fumarate, iron(III), or dimethyl sulfoxide (DMSO) as electron acceptors. Biochemical assays demonstrated the expression of different c-type cytochromes as well as decreased fumarate reductase activity in the mutant transferred to anoxic growth conditions. Transcriptomic studies showed the inability of the mutant to up-express and down-express genes, including c-type cytochromes (e.g., SO4047/SO4048, SO3285/SO3286), reductases (e.g., SO0768, SO1427), and potential regulators (e.g., SO1329). The complemented strain was able to grow when transferred from aerobic to anoxic growth conditions with the tested electron acceptors. The modeled structure for the SO3389 PAS domains was highly similar to the crystal structures of FAD-binding PAS domains that are known O2/redox sensors. Based on physiological, genomic, and bioinformatic results, we suggest that the sensory box protein, SO3389, is an O2/redox sensor that is involved in optimization of aerobic growth and transitions to anoxia in S

  3. Extracellular respiration of dimethyl sulfoxide by Shewanella oneidensis strain MR-1

    PubMed Central

    Gralnick, Jeffrey A.; Vali, Hojatollah; Lies, Douglas P.; Newman, Dianne K.

    2006-01-01

    Shewanella species are renowned for their respiratory versatility, including their ability to respire poorly soluble substrates by using enzymatic machinery that is localized to the outside of the cell. The ability to engage in “extracellular respiration” to date has focused primarily on respiration of minerals. Here, we identify two gene clusters in Shewanella oneidensis strain MR-1 that each contain homologs of genes required for metal reduction and genes that are predicted to encode dimethyl sulfoxide (DMSO) reductase subunits. Molecular and genetic analyses of these clusters indicate that one (SO1427–SO1432) is required for anaerobic respiration of DMSO. We show that DMSO respiration is an extracellular respiratory process through the analysis of mutants defective in type II secretion, which is required for transporting proteins to the outer membrane in Shewanella. Moreover, immunogold labeling of DMSO reductase subunits reveals that they reside on the outer leaflet of the outer membrane under anaerobic conditions. The extracellular localization of the DMSO reductase in S. oneidensis suggests these organisms may perceive DMSO in the environment as an insoluble compound. PMID:16537430

  4. Extracellular respiration of dimethyl sulfoxide by Shewanella oneidensis strain MR-1.

    PubMed

    Gralnick, Jeffrey A; Vali, Hojatollah; Lies, Douglas P; Newman, Dianne K

    2006-03-21

    Shewanella species are renowned for their respiratory versatility, including their ability to respire poorly soluble substrates by using enzymatic machinery that is localized to the outside of the cell. The ability to engage in "extracellular respiration" to date has focused primarily on respiration of minerals. Here, we identify two gene clusters in Shewanella oneidensis strain MR-1 that each contain homologs of genes required for metal reduction and genes that are predicted to encode dimethyl sulfoxide (DMSO) reductase subunits. Molecular and genetic analyses of these clusters indicate that one (SO1427-SO1432) is required for anaerobic respiration of DMSO. We show that DMSO respiration is an extracellular respiratory process through the analysis of mutants defective in type II secretion, which is required for transporting proteins to the outer membrane in Shewanella. Moreover, immunogold labeling of DMSO reductase subunits reveals that they reside on the outer leaflet of the outer membrane under anaerobic conditions. The extracellular localization of the DMSO reductase in S. oneidensis suggests these organisms may perceive DMSO in the environment as an insoluble compound.

  5. Molecular Underpinnings of Fe(III) Oxide Reduction by Shewanella oneidensis MR-1

    SciTech Connect

    Shi, Liang; Rosso, Kevin M.; Clarke, Thomas A.; Richardson, David J.; Zachara, John M.; Fredrickson, Jim K.

    2012-02-15

    In the absence of O2 and other electron acceptors, the Gram-negative bacterium Shewanella oneidensis MR-1 can use ferric [Fe(III)] (oxy)(hydr)oxide minerals as the terminal electron acceptors for anaerobic respiration. At circumneutral pH and in the absence of strong complexing ligands, Fe(III) oxides are relatively insoluble and thus are external to the bacterial cells. S. oneidensis MR-1 and related strains of metal-reducing Shewanella have evolved the machinery (i.e., metal-reducing or Mtr pathway) for transferring electrons from the inner-membrane, through the periplasm and across the outer-membrane to the surface of extracellular Fe(III) oxides. The protein components identified to date for the Mtr pathway include CymA, MtrA, MtrB, MtrC and OmcA. CymA is an inner-membrane tetraheme c-type cytochrome (c-Cyt) that belongs to the NapC/NrfH family of quinol dehydrogenases. It is proposed that CymA oxidizes the quinol in the inner-membrane and transfers the released electrons to redox proteins in the periplasm. Although the periplasmic proteins receiving electrons from CymA during Fe(III) oxidation have not been identified, they are believed to relay the electrons in the periplasm to MtrA. A decaheme c-Cyt, MtrA is thought to be embedded in the trans outer-membrane and porin-like protein MtrB. Together, MtrAB deliver the electrons through the outer-membrane to the MtrC and OmcA on the outmost bacterial surface. MtrC and OmcA are the outer-membrane decaheme c-Cyts that are translocated across the outer-membrane by the bacterial type II secretion system. Functioning as terminal reductases, MtrC and OmcA can bind the surface of Fe(III) oxides and transfer electrons directly to these minerals via their solvent-exposed hemes. To increase their reaction rates, MtrC and OmcA can use the flavins secreted by S. oneidensis MR-1 cells as diffusible co-factors for reduction of Fe(III) oxides. Because of their extracellular location and broad redox potentials, MtrC and OmcA can

  6. Integrated Analysis of Protein Complexes and Regulatory Networks Involved in Anaerobic Energy Metabolism of Shewanella Oneidensis MR-1

    SciTech Connect

    Tiedje, James M.

    2005-06-01

    Anaerobic Nitrate Reduction. Nitrate is an extensive co-contaminant at some DOE sites making metal and radionuclide reduction problematic. Hence, we sought to better understand the nitrate reduction pathway and its control in S. oneidensis MR-1. It is not known whether the nitrate reduction is by denitrification or dissimilatory nitrate reduction into ammonium (DNRA). By both physiological and genetic evidence, we proved that DNRA is the nitrate reduction pathway in this organism. Using the complete genome sequence of S. oneidensis MR-1, we identified a gene encoding a periplasmic nitrate reductase based on its 72% sequence identity with the napA gene in E. coli. Anaerobic growth of MR-1 on nitrate was abolished in a site directed napA mutant, indicating that NapA is the only nitrate reductase present. The anaerobic expression of napA and nrfA, a homolog of the cytochrome b552 nitrite reductase in E. coli, increased with increasing nitrate concentration until a plateau was reached at 3 mM KNO3. This indicates that these genes are not repressed by increasing concentrations of nitrate. The reduction of nitrate can generate intermediates that can be toxic to the microorganism. To determine the genetic response of MR-1 to high concentrations of nitrate, DNA microarrays were used to obtain a complete gene expression profile of MR-1 at low (1 mM) versus high (40 mM) nitrate concentrations. Genes encoding transporters and efflux pumps were up-regulated, perhaps as a mechanism to export toxic compounds. In addition, the gene expression profile of MR-1, grown anaerobically with nitrate as the only electron acceptor, suggested that this dissimilatory pathway contributes to N assimilation. Hence the nitrate reduction pathway could serve a dual purpose. The role of EtrA, a homolog of Fnr (global anaerobic regulator in E. coli) was examined using an etrA deletion mutant we constructed, S. oneidensis EtrA7-1.

  7. Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1

    PubMed Central

    Clark, Iain C.; Melnyk, Ryan A.; Youngblut, Matthew D.; Carlson, Hans K.; Iavarone, Anthony T.

    2015-01-01

    ABSTRACT Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. PMID:25991681

  8. Investigations of Structure and Metabolism within Shewanella oneidensis MR-1 Biofilms

    SciTech Connect

    Mclean, Jeffrey S.; Majors, Paul D.; Reardon, Catherine L.; Bilskis, Christina L.; Reed, Samantha B.; Romine, Margaret F.; Fredrickson, Jim K.

    2008-07-01

    Biofilms are known to possess spatially and temporally varying metabolite concentration profiles at the macroscopic and microscopic scales. This results in varying growth environments within that may ultimately drive species diversity, determine biofilm structure and also the spatial arrangement of the community members. Using noninvasive nuclear magnetic resonance (NMR) microscopic imaging/spectroscopy and confocal imaging, we investigated anaerobic reduction kinetics, structural variation, and the stratification of metabolism within live biofilms of the facultative anaerobic dissimilatory metal-reducing Shewanella oneidensis strain MR-1. Biofilms were pregrown using a defined minimal media in a homebuilt constant depth film fermenter and subsequently transferred to an in-magnet sample chamber under laminar flow for NMR measurements. The sample was subjected to various, rapidly switched substrate/ anaerobic electron acceptor combinations (fumarate, dimethyl sulfoxide, and nitrate electron acceptors). Localized NMR spectroscopy was used to non-invasively monitored the spectra of hydrogen-containing metabolites at high temporal resolution (4.5 min) under oxygen-limited conditions. Anaerobic reduction was immediately observed upon switching feed solutions indicate that no gene induction (transcriptional response) was needed for MR-1 to switch between fumarate, dimethyl sulfoxide (DMSO) and nitrate electron acceptors. In parallel experiments, confocal microscopy was used with constitutively expressed fluorescent reporters to independently investigate structural changes in response to the availability of electron acceptor and also the outcome of metabolic competition under oxygen-limited conditions. A clearer understanding of the metabolic diversity and plasticity of the biofilm mode of growth as well as how this possibly translates to the environmental fitness is made possible through the use of non-invasive and non-destructive techniques such as described here.

  9. The roles of CymA in support of the respiratory flexibility of Shewanella oneidensis MR-1

    SciTech Connect

    Marritt, Sophie; McMillan, Duncan G.; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Richardson, David J.; Jeuken, Lars J.; Butt, Julea N.

    2012-12-01

    Shewanella species are isolated from the oxic/anoxic regions of seawater and aquatic sediments where redox conditions fluctuate in time and space. Colonization of these environments is by virtue of flexible respiratory chains, many of which are notable for the ability to reduce extracellular substrates including the Fe(III) and Mn(IV) contained in oxide and phyllosilicate minerals. Shewanella oneidensis MR-1 serves as a model organism to consider the biochemical basis of this flexibility. In the present paper, we summarize the various systems that serve to branch the respiratory chain of S. oneidensis MR-1 in order that electrons from quinol oxidation can be delivered the various terminal electron acceptors able to support aerobic and anaerobic growth. This serves to highlight several unanswered questions relating to the regulation of respiratory electron transport in Shewanella and the central role(s) of the tetrahaem-containing quinol dehydrogenase CymA in that process.

  10. Probing Electron Transfer Mechanisms in Shewanella oneidensis MR-1 using a Nanoelectrode Platform and Single-Cell Imaging

    DTIC Science & Technology

    2010-01-01

    investigate extracellu- lar electron transfer in Shewanella oneidensisMR-1,where an array of nanoholes precludes or single window allows for direct...the single-cell level (Fig. 1B) highlights the re- lative sizes of the nanohole and window openings in the insulating layer deposited over electrodes...relative to individual bacteria such as Shewanella. The nanoholes are sufficiently small to preclude direct contact of the bacterial cell body to the

  11. Promoted reduction of tellurite and formation of extracellular tellurium nanorods by concerted reaction between iron and Shewanella oneidensis MR-1.

    PubMed

    Kim, Dong-Hun; Kim, Min-Gyu; Jiang, Shenghua; Lee, Ji-Hoon; Hur, Hor-Gil

    2013-08-06

    The reduction of tellurite (Te(IV)) by dissimilatory metal reducing bacterium, Shewanella oneidensis MR-1, was promoted in the presence of Fe(III) in comparison with Te(IV) bioreduction in the absence of Fe(III). Electron microscopic analyses revealed that iron promoted Te(IV) reduction led to form exclusively extracellular crystalline Te(0) nanorods, as compared to the mostly intracellular formation of Te(0) nanorods in the absence of Fe(III). The Te K-edge X-ray absorption spectrometric analyses demonstrated that S. oneidensis MR-1 in the presence of Fe(III) reduced Te(IV) to less harmful metallic Te(0) nanorods through the precipitation of tellurite (Te(IV)Ox) complex by the bacterial respiration of Fe(III) to Fe(II) under anaerobic conditions. However, Fe(II) ion itself was only able to precipitate the solid tellurite (Te(IV)Ox) complex from the Te(IV) solution, which was not further reduced to Te(0). The results clearly indicated that bacterial S. oneidensis MR-1 plays important roles in the reduction and crystallization of Te(0) nanorods by as yet undetermined biochemical mechanisms. As compared to the slow bacterial Te(IV) reduction in the absence of Fe(III), the rapid reduction of Te(IV) to Te(0) by the concerted biogeochemical reaction between Fe(II) and S. oneidensis MR-1 could be applied for the sequestration and detoxification of Te(IV) in the environments as well as for the preparation of extracellular Te(0) nanorod structures.

  12. Comparison of uranium(VI) removal by Shewanella oneidensis MR-1 in flow and batch reactors

    SciTech Connect

    Sani, Rajesh K.; Peyton, Brent M.; Dohnalkova, Alice

    2008-06-01

    To better understand the interactions among metal contaminants, nutrients, and microorganisms in subsurface under fracture-flow conditions, iron-reducing biofilms (pure cultures of Shewanella oneidensis MR-1) were grown in six fracture flow reactors (FFRs) of different geometries. The spatial and temporal distribution of nutrients, contaminant, and bacteria were examined using a tracer dye (brilliant blue FCF) and microscopy. The results showed that plugging by bacterial cells depended on the geometry of the reactor; and iron-reducing biofilms grown in FFRs had a definite U(VI)-reduction capacity. To find out the U(VI)-reduction capacity of iron-reducing biofilms, batch experiments of U(VI) reduction were performed in repetitive addition mode. U(VI)-reduction rates of stationary phase grown iron-reducing cultures with and without spent medium decreased after each U(VI) addition. At the end of the fourth U(VI)-addition, stationary phase iron-reducing cultures treated with U(VI) with and without spent medium yielded grey and black precipitates, respectively. These grey and black U precipitates were analyzed using High Resolution-Transmission Electron Microscopy, Energy-dispersive X-ray spectroscopy, and X-ray diffraction. Data for randomly selected area of black and grey U precipitates showed that reduced U particles (3-6 nm) were crystalline and amorphous in nature, respectively. This information obtained in this study could be used to develop substrate addition strategies for metal immobilization in subsurface fracture flow systems.

  13. Laboratory investigation of high pressure survival in Shewanella oneidensis MR-1 into the gigapascal pressure range

    PubMed Central

    Hazael, Rachael; Foglia, Fabrizia; Kardzhaliyska, Liya; Daniel, Isabelle; Meersman, Filip; McMillan, Paul

    2014-01-01

    The survival of Shewanella oneidensis MR-1 at up to 1500 MPa was investigated by laboratory studies involving exposure to high pressure followed by evaluation of survivors as the number (N) of colony forming units (CFU) that could be cultured following recovery to ambient conditions. Exposing the wild type (WT) bacteria to 250 MPa resulted in only a minor (0.7 log N units) drop in survival compared with the initial concentration of 108 cells/ml. Raising the pressure to above 500 MPa caused a large reduction in the number of viable cells observed following recovery to ambient pressure. Additional pressure increase caused a further decrease in survivability, with approximately 102 CFU/ml recorded following exposure to 1000 MPa (1 GPa) and 1.5 GPa. Pressurizing samples from colonies resuscitated from survivors that had been previously exposed to high pressure resulted in substantially greater survivor counts. Experiments were carried out to examine potential interactions between pressure and temperature variables in determining bacterial survival. One generation of survivors previously exposed to 1 GPa was compared with WT samples to investigate survival between 37 and 8°C. The results did not reveal any coupling between acquired high pressure resistance and temperature effects on growth. PMID:25452750

  14. Structure of biogenic uraninite produced by Shewanella oneidensis strain MR-1

    SciTech Connect

    Schofield, Eleanor J.; Veeramani, Harish; Sharp, Jonathan; Suvorova, Elena; Bernier-Latmani, Rizlan; Mehta, Apurva; STAHLMAN, JONATHAN O.; Webb, Samuel M.; Clark, David L.; Conradson, Steven D.; Ilton, Eugene S.; Bargar, John R.

    2008-11-01

    The stability of biogenic uraninite with respect to oxidation is seminal to the success of in-situ bioreduction strategies for remediation of subsurface U(VI) contamination. The properties and hence stability of uraninite are dependent on its size, structure and composition. In this study, the local-, intermediate-, and long-range molecular-scale structure of nanoscale uraninite produced by Shewanella oneidensis strain MR-1 was investigated using EXAFS, SR-based powder diffraction and TEM. The uraninite products were found to be structurally homologous with stoichiometric UO2 under all conditions considered. Significantly, there was no evidence for lattice strain of the biogenic uraninite nanoparticles. The fresh nanoparticles were found to exhibit a well-ordered interior core of diameter ca 1 nm and an outer region of thickness ca ~ 1 nm in which the structure is locally distorted. The lack of nanoparticle strain and structural homology with stoichiometric UO2 suggests that established thermodynamic parameters for the latter material are an appropriate starting point to model the behavior of nano-biogenic uraninite. The detailed structural analysis in this study provides an essential foundation for subsequent investigations of more environmentally relevant samples.

  15. Extracellular biosynthesis of copper sulfide nanoparticles by Shewanella oneidensis MR-1 as a photothermal agent.

    PubMed

    Zhou, Nan-Qing; Tian, Li-Jiao; Wang, Yu-Cai; Li, Dao-Bo; Li, Pan-Pan; Zhang, Xing; Yu, Han-Qing

    2016-12-01

    Photothermal therapy (PTT) is a minimally invasive and effective cancer treatment method and has a great potential for innovating the conventional chemotherapy approaches. Copper sulfide (CuS) exhibits photostability, low cost, and high absorption in near infrared region, and is recognized as an ideal candidate for PTT. However, CuS, as a photothermal agent, is usually synthesized with traditional chemical approaches, which require high temperature, additional stabilization and hydrophilic modification. Herein, we report, for the first time, the preparation of CuS nanoparticles as a photothermal agent by a dissimilatory metal reducing bacterium Shewanella. oneidensis MR-1. The prepared nanoparticles are homogenously shaped, hydrophilic, small-sized (∼5nm) and highly stable. Furthermore, the biosynthesized CuS nanoparticles display a high photothermal conversion efficiency of 27.2% because of their strong absorption at 1100nm. The CuS nanoparticles could be effectively used as a PTT agent under the irradiation of 1064nm. This work provides a simple, eco-friendly and cost-effective approach for fabricating PTT agents.

  16. Multi-heme Cytochromes in Shewanella oneidensis MR-1: Structures, functions and opportunities

    SciTech Connect

    Breuer, Marian; Rosso, Kevin M.; Blumberger, Jochen; Butt, Julea N.

    2014-11-05

    Multi-heme cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometers. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-heme cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-heme cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward there are opportunities to engage multi-heme cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-heme cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-heme cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies.

  17. Anaerobic Central Metabolic Pathways in Shewanella oneidensis MR-1 Reinterpreted in the Light of Isotopic Metabolite Labeling▿

    PubMed Central

    Tang, Yinjie J.; Meadows, Adam L.; Kirby, James; Keasling, Jay D.

    2007-01-01

    It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions. PMID:17114268

  18. A biochemical approach to study the role of the terminal oxidases in aerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    Le Laz, Sébastien; Kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2014-01-01

    The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed.

  19. Transient storage of electrical charge in biofilms of Shewanella oneidensis MR-1 growing in a microbial fuel cell.

    PubMed

    Uría, Naroa; Muñoz Berbel, Xavier; Sánchez, Olga; Muñoz, Francesc Xavier; Mas, Jordi

    2011-12-01

    Current output of microbial fuel cells (MFCs) depends on a number of engineering variables mainly related to the design of the fuel cell reactor and the materials used. In most cases the engineering of MFCs relies on the premise that for a constant biomass, current output correlates well with the metabolic activity of the cells. In this study we analyze to what extent, MFC output is also affected by the mode of operation, emphasizing how discontinuous operation can affect temporal patterns of current output. The experimental work has been carried out with Shewanella oneidensis MR-1, grown in conventional two-chamber MFCs subject to periodic interruptions of the external circuit. Our results indicate that after closure of the external circuit, current intensity shows a peak that decays back to basal values. The result suggests that the MFC has the ability to store charge during open circuit situations. Further studies using chronoamperometric analyses were carried out using isolated biofilms of Shewanella oneidensis MR-1 developed in a MFC and placed in an electrochemistry chamber in the presence of an electron donor. The results of these studies indicate that the amount of excess current over the basal level released by the biofilm after periods of circuit disconnection is proportional to the duration of the disconnection period up to a maximum of approximately 60 min. The results indicate that biofilms of Shewanella oneidensis MR-1 have the ability to store charge when oxidizing organic substrates in the absence of an external acceptor.

  20. Analyses of current-generating mechanisms of Shewanella loihica PV-4 and Shewanella oneidensis MR-1 in microbial fuel cells.

    PubMed

    Newton, Gregory J; Mori, Shigeki; Nakamura, Ryuhei; Hashimoto, Kazuhito; Watanabe, Kazuya

    2009-12-01

    Although members of the genus Shewanella have common features (e.g., the presence of decaheme c-type cytochromes [c-cyts]), they are widely variable in genetic and physiological features. The present study compared the current-generating ability of S. loihica PV-4 in microbial fuel cells (MFCs) with that of well-characterized S. oneidensis MR-1 and examined the roles of c-cyts in extracellular electron transfer. We found that strains PV-4 and MR-1 exhibited notable differences in current-generating mechanisms. While the MR-1 MFCs maintained a constant current density over time, the PV-4 MFCs continued to increase in current density and finally surpassed the MR-1 MFCs. Coulombic efficiencies reached 26% in the PV-4 MFC but 16% in the MR-1 MFCs. Although both organisms produced quinone-like compounds, anode exchange experiments showed that anode-attached cells of PV-4 produced sevenfold more current than planktonic cells in the same chamber, while planktonic cells of MR-1 produced twice the current of the anode-attached cells. Examination of the genome sequence indicated that PV-4 has more c-cyt genes in the metal reductase-containing locus than MR-1. Mutational analysis revealed that PV-4 relied predominantly on a homologue of the decaheme c-cyt MtrC in MR-1 for current generation, even though it also possesses two homologues of the decaheme c-cyt OmcA in MR-1. These results suggest that current generation in a PV-4 MFC is in large part accomplished by anode-attached cells, in which the MtrC homologue constitutes the main path of electrons toward the anode.

  1. Multi-haem cytochromes in Shewanella oneidensis MR-1: structures, functions and opportunities

    PubMed Central

    Breuer, Marian; Rosso, Kevin M.; Blumberger, Jochen; Butt, Julea N.

    2015-01-01

    Multi-haem cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometres. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-haem cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-haem cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward, there are opportunities to engage multi-haem cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence, it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-haem cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-haem cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies. PMID:25411412

  2. Pyruvate and lactate metabolism by Shewanella oneidensis MR-1 under fermentation, oxygen limitation, and fumarate respiration conditions.

    PubMed

    Pinchuk, Grigoriy E; Geydebrekht, Oleg V; Hill, Eric A; Reed, Jennifer L; Konopka, Allan E; Beliaev, Alexander S; Fredrickson, Jim K

    2011-12-01

    Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O(2), lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions, S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O(2) limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent d-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.

  3. Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

    SciTech Connect

    Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

    2011-12-01

    Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of wide range of electron acceptors. Here, we quantitatively assessed lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor limited growth on lactate with O2; lactate with fumarate; and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the TCA cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under O2 limitation but was required for anaerobic growth likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

  4. Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation

    PubMed Central

    Barchinger, Sarah E.; Pirbadian, Sahand; Baker, Carol S.; Leung, Kar Man; Burroughs, Nigel J.; El-Naggar, Mohamed Y.

    2016-01-01

    ABSTRACT In limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates that S. oneidensis MR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of the mtrA and mtrC homologs mtrF and mtrD either remains unaffected or decreases under these conditions. The ompW gene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream of mtrC and omcA. The transcriptome and mutant analyses of S. oneidensis MR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires. IMPORTANCE Shewanella oneidensis MR-1 has the capacity to transfer electrons to its external surface

  5. Enhanced photocurrent production by the synergy of hematite nanowire-arrayed photoanode and bioengineered Shewanella oneidensis MR-1.

    PubMed

    Zhu, Gaolong; Yang, Yun; Liu, Juan; Liu, Feng; Lu, Anhuai; He, Weidong

    2017-03-08

    Coupling the light-harvesting capabilities of semiconductors with the catalytic power of bacteria is a promising way to increase the efficiency of bioelectrochemical systems. Here, we reported the enhanced photocurrents produced by the synergy of hematite nanowire-arrayed photoanode and the bio-engineered Shewanella oneidensis MR-1 in a solar-assisted microbial photoelectrochemical system (solar MPS) under the visible light. To increase the supply of bioelectrons, the D-lactate transporter, SO1522, was overexpressed in the recombinant S. oneidensis (T-SO1522) that could digest D-lactate 61% faster than the wild-type S. oneidenesis. Without light illumination, the addition of either the wild-type or the recombinant S. oneidensis to the system did not induce any obvious increase in the current output. However, under one-sun illumination, the photocurrent of the abiotic control was 16±2 μA cm(-2) at 0.8V vs. Ag/AgCl, and the addition of the wild-type S. oneidensis and the recombinant S. oneidensis increased the photocurrent to 70±6 and 95±8 μA cm(-2), respectively, at 0.8V vs. Ag/AgCl. Moreover, the solar MPS with T-SO1522 presented quick and repeatable responses to the on/off illumination cycles, and had relatively stable photocurrent generation in the 273-h operation. Scanning electron microscope (SEM) images showed that the cell density on the hematite photoelectrode was similar between the recombinant and the wild-type S. oneidensis. These findings revealed the pronounced influence of metabolic rates on the light-to-electricity conversion in the complex photocatalyst-electricigen hybrid system, which is important to promote the development of the solar MPS for electricity production and wastewater treatment.

  6. Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in Shewanella oneidensis MR-1

    SciTech Connect

    Cruz-Garza, Claribel; Murray, Alison E.; Rodrigues, Jorge L.M.; Gralnick, Jeffrey A.; McCue, Lee Ann; Romine, Margaret F.; Loffler, F. E.; Tiedje, James M.

    2011-03-30

    EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is not well understood. The expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down regulated at least 2-fold and the EtrA7-1 mutant grew poorly with fumarate and dimethyl sulfoxide (DMSO), suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down regulation of genes implicated in aerobic metabolism. In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and confers physiological advantages to strain MR-1 under certain growth conditions. In conjunction with other regulators, EtrA fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1.

  7. Catabolic and regulatory systems in Shewanella oneidensis MR-1 involved in electricity generation in microbial fuel cells

    PubMed Central

    Kouzuma, Atsushi; Kasai, Takuya; Hirose, Atsumi; Watanabe, Kazuya

    2015-01-01

    Shewanella oneidensis MR-1 is a facultative anaerobe that respires using a variety of inorganic and organic compounds. MR-1 is also capable of utilizing extracellular solid materials, including anodes in microbial fuel cells (MFCs), as electron acceptors, thereby enabling electricity generation. As MFCs have the potential to generate electricity from biomass waste and wastewater, MR-1 has been extensively studied to identify the molecular systems that are involved in electricity generation in MFCs. These studies have demonstrated the importance of extracellular electron-transfer (EET) pathways that electrically connect the quinone pool in the cytoplasmic membrane to extracellular electron acceptors. Electricity generation is also dependent on intracellular catabolic pathways that oxidize electron donors, such as lactate, and regulatory systems that control the expression of genes encoding the components of catabolic and electron-transfer pathways. In addition, recent findings suggest that cell-surface polymers, e.g., exopolysaccharides, and secreted chemicals, which function as electron shuttles, are also involved in electricity generation. Despite these advances in our knowledge on the EET processes in MR-1, further efforts are necessary to fully understand the underlying intra- and extracellular molecular systems for electricity generation in MFCs. We suggest that investigating how MR-1 coordinates these systems to efficiently transfer electrons to electrodes and conserve electrochemical energy for cell proliferation is important for establishing the biological basis for MFCs. PMID:26136738

  8. Comparative Analysis of Differentially Expressed Genes in Shewanella oneidensis MR-1 following Exposure to UVC, UVB, and UVA Radiation†

    PubMed Central

    Qiu, Xiaoyun; Sundin, George W.; Wu, Liyou; Zhou, Jizhong; Tiedje, James M.

    2005-01-01

    We previously reported that Shewanella oneidensis MR-1 is highly sensitive to UVC (254 nm), UVB (290 to 320 nm), and UVA (320 to 400 nm). Here we delineated the cellular response of MR-1 to UV radiation damage by analyzing the transcriptional profile during a 1-h recovering period after UVC, UVB, and UVA exposure at a dose that yields about a 20% survival rate. Although the SOS response was observed with all three treatments, the induction was more robust in response to short-wavelength UV radiation (UVB and UVC). Similarly, more prophage-related genes were induced by short-wavelength UV radiation. MR-1 showed an active detoxification mechanism in response to UVA, which included the induction of antioxidant enzymes and iron-sequestering proteins to scavenge reactive oxygen species. In addition, a great number of genes encoding multidrug and heavy metal efflux pumps were induced following UVA irradiation. Our data suggested that activation of prophages appears the major lethal factor in MR-1 following UVC or UVB irradiation, whereas oxidative damage contributes greatly to the high UVA sensitivity in MR-1. PMID:15866945

  9. Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

    SciTech Connect

    Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

    2011-12-30

    Shewanella oneidensis MR-1 is a facultative anaerobe growing by coupling organic matter oxidation to reduction of wide range of electron acceptors. Here we quantitatively assessed lactate and pyruvate metabolism of these bacteria under three distinct conditions: electron acceptor limited growth on lactate with O2 and fumarate, and pyruvate fermentation, which does not sustain growth but allows cells to survive for prolonged period. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of all ATP needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute much to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, and TCA cycle did not contribute significantly to substrate oxidation. Pyruvate dehydrogenase reaction was not involved in lactate metabolism under O2 limitation, however was important for anaerobic growth probably supplying reducing equivalents for biosynthesis. Unexpectedly, obtained results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination between substrate-level phosphorylation and a respiratory process, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). Based on involved enzymes localization we hypothesize that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

  10. Constraint-Based Model of Shewanella oneidensis MR-1 Metabolism: A Tool for Data Analysis and Hypothesis Generation

    PubMed Central

    Hill, Eric A.; Geydebrekht, Oleg V.; De Ingeniis, Jessica; Zhang, Xiaolin; Osterman, Andrei; Scott, James H.; Reed, Samantha B.; Romine, Margaret F.; Konopka, Allan E.; Beliaev, Alexander S.; Fredrickson, Jim K.

    2010-01-01

    Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems

  11. In vitro enzymatic reduction kinetics of mineral oxides by membrane fractions from Shewanella oneidensis MR-1

    NASA Astrophysics Data System (ADS)

    Ruebush, Shane S.; Icopini, Gary A.; Brantley, Susan L.; Tien, Ming

    2006-01-01

    This study documents the first example of in vitro solid-phase mineral oxide reduction by enzyme-containing membrane fractions. Previous in vitro studies have only reported the reduction of aqueous ions. Total membrane (TM) fractions from iron-grown cultures of Shewanella oneidensis MR-1 were isolated and shown to catalyze the reduction of goethite, hematite, birnessite, and ramsdellite/pyrolusite using formate. In contrast, nicotinamide adenine dinucleotide (NADH) and succinate cannot function as electron donors. The significant implications of observations related to this cell-free system are: (i) both iron and manganese mineral oxides are reduced by the TM fraction, but aqueous U(VI) is not; (ii) TM fractions from anaerobically grown, but not aerobically grown, cells can reduce the mineral oxides; (iii) electron shuttles and iron chelators are not needed for this in vitro reduction, documenting conclusively that reduction can occur by direct contact with the mineral oxide; (iv) electron shuttles and EDTA stimulate the in vitro Fe(III) reduction, documenting that exogenous molecules can enhance rates of enzymatic mineral reduction; and (v) multiple membrane components are involved in solid-phase oxide reduction. The membrane fractions, consisting of liposomes of cytoplasmic and outer membrane segments, contain at least 100 proteins including the enzyme that oxidizes formate, formate dehydrogenase. Mineral oxide reduction was inhibited by the addition of detergent Triton X-100, which solubilizes membranes and their associated proteins, consistent with the involvement of multiple electron carriers that are disrupted by detergent addition. In contrast, formate dehydrogenase activity was not inhibited by Triton X-100. The addition of anthraquinone-2,6-disulfonate (AQDS) and menaquinone-4 was unable to restore activity; however, menadione (MD) restored 33% of the activity. The addition of AQDS and MD to reactions without added detergent increased the rate of goethite

  12. In Vitro Enzymatic Reduction Kinetics of Mineral Oxides by Membrane Fractions from Shewanella oneidensis MR-1

    SciTech Connect

    Ruebush,S.; Icopini, G.; Brantley, S.; Tien, M.

    2006-01-01

    This study documents the first example of in vitro solid-phase mineral oxide reduction by enzyme-containing membrane fractions. Previous in vitro studies have only reported the reduction of aqueous ions. Total membrane (TM) fractions from iron-grown cultures of Shewanella oneidensis MR-1 were isolated and shown to catalyze the reduction of goethite, hematite, birnessite, and ramsdellite/pyrolusite using formate. In contrast, nicotinamide adenine dinucleotide (NADH) and succinate cannot function as electron donors. The significant implications of observations related to this cell-free system are: (i) both iron and manganese mineral oxides are reduced by the TM fraction, but aqueous U(VI) is not; (ii) TM fractions from anaerobically grown, but not aerobically grown, cells can reduce the mineral oxides; (iii) electron shuttles and iron chelators are not needed for this in vitro reduction, documenting conclusively that reduction can occur by direct contact with the mineral oxide; (iv) electron shuttles and EDTA stimulate the in vitro Fe(III) reduction, documenting that exogenous molecules can enhance rates of enzymatic mineral reduction; and (v) multiple membrane components are involved in solid-phase oxide reduction. The membrane fractions, consisting of liposomes of cytoplasmic and outer membrane segments, contain at least 100 proteins including the enzyme that oxidizes formate, formate dehydrogenase. Mineral oxide reduction was inhibited by the addition of detergent Triton X-100, which solubilizes membranes and their associated proteins, consistent with the involvement of multiple electron carriers that are disrupted by detergent addition. In contrast, formate dehydrogenase activity was not inhibited by Triton X-100. The addition of anthraquinone-2,6-disulfonate (AQDS) and menaquinone-4 was unable to restore activity; however, menadione (MD) restored 33% of the activity. The addition of AQDS and MD to reactions without added detergent increased the rate of goethite

  13. Siderophores are not involved in Fe(III) solubilization during anaerobic Fe(III) respiration by Shewanella oneidensis MR-1.

    PubMed

    Fennessey, Christine M; Jones, Morris E; Taillefert, Martial; DiChristina, Thomas J

    2010-04-01

    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.

  14. An iTRAQ characterisation of the role of TolC during electron transfer from Shewanella oneidensis MR-1.

    PubMed

    Fowler, Gregory J S; Pereira-Medrano, Ana G; Jaffe, Stephen; Pasternak, Grzegorz; Pham, Trong Khoa; Ledezma, Pablo; Hall, Simon T E; Ieropoulos, Ioannis A; Wright, Phillip C

    2016-11-01

    Anodophilic bacteria have the ability to generate electricity in microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigated the anode-specific responses of Shewanella oneidensis MR-1, an exoelectroactive Gammaproteobacterium, using for the first time iTRAQ and 2D-LC MS/MS driven membrane proteomics to compare protein abundances in S. oneidensis when generating power in MFCs, and growing in a continuous culture. The regulated dataset produced was enriched in membrane proteins. Proteins shown to be more abundant in anaerobic electroactive anodic cells included efflux pump TolC and an uncharacterised tetratricopeptide repeat (TPR) protein, whilst the TonB2 system and associated uncharacterised proteins such as TtpC2 and DUF3450 were more abundant in microaerobic planktonic cells. In order to validate the iTRAQ data, the functional role for TolC was examined using a δTolC knockout mutant of S. oneidensis. Possible roles for the uncharacterised proteins were identified using comparative bioinformatics. We demonstrate that employing an insoluble extracellular electron acceptor requires multiple proteins involved in cell surface properties. All MS and processed data are available via ProteomeXchange with identifier PXD004090.

  15. Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

    PubMed Central

    Pirbadian, Sahand; Barchinger, Sarah E.; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A.; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad A.; Shi, Liang; Gorby, Yuri A.; Golbeck, John H.; El-Naggar, Mohamed Y.

    2014-01-01

    Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic–abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution. PMID:25143589

  16. Iron reduction in the DAMO/Shewanella oneidensis MR-1 coculture system and the fate of Fe(II).

    PubMed

    Fu, Liang; Li, Shan-Wei; Ding, Zhao-Wei; Ding, Jing; Lu, Yong-Ze; Zeng, Raymond J

    2016-01-01

    Dissimilatory iron reduction and anaerobic methane oxidation processes play important roles in the global iron and carbon cycle, respectively. This study explored the ferrihydrite reduction process with methane as a carbon source in a coculture system of denitrifying anaerobic methane oxidation (DAMO) microbes enriched in laboratory and Shewanella oneidensis MR-1, and then characterized the reduced products. Ferrihydrite reduction was also studied in the DAMO and Shewanella systems alone. The ferrihydrite was reduced slightly (<13.3%) in the separate systems, but greatly (42.0-88.3%) in the coculture system. Isotope experiment of (13)CH4 addition revealed that DAMO microbes coupled to S. oneidensis MR-1 in a ferric iron reduction process with (13)CH4 consumption and (13)CO2 production. Compared with ferrihydrite, the reduced products showed increased crystallinity (from amorphous state to crystallinity 77.1%) and magnetism (from paramagnetic to ferromagnetic). The produced ferrous iron was formed into minerals primarily composed of siderite with a small amount vivianite and magnetite. A portion of products covered the cell surface and hindered further reactions. The results presented herein widen the current understanding of iron metabolism and mineralization in the ocean, and show that the coculture systems of DAMO microbes and Shewanella have the potential to be globally important to iron reduction and methane oxidation.

  17. Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

    PubMed

    Pirbadian, Sahand; Barchinger, Sarah E; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Shi, Liang; Gorby, Yuri A; Golbeck, John H; El-Naggar, Mohamed Y

    2014-09-02

    Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

  18. Recombinant engineering of Shewanella oneidensis MR-1 c-type cytochromes in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shewanella oneidensis is known to respire with extracellular solid metal oxides (i.e., iron, manganese, uranium) as a terminal electron acceptor. It has become the focus of intensive research not only due to its important bioremediation features, but also as a potential organism for biological elec...

  19. Confirmation of the expression of a large set of conserved hypothetical proteins in Shewanella oneidensis MR-1.

    PubMed

    Elias, Dwayne A; Monroe, Matthew E; Smith, Richard D; Fredrickson, James K; Lipton, Mary S

    2006-08-01

    High-throughput "omic" technologies have allowed for a relatively rapid, yet comprehensive analysis of the global expression patterns within an organism in response to perturbations. In the current study, 9503 different tryptic peptides were identified with high confidence from capillary liquid chromatography-mass spectrometry analysis of 26 chemostat cultures of Shewanella oneidensis MR-1 under various conditions. Using at least one distinctive and a total of two total peptide identifications per protein, we detected the expression of 758 conserved hypothetical proteins. This included 359 such proteins previously described [Kolker, E., Picone, A.F., Galperin, M.Y., Romine, M.F., Higdon, R., Makarova, K.S., Kolker, N., Anderson, G.A., Qiu, X., Auberry, K.J., Babnigg, G., Beliaev, A.S., Edlefsen, P., Elias, D.A., Gorby, Y.A., Holzman, T., Klappenbach, J.A., Konstantinidis, K.T., Land, M.L., Lipton, M.S., McCue, L.A., Monroe, M., Pasa-Tolic, L., Pinchuk, G., Purvine, S., Serres, M.H., Tsapin, S., Zakrajsek, B.A., Zhu, W., Zhou, J., Larimer, F.W., Lawrence, C.E., Riley, M., Collart, F.R., Yates, J.R., III, Smith, R.D., Giometti, C.S., Nealson, K.H., Fredrickson, J.K., Tiedje, J.M., 2005. Global profiling of Shewanella oneidensis MR-1: expression of hypothetical genes and improved functional annotations. Proc Natl Acad Sci U S A 102, 2099-2104] with an additional 399 reported herein for the first time. The latter 399 proteins ranged from 5.3 to 208.3 kDa, with 44 being of 100 amino acid residues or less. Using a combination of information including peptide detection in cells grown under specific culture conditions and predictive algorithms such as PSORT and PSORT-B, possible/plausible functions are proposed for some conserved hypothetical proteins. Such proteins were found not only to be expressed, but 19 were only expressed under certain culturing conditions, thereby providing insight into potential functions. These findings also impact the genomic annotation for S

  20. H₂-dependent azoreduction by Shewanella oneidensis MR-1: involvement of secreted flavins and both [Ni-Fe] and [Fe-Fe] hydrogenases.

    PubMed

    Le Laz, Sébastien; Kpebe, Arlette; Lorquin, Jean; Brugna, Myriam; Rousset, Marc

    2014-03-01

    In this paper, the hydrogen (H2)-dependent discoloration of azo dye amaranth by Shewanella oneidensis MR-1 was investigated. Experiments with hydrogenase-deficient strains demonstrated that periplasmic [Ni-Fe] hydrogenase (HyaB) and periplasmic [Fe-Fe] hydrogenase (HydA) are both respiratory hydrogenases of dissimilatory azoreduction in S. oneidensis MR-1. These findings suggest that HyaB and HydA can function as uptake hydrogenases that couple the oxidation of H2 to the reduction of amaranth to sustain cellular growth. This constitutes to our knowledge the first report of the involvement of [Fe-Fe] hydrogenase in a bacterial azoreduction process. Assays with respiratory inhibitors indicated that a menaquinone pool and different cytochromes were involved in the azoreduction process. High-performance liquid chromatography analysis revealed that flavin mononucleotide and riboflavin were secreted in culture supernatant by S. oneidensis MR-1 under H2-dependent conditions with concentration of 1.4 and 2.4 μmol g protein(-1), respectively. These endogenous flavins were shown to significantly accelerate the reduction of amaranth at micromolar concentrations acting as electron shuttles between the cell surface and the extracellular azo dye. This work may facilitate a better understanding of the mechanisms of azoreduction by S. oneidensis MR-1 and may have practical applications for microbiological treatments of dye-polluted industrial effluents.

  1. Roles of 3,3′,4′,5-tetrachlorosalicylanilide in regulating extracellular electron transfer of Shewanella oneidensis MR-1

    PubMed Central

    Wang, Yong-Peng; Yu, Sheng-Song; Zhang, Hai-Ling; Li, Wen-Wei; Cheng, Yuan-Yuan; Yu, Han-Qing

    2015-01-01

    Microbial extracellular electron transfer (EET) is critically involved in many pollutant conversion processes in both natural environment and engineered bioelectrochemical systems (BES), but typically with limited efficiency and poor controllability. In this study, we discover an important role of uncouplers in affecting the microbial energy metabolism and EET. Dose of lower-concentration 3,3′,4′,5-tetrachlorosalicylanilide (TCS) in the anolyte promoted the current generation and substrate degradation of an MFC inoculated with Shewanella oneidensis MR-1. However, higher TCS dosage caused obvious microbial inhibition. Our results suggest a previously unknown role of uncouplers in regulating the microbial EET. In addition, the underlying mechanisms of such processes are investigated. This work broadens our view about the EET behaviors of microorganisms in real water environment where uncouplers are usually present, and suggests a possible new approach to regulate microbial EET in BES. PMID:25612888

  2. Shewanella oneidensis MR-1 H-NOX Regulation of a Histidine Kinase by Nitric Oxide

    PubMed Central

    Price, Mark S.; Chao, Lily; Marletta, Michael A.

    2008-01-01

    Nitric oxide (NO) signaling in animals controls processes such as smooth muscle relaxation and neurotransmission by activation of soluble guanylate cyclase (sGC). Prokaryotic homologs of the sGC heme domain, called H-NOX domains, have been identified and are generally found in a predicted operon in conjunction with a histidine kinase. Here, we show that an H-NOX protein (SO2144) from Shewanella oneidensis, directly interacts with the sensor histidine kinase (SO2145), binds NO in a 5-coordinate complex similar to mammalian sGC, and in that form inhibits the activity of a histidine kinase (SO2145). We also describe the first account of NO formation by S. oneidensis under anaerobic growth conditions derived from nitrate and nitrite. These observations suggest that the S. oneidensis H-NOX and histidine kinase pair function as part of a novel two-component signaling pathway that is responsive to NO formation from higher nitrogen oxides used as electron acceptors when oxygen is low and thereby functioning as an environmental sensor. PMID:17988156

  3. The role of Shewanella oneidensis MR-1 outer surface structures in extracellular electron transfer

    SciTech Connect

    Bouhenni, Rachida; Vora, Gary J.; Biffinger, Justin C.; Shirodkar, Sheetal; Brockman, K. L.; Ray, Ricky; Wu, Peter; Johnson, Brandy J.; Biddle, E. M.; Marshall, Matthew J.; Fitzgerald, Lisa A.; Little, Brenda; Fredrickson, Jim K.; Beliaev, Alex S.; Ringeisen, Bradley R.; Saffarini, Daad

    2010-04-20

    Shewanella oneidensis is a facultative anaerobe that uses more than 14 different terminal electron acceptors for respiration. These include metal oxides and hydroxyoxides, and toxic metals such as uranium and chromium. Mutants deficient in metal reduction were isolated using the mariner transposon derivative, minihimar RB1. These included mutants with transposon insertions in the prepilin peptidase and type II secretion system genes. All mutants were deficient in Fe(III) and Mn(IV) reduction, and exhibited slow growth when DMSO was used as the electron acceptor. The genome sequence of S. oneidensis contains one prepilin peptidase gene, pilD. A similar prepilin peptidase that may function in the processing of type II secretion prepilins was not found. Single and multiple chromosomal deletions of four putative type IV pilin genes did not affect Fe(III) and Mn(IV) reduction. These results indicate that PilD in S. oneidensis is responsible for processing both type IV and type II secretion prepilin proteins. Type IV pili do not appear to be required for Fe(III) and Mn(IV) reduction.

  4. Shewanella oneidensis MR-1 chemotaxis proteins and electron-transport chain components essential for congregation near insoluble electron acceptors.

    PubMed

    Harris, H Wayne; El-Naggar, Mohamed Y; Nealson, Kenneth H

    2012-12-01

    Shewanella oneidensis MR-1 cells utilize a behaviour response called electrokinesis to increase their speed in the vicinity of IEAs (insoluble electron acceptors), including manganese oxides, iron oxides and poised electrodes [Harris, El-Naggar, Bretschger, Ward, Romine, Obraztsova and Nealson (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 326-331]. However, it is not currently understood how bacteria remain in the vicinity of the IEA and accumulate both on the surface and in the surrounding medium. In the present paper, we provide results indicating that cells that have contacted the IEAs swim faster than those that have not recently made contact. In addition, fast-swimming cells exhibit an enhancement of swimming reversals leading to rapid non-random accumulation of cells on, and adjacent to, mineral particles. We call the observed accumulation near IEAs 'congregation'. Congregation is eliminated by the loss of a critical gene involved with EET (extracellular electron transport) (cymA, SO_4591) and is altered or eliminated in several deletion mutants of homologues of genes that are involved with chemotaxis or energy taxis in Escherichia coli. These genes include chemotactic signal transduction protein (cheA-3, SO_3207), methyl-accepting chemotaxis proteins with the Cache domain (mcp_cache, SO_2240) or the PAS (Per/Arnt/Sim) domain (mcp_pas, SO_1385). In the present paper, we report studies of S. oneidensis MR-1 that lend some insight into how microbes in this group can 'sense' the presence of a solid substrate such as a mineral surface, and maintain themselves in the vicinity of the mineral (i.e. via congregation), which may ultimately lead to attachment and biofilm formation.

  5. Use of SWATH mass spectrometry for quantitative proteomic investigation of Shewanella oneidensis MR-1 biofilms grown on graphite cloth electrodes.

    PubMed

    Grobbler, Christy; Virdis, Bernardino; Nouwens, Amanda; Harnisch, Falk; Rabaey, Korneel; Bond, Philip L

    2015-03-01

    Quantitative proteomics from low biomass, biofilm samples is not well documented. In this study we show successful use of SWATH-MS for quantitative proteomic analysis of a microbial electrochemically active biofilm. Shewanella oneidensis MR-1 was grown on carbon cloth electrodes under continuous anodic electrochemical polarizations in a bioelectrochemical system (BES). Using lactate as the electron donor, anodes serving as terminal microbial electron acceptors were operated at three different electrode potentials (+0.71 V, +0.21 V & -0.19 V vs. SHE) and the development of catalytic activity was monitored by measuring the current traces over time. Once maximum current was reached (usually within 21-29 h) the electrochemical systems were shut off and biofilm proteins were extracted from the electrodes for proteomic assessment. SWATH-MS analysis identified 704 proteins, and quantitative comparison was made of those associated with tricarboxcylic acid (TCA) cycle. Metabolic differences detected between the biofilms suggested a branching of the S. oneidensis TCA cycle when grown at the different electrode potentials. In addition, the higher abundance of enzymes involved in the TCA cycle at higher potential indicates an increase in metabolic activity, which is expected given the assumed higher energy gains. This study demonstrates high numbers of identifications on BES biofilm samples can be achieved in comparison to what is currently reported. This is most likely due to the minimal preparation steps required for SWATH-MS.

  6. Shewanella oneidensis MR-1 Nanowires are Outer Membrane and Periplasmic Extensions of the Extracellular Electron Transport Components

    SciTech Connect

    Pirbadian, S.; Barchinger, S. E.; Leung, K. M.; Byun, H. S.; Jangir, Y.; Bouhenni, Rachida; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad; Shi, Liang; Gorby, Yuri A.; Golbeck, J. H.; El-Naggar, Mohamed Y.

    2014-08-20

    Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella neidensis MR-1. Using live fluorescence measurements, immunolabeling, and quantitative gene expression analysis, we report that S. oneidensis MR-1 nanowires are extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures, as previously thought. These bacterial nanowires were also associated with outer membrane vesicles and vesicle chains, structures ubiquitous in gram-negative bacteria. Redoxfunctionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

  7. Transcriptome analysis reveals response regulator SO2426-mediated gene expression in Shewanella oneidensis MR-1 under chromate challenge

    PubMed Central

    Chourey, Karuna; Wei, Wei; Wan, Xiu-Feng; Thompson, Dorothea K

    2008-01-01

    Background Shewanella oneidensis MR-1 exhibits diverse metal ion-reducing capabilities and thus is of potential utility as a bioremediation agent. Knowledge of the molecular components and regulatory mechanisms dictating cellular responses to heavy metal stress, however, remains incomplete. In a previous work, the S. oneidensis so2426 gene, annotated as a DNA-binding response regulator, was demonstrated to be specifically responsive at both the transcript and protein levels to acute chromate [Cr(VI)] challenge. To delineate the cellular function of SO2426 and its contribution to metal stress response, we integrated genetic and physiological approaches with a genome-wide screen for target gene candidates comprising the SO2426 regulon. Results Inactivation of so2426 by an in-frame deletion resulted in enhanced chromate sensitivity and a reduced capacity to remove extracellular Cr(VI) relative to the parental strain. Time-resolved microarray analysis was used to compare transcriptomic profiles of wild-type and SO2426-deficient mutant S. oneidensis under conditions of chromate exposure. In total, 841 genes (18% of the arrayed genome) were up- or downregulated at least twofold in the Δso2426 mutant for at least one of six time-point conditions. Hierarchical cluster analysis of temporal transcriptional profiles identified a distinct cluster (n = 46) comprised of co-ordinately regulated genes exhibiting significant downregulated expression (p < 0.05) over time. Thirteen of these genes encoded proteins associated with transport and binding functions, particularly those involved in Fe transport and homeostasis (e.g., siderophore biosynthetic enzymes, TonB-dependent receptors, and the iron-storage protein ferritin). A conserved hypothetical operon (so1188-so1189-so1190), previously identified as a potential target of Fur-mediated repression, as well as a putative bicyclomycin resistance gene (so2280) and cation efflux family protein gene (so2045) also were repressed in the

  8. Trace Element Speciation and Distribution Study at Shewanella oneidensis MR-1 Biofilm/Mineral/Water Interfaces

    NASA Astrophysics Data System (ADS)

    Gelabert, A.; Wang, Y.; Gescher, J.; Ha, J.; Cordova, C. D.; Singer, D. M.; Spormann, A. M.; Trainor, T. P.; Eng, P. J.; Brown, G. E.

    2006-12-01

    Fe- and Al-(oxyhydr)oxides are among the most reactive mineral surfaces contacted by surface and ground waters, and thus they constitute important sorbents for heavy metal and metalloid ions. As microbial biofilms may be present as coatings on these minerals, they are likely to induce major changes in surface charges and sorption capacities for metal(loid) ions compared to biofilm-free mineral surfaces. In addition, the micro- environments in biofilms can be quite different from those in bulk solutions, which can enhance (or inhibit) metal adsorption on mineral surfaces and produce biominerals that are not predicted by equilibrium thermodynamics based on the bulk solution values. In order to provide a more quantitative understanding of these effects, we have carried out a study of the interaction of Zn(II), Pb(II), and As(V) with Shewanella oneidensis (wild type, EPS-deficient mutant, and ppx- and ppk-deficient mutants) grown on highly polished and oriented single crystal surfaces of α-Al2O3 (1-102) and α-Fe2O3 (0001). This gram-negative bacterium commonly found in soil and sediments can use a wide range of electron donors and terminal electron acceptors including Fe(III) and Mn(IV) oxides under anaerobic conditions. In-situ ATR-FTIR analyses and potentiometric titrations of S. oneidensis biofilm collected from a glass bead-filled column inoculated with S. oneidensis were conducted in order to determine the nature of functional groups present on the bacterial surfaces, to quantify the site densities and protonation constants for these groups, and to determine the electrostatic parameters for S. oneidensis surfaces. GI-XAFS analyses performed on BL 11-2 at SSRL, together with macroscopic metal adsorption experiments as a function of pH (2 to 6.5), metal concentration (10-3 to 10-7 M), and ionic strength (10-1 to 10-3 M), were used to determine ion speciation and local coordination environments in the biofilm and to develop a surface complexation model describing

  9. Evaluation of the effects of various culture condition on Cr (VI)reduction by Shewanella oneidensis MR-1 in a novel high-throughputmini-bioreactor

    SciTech Connect

    Tang, Yinjie J.; Laidlaw, David; Gani, Kishen; Keasling, Jay D.

    2006-03-16

    The growth and Cr(VI) reduction by Shewanella oneidensisMR-1 was examined using a mini-bioreactor system that independentlymonitors and controls pH, dissolved oxygen, and temperature for each ofits 24, 10-mL reactors. Independent monitoring and control of eachreactor in the cassette allows the exploration of a matrix ofenvironmental conditions known to influence S. oneidensis chromiumreduction. S. oneidensis MR-1 grew in minimal medium without amino acidor vitamin supplementation under aerobic conditions but required serineand glycine supplementation under anaerobic conditions. Growth wasinhibited by dissolved oxygen concentrations>80 percent. Lactatetransformation to acetate was enhanced by low concentration of dissolvedoxygen during the logarithmic growth phase. Between 11 and 35oC, thegrowth rate obeyed the Arrhenius reaction rate-temperature relationship,with a maximum growth rate occurring at 35oC. S. oneidensis MR-1 was ableto grow over a wide range of pH (6-9). At neutral pH and temperaturesranging from 30-35oC, S. oneidensis MR-1 reduced 100 mu M Cr(VI) toCr(III) within 20 minutes in the exponential growth phase, and the growthrate was not affected by the addition of chromate; it reduced chromateeven faster at temperatures between 35 and 39oC. At low temperatures(<25oC), acidic (pH<6.5), or alkaline (pH>8.5) conditions, 100mu M Cr(VI) strongly inhibited growth and chromate reduction. Themini-bioreactor system enabled the rapid determination of theseparameters reproducibly and easily by performing very few experiments.Besides its use for examining parameters of interest to environmentalremediation, the device will also allow one to quickly assess parametersfor optimal production of recombinant proteins or secondarymetabolites

  10. Substrate-level phosphorylation is the primary source of energy conservation during anaerobic respiration of Shewanella oneidensis strain MR-1.

    PubMed

    Hunt, Kristopher A; Flynn, Jeffrey M; Naranjo, Belén; Shikhare, Indraneel D; Gralnick, Jeffrey A

    2010-07-01

    It is well established that respiratory organisms use proton motive force to produce ATP via F-type ATP synthase aerobically and that this process may reverse during anaerobiosis to produce proton motive force. Here, we show that Shewanella oneidensis strain MR-1, a nonfermentative, facultative anaerobe known to respire exogenous electron acceptors, generates ATP primarily from substrate-level phosphorylation under anaerobic conditions. Mutant strains lacking ackA (SO2915) and pta (SO2916), genes required for acetate production and a significant portion of substrate-level ATP produced anaerobically, were tested for growth. These mutant strains were unable to grow anaerobically with lactate and fumarate as the electron acceptor, consistent with substrate-level phosphorylation yielding a significant amount of ATP. Mutant strains lacking ackA and pta were also shown to grow slowly using N-acetylglucosamine as the carbon source and fumarate as the electron acceptor, consistent with some ATP generation deriving from the Entner-Doudoroff pathway with this substrate. A deletion strain lacking the sole F-type ATP synthase (SO4746 to SO4754) demonstrated enhanced growth on N-acetylglucosamine and a minor defect with lactate under anaerobic conditions. ATP synthase mutants grown anaerobically on lactate while expressing proteorhodopsin, a light-dependent proton pump, exhibited restored growth when exposed to light, consistent with a proton-pumping role for ATP synthase under anaerobic conditions. Although S. oneidensis requires external electron acceptors to balance redox reactions and is not fermentative, we find that substrate-level phosphorylation is its primary anaerobic energy conservation strategy. Phenotypic characterization of an ackA deletion in Shewanella sp. strain MR-4 and genomic analysis of other sequenced strains suggest that this strategy is a common feature of Shewanella.

  11. Genes That Enhance the Ecological Fitness of Shewanella oneidensis MR-1 in Sediments Reveal the Value of Antibiotic Resistance▿ †

    PubMed Central

    Groh, Jennifer L.; Luo, Qingwei; Ballard, Jimmy D.; Krumholz, Lee R.

    2007-01-01

    Environmental bacteria persist in various habitats, yet little is known about the genes that contribute to growth and survival in their respective ecological niches. Signature-tagged mutagenesis (STM) of Shewanella oneidensis MR-1 coupled with a screen involving incubations of mutant strains in anoxic aquifer sediments allowed us to identify 47 genes that enhance fitness in sediments. Gene functions inferred from annotations provide us with insight into physiological and ecological processes that environmental bacteria use while growing in sediment ecosystems. Identification of the mexF gene and other potential membrane efflux components by STM demonstrated that homologues of multidrug resistance genes present in pathogens are required for sediment fitness of nonpathogenic bacteria. Further studies with a mexF deletion mutant demonstrated that the multidrug resistance pump encoded by mexF is required for resistance to antibiotics, including chloramphenicol and tetracycline. Chloramphenicol-adapted cultures exhibited mutations in the gene encoding a TetR family regulatory protein, indicating a role for this protein in regulating expression of the mexEF operon. The relative importance of mexF for sediment fitness suggests that antibiotic efflux may be a required process for bacteria living in sediment systems. PMID:17114320

  12. Ferrous Phosphate Surface Precipitates Resulting from the Reduction of Intragrain 6-line Ferrihydrite by Shewanella oneidensis MR-1

    SciTech Connect

    Peretyazhko, Tetyana; Zachara, John M.; Kennedy, David W.; Fredrickson, Jim K.; Arey, Bruce W.; McKinley, James P.; Wang, Chong M.; Dohnalkova, Alice; Xia, Yuanxian

    2010-07-01

    The reductive biotransformation of 6-line ferrihydrite located within porous silica (intragrain ferrihydrite) by Shewanella oneidensis MR-1 was investigated and compared to the behavior of 6-line ferrihydrite in suspension (free ferrihydrite). The effect of buffer type (PIPES and NaHCO3) and phosphate (P) on the extent of reduction and formation of Fe(II) secondary phases was investigated under anoxic conditions. Electron microscopy and micro X-ray diffraction were applied to evaluate the morphology and mineralogy of the biogenic precipitates and to study the distribution of microorganisms on the surface of porous silica after bioreduction. Kinetic reduction experiments with free and intragrain ferrihydrite revealed contrasting behaviour with respect to the buffer and presence of P. The overall amount of intragrain ferrihydrite reduction was less than that of free ferrihydrite [at 5 mmol L-1 Fe(III)T]. In the intragrain ferrihydrite suspensions, 200-300 µmol L-1 dissolved Fe(III) was released during the initial stages of incubation; no Fe(III)aq was detected in the free ferrihydrite suspensions. Reductive mineralization was not observed in the intragrain ferrihydrite incubations without P, and all biogenic Fe(II) concentrated in the aqueous phase. Distinctive surface precipitates of Fe(II) phosphates with spherical morphology were observed on porous silica when P was present. These precipitates were well colonized by microorganisms and fragments of extracellular materials at the end of incubation.

  13. Fate of Fe and Cd upon microbial reduction of Cd-loaded polyferric flocs by Shewanella oneidensis MR-1.

    PubMed

    Li, Chenchen; Yi, Xiaoyun; Dang, Zhi; Yu, Hui; Zeng, Tao; Wei, Chaohai; Feng, Chunhua

    2016-02-01

    Polyferric sulphate has been widely used for emergent control on incidental release of heavy metals such as Cd to surface water, causing precipitation of Cd-loaded polyferric flocs to the sediment. To date, little is known about whether the dissolution of the flocs in the presence of dissimilatory iron reducing bacteria (DIRB) can occur and how the dissolution influences the fate of Fe and Cd in the sediment. Here, we demonstrated that Shewanella oneidensis MR-1, as representative DIRB, has the ability to reduce the flocs, resulting in the release of Fe(2+) and Cd(2+) to the solution. Batch experiment results showed that the concentrations of Fe(2+) and Cd(2+)reached the maximum values at 48 h and then decreased over the remaining incubation time. The characterizations on the solid phase by the scanning electron microscopy coupled with energy dispersive spectrometer, X-ray diffraction, and X-ray photoelectron spectroscopy technologies revealed the formation of iron minerals such as goethite and magnetite as a consequence of microbial Fe(III) reduction. The newly formed iron minerals played a significant role in re-immobilizing Cd by sorption. These results imply that microbial reduction of polyferric flocs is an important contributor to the transport and transformation of metals in the sediment-water interface.

  14. Control of Formation and Cellular Detachment from Shewanella oneidensis MR-1 Biofilms by Cyclic di-GMP

    SciTech Connect

    Thormann, Kai M.; Duttler, Stefanie; Saville, Renee; Hyodo, Mamoru; Shukla, Soni; Hayakawa, Yoshihiro; Spormann, Alfred M.

    2006-04-01

    Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral -reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3',5')guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a Delta mxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

  15. Expression of Shewanella oneidensis MR-1 [FeFe]-Hydrogenase Genes in Anabaena sp. Strain PCC 7120

    PubMed Central

    Gärtner, Katrin; Lechno-Yossef, Sigal; Cornish, Adam J.; Wolk, C. Peter

    2012-01-01

    H2 generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H2 from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O2 sensitive. Certain filamentous cyanobacteria protect nitrogenase from O2 by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter PhetN. Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O2. PMID:23023750

  16. Involvement of a membrane-bound class III adenylate cyclase in regulation of anaerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    Charania, M A; Brockman, K L; Zhang, Y; Banerjee, A; Pinchuk, G E; Fredrickson, J K; Beliaev, A S; Saffarini, D A

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  17. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K. L.; Zhang, Y.; Banerjee, A.; Pinchuk, Grigoriy E.; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  18. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K.; Zhang, Yang; Banerjee, A.; Pinchuk, Grigoriy; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, S. oneidensis MR-1 uses the cAMP receptor protein, CRP, for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP.

  19. Antibacterial activity of graphene-modified anode on Shewanella oneidensis MR-1 biofilm in microbial fuel cell

    NASA Astrophysics Data System (ADS)

    Chen, Jie; Deng, Feng; Hu, Yongyou; Sun, Jian; Yang, Yonggang

    2015-09-01

    To clearly illustrate the antibacterial activity of graphene on anodic exoelectrogen, the growth of a Shewanella oneidensis MR-1 biofilm on graphene-modified anodes (GMAs) and bare graphite anodes (BGs) were compared. The GMAs with different amounts of graphene were obtained by the cyclic voltammetric electrodeposition of 5, 20 and 40 potential cycles (5-G, 20-G and 40-G). Confocal scanning laser microscopy and cyclic voltammetry results demonstrated that graphene exhibited an obvious antibacterial effect for initial Shewanella MR biofilm growth. After 5 h of inoculation, 40-G, 20-G and 5-G had 6.3, 8.8 and 13.9% lower levels of biofilm viability, respectively, compared to BG, and all three exhibited approximately 70% lower electrochemical activity compared to BG. However, 18 h later, the biofilm on the GMAs exhibited much higher viability than that of the BG, and the electrochemical activity increased to a similar level. This study revealed the dual effect of graphene, including the antibacterial activity on biofilms and the enhancement of bacterial attachment and electron transfer.

  20. Anaerobic central metabolic pathways in Shewanella oneidensis MR-1interpreted in the light of isotopic metabolite labeling, enzymeactivities and genome annotation

    SciTech Connect

    Tang, Yinjie J.; Meadows, Adam L.; Kirby, James; Keasling, Jay D.

    2006-06-27

    It has been proposed that during growth under anaerobic oroxygen-limited conditions Shewanella oneidensis MR-1 uses theserine-isocitrate lyase pathway common to many methylotrophic anaerobes,in which formaldehyde produced from pyruvate is condensed with glycine toform serine. The serine is then transformed through hydroxypyruvate andglycerate to enter central metabolism at phosphoglycerate. To examine itsuse of the serine-isocitrate lyase pathway under anaerobic conditions, wegrew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source witheither trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor.Analysis of cellular metabolites indicates that a large percentage(>75 percent) of lactate was partially oxidized to either acetate orpyruvate. The 13C isotope distributions in amino acids and other keymetabolites indicate that, under anaerobic conditions, a complete serinepathway is not present, and lactate is oxidized via a highly reversibleserine degradation pathway. The labeling data also suggest significantactivity in the anaplerotic (malic enzyme and phosphoenolpyruvatecarboxylase) and glyoxylate shunt (isocitrate lyase and malate synthase)reactions. Although the tricarboxylic acid (TCA) cycle is often observedto be incomplete in many other anaerobes (absence of 2-oxoglutaratedehydrogenase activity), isotopic labeling supports the existence of acomplete TCA cycle in S. oneidensis MR-1 under TMAO reductioncondition.

  1. Accumulation of amorphous Cr(III)-Te(IV) nanoparticles on the surface of Shewanella oneidensis MR-1 through reduction of Cr(VI).

    PubMed

    Kim, Dong-Hun; Park, Sunhwa; Kim, Min-Gyu; Hur, Hor-Gil

    2014-12-16

    Industrial effluents constitute a major source of metal pollution of aquatic bodies. Moreover, due to their environmental persistence, toxic metal pollution is of special concern. Microbial reduction is considered a promising strategy for toxic metal removal among the several methods available for metal remediation. Here, we describe the coremediation of toxic Cr(VI) and Te(IV) by the dissimilatory metal reducing bacterium-Shewanella oneidensis MR-1. In the presence of both Cr(VI) and Te(IV), S. oneidensis MR-1 reduced Cr(VI) to the less toxic Cr(III) form, but not Te(IV) to Te(0). The reduced Cr(III) ions complexed rapidly with Te(IV) ions and were precipitated from the cell cultures. Electron microscopic analyses revealed that the Cr-Te complexed nanoparticles localized on the bacterial outer membranes. K-edge X-ray absorption spectrometric analyses demonstrated that Cr(III) produced by S. oneidensis MR-1 was rapidly complexed with Te(IV) ions, followed by formation of amorphous Cr(III)-Te(IV) nanoparticles on the cell surface. Our results could be applied for the simultaneous sequestration and detoxification of both Cr(VI) and Te(IV) as well as for the preparation of nanomaterials through environmental friendly processes.

  2. Elucidating the Molecular Basis and Regulation of Chromium (VI) Reduction by Shewanella oneidensis MR-1 Using Biochemical, Genomic, and Proteomic Approaches

    SciTech Connect

    Hettich, Robert L.

    2006-10-30

    Although microbial metal reduction has been investigated intensively from physiological and biochemical perspectives, little is known about the genetic basis and regulatory mechanisms underlying the ability of certain bacteria to transform, detoxify, or immobilize a wide array of heavy metals contaminating DOE-relevant environments. The major goal of this work is to elucidate the molecular components comprising the chromium(VI) response pathway, with an emphasis on components involved in Cr(VI) detoxification and the enzyme complex catalyzing the terminal step in Cr(VI) reduction by Shewanella oneidensis MR-1. We have identified and characterized (in the case of DNA-binding response regulator [SO2426] and a putative azoreductase [SO3585]) the genes and gene products involved in the molecular response of MR-1 to chromium(VI) stress using whole-genome sequence information for MR-1 and recently developed proteomic technology, in particular liquid chromatographymass spectrometry (LC-MS), in conjunction with conventional protein purification and characterization techniques. The proteome datasets were integrated with information from whole-genome expression arrays for S. oneidensis MR-1 (as illustrated in Figure 1). The genes and their encoded products identified in this study are of value in understanding metal reduction and bacterial resistance to metal toxicity and in developing effective metal immobilization strategies.

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

    PubMed Central

    Newton, Gregory J.; Mori, Shigeki; Nakamura, Ryuhei; Hashimoto, Kazuhito; Watanabe, Kazuya

    2009-01-01

    Although members of the genus Shewanella have common features (e.g., the presence of decaheme c-type cytochromes [c-cyts]), they are widely variable in genetic and physiological features. The present study compared the current-generating ability of S. loihica PV-4 in microbial fuel cells (MFCs) with that of well-characterized S. oneidensis MR-1 and examined the roles of c-cyts in extracellular electron transfer. We found that strains PV-4 and MR-1 exhibited notable differences in current-generating mechanisms. While the MR-1 MFCs maintained a constant current density over time, the PV-4 MFCs continued to increase in current density and finally surpassed the MR-1 MFCs. Coulombic efficiencies reached 26% in the PV-4 MFC but 16% in the MR-1 MFCs. Although both organisms produced quinone-like compounds, anode exchange experiments showed that anode-attached cells of PV-4 produced sevenfold more current than planktonic cells in the same chamber, while planktonic cells of MR-1 produced twice the current of the anode-attached cells. Examination of the genome sequence indicated that PV-4 has more c-cyt genes in the metal reductase-containing locus than MR-1. Mutational analysis revealed that PV-4 relied predominantly on a homologue of the decaheme c-cyt MtrC in MR-1 for current generation, even though it also possesses two homologues of the decaheme c-cyt OmcA in MR-1. These results suggest that current generation in a PV-4 MFC is in large part accomplished by anode-attached cells, in which the MtrC homologue constitutes the main path of electrons toward the anode. PMID:19837834

  4. Ferrous phosphate surface precipitates resulting from the reduction of intragrain 6-line ferrihydrite by Shewanella oneidensis MR-1

    SciTech Connect

    Peretyazhko, Tetyana; Zachara, John M.; Kennedy, David W.; Fredrickson, Jim K.; Arey, Bruce W.; McKinley, James P.; Wang, Chong M.; Dohnalkova, Alice; Xia, Yuanxian

    2010-07-01

    The reductive biotransformation of 6-line ferrihydrite located within porous silica (intragrain ferrihydrite) by Shewanella oneidensis MR-1 was investigated and compared to the behavior of 6-line ferrihydrite in suspension (free ferrihydrite). The effect of buffer type (PIPES and NaHCO3), phosphate (P), and an electron shuttle (AQDS) on the extent of reduction and formation of Fe(II) secondary phases was investigated under anoxic conditions. Electron microscopy and micro X-ray diffraction were applied to evaluate the morphology and mineralogy of the biogenic precipitates and to study the distribution of microorganisms on the surface of porous silica after bioreduction. Kinetic reduction experiments with free and intragrain ferrihydrite revealed contrasting behaviour with respect to the buffer and presence of P. The overall amount of intragrain ferrihydrite reduction was less than that of free ferrihydrite [at 5 mmol L-1 Fe(III)T]. Reductive mineralization was not observed in the intragrain ferrihydrite incubations without P, and all biogenic Fe(II) concentrated in the aqueous phase. Irrespective of buffer and AQDS addition, rosettes of Fe(II) phosphate of approximate 20-30 μm size were observed on porous silica when P was present. The rosettes grew not only on the silica surface but also within it, forming a coherent spherical structure. These precipitates were well colonized by microorganisms and contained extracellular materials at the end of incubation. Microbial extracellular polymeric substances may have adsorbed Fe(II) promoting Fe(II) phosphate nucleation with subsequent crystal growth proceeding in different directions from a common center.

  5. Probing electron transfer mechanisms in Shewanella oneidensis MR-1 using a nanoelectrode platform and single-cell imaging.

    PubMed

    Jiang, Xiaocheng; Hu, Jinsong; Fitzgerald, Lisa A; Biffinger, Justin C; Xie, Ping; Ringeisen, Bradley R; Lieber, Charles M

    2010-09-28

    Microbial fuel cells (MFCs) represent a promising approach for sustainable energy production as they generate electricity directly from metabolism of organic substrates without the need for catalysts. However, the mechanisms of electron transfer between microbes and electrodes, which could ultimately limit power extraction, remain controversial. Here we demonstrate optically transparent nanoelectrodes as a platform to investigate extracellular electron transfer in Shewanella oneidensis MR-1, where an array of nanoholes precludes or single window allows for direct microbe-electrode contacts. Following addition of cells, short-circuit current measurements showed similar amplitude and temporal response for both electrode configurations, while in situ optical imaging demonstrates that the measured currents were uncorrelated with the cell number on the electrodes. High-resolution imaging showed the presence of thin, 4- to 5-nm diameter filaments emanating from cell bodies, although these filaments do not appear correlated with current generation. Both types of electrodes yielded similar currents at longer times in dense cell layers and exhibited a rapid drop in current upon removal of diffusible mediators. Reintroduction of the original cell-free media yielded a rapid increase in current to ∼80% of original level, whereas imaging showed that the positions of > 70% of cells remained unchanged during solution exchange. Together, these measurements show that electron transfer occurs predominantly by mediated mechanism in this model system. Last, simultaneous measurements of current and cell positions showed that cell motility and electron transfer were inversely correlated. The ability to control and image cell/electrode interactions down to the single-cell level provide a powerful approach for advancing our fundamental understanding of MFCs.

  6. Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1.

    PubMed

    Alves, Mónica N; Neto, Sónia E; Alves, Alexandra S; Fonseca, Bruno M; Carrêlo, Afonso; Pacheco, Isabel; Paquete, Catarina M; Soares, Cláudio M; Louro, Ricardo O

    2015-01-01

    The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB-OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC.

  7. Role of outer membrane c-type cytochromes MtrC and OmcA in Shewanella oneidensis MR-1 cell production, accumulation and detachment during respiration on hematite

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The iron-reducing bacterium Shewanella oneidensis MR-1 has the capacity to contribute to iron cycling over the long term by respiring on crystalline iron oxides such as hematite when poorly crystalline phases are depleted. The ability of outer membrane cytochromes OmcA and MtrC of MR-1 to bind to an...

  8. Elucidating the Molecular Basis and Regulation of Chromium(VI) Reduction by Shewanella oneidensis MR-1 and Resistance to Metal Toxicity Using Integrated Biochemical, Genomic, and Proteomic Approaches

    SciTech Connect

    Dorothea K. Thompson; Steven D. Brown; Robert L. Hettich; Nathan VerBerkmoes; Jizhong Zhou

    2004-03-17

    The mediation of metal reduction by microorganisms has been investigated intensively from physiological and biochemical perspectives; however, little is known about the genetic basis and regulatory mechanisms underlying the ability of certain bacteria to transform or immobilize a wide array of heavy metals contaminating DOE field sites. Chromium(VI), for example, is one of several risk-driving contaminants at DOE sites and has been targeted by the DOE for bioremediation research. The bacterium Shewanella oneidensis MR-1 can potentially be used to immobilize chromium, a toxic and mutagenic metal, by reducing soluble Cr(VI) to the insoluble and less bioavailable form of Cr(III), thus facilitating its removal from contained-storage and natural sites. The overall goal of this study is to integrate targeted biochemical and proteomic analyses with genome-wide gene expression profiling to examine the molecular basis and regulation of chromium(VI) reduction by Shewanella oneidensis MR-1. Towards this goal, we will (1) isolate and identify the terminal chromium(VI) reductase and the gene(s) encoding this activity using whole-genome sequence information for MR-1 and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with conventional protein purification and characterization techniques; (2) verify the function of the gene(s) encoding the terminal Cr(VI) reductase and compare whole transcriptome data with whole proteome data in order to understand the regulation of chromium reduction; and (3) investigate the molecular stress response and adaptation of S. oneidensis to toxic levels of soluble Cr(VI) and other heavy metals. This research will provide important information on the functional components and regulatory mechanisms of microbial metal reduction, which should prove valuable in developing effective assessment strategies for in situ bioremediation and genetically engineering desired bacteria for enhanced bioremediation.

  9. Kinetics of Dissimilatory Iron Reduction in Shewanella oneidensis MR-1: Scaling From Purified Proteins to Whole Cell Cultures

    NASA Astrophysics Data System (ADS)

    Brantley, S.; Ross, D.; Tien, M.

    2006-12-01

    To predict biogeochemical reactions in the field, it will be necessary to predict rates and processes from fundamental biochemical observations. Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe that has a versatile respiration system capable of utilizing a large number of terminal electron acceptors. Genetic studies have identified most if not all of the proteins involved in iron reduction; however, the mechanism of iron reduction is yet to be determined. Our research is focused on how this bacterium is able to utilize solid iron forms as its terminal electron acceptor; we are also investigating the reduction of chelated iron forms such as EDTA, NTA and ferric citrate for kinetic comparison. Solid iron oxides are the predominant form which iron is found in the earth's crust under aerobic and circumneutral pH conditions. In order to fully understand how this process works, we have taken a biochemical/kinetics approach. We have measured kinetic constants at three different scales: transient-state kinetic studies using a stop flow, steady-state kinetic from isolated membrane fractions and, whole cell kinetic. With the stop flow, we have measured rate constants between hemeproteins OmcA, MtrA and CctA with EDTA-Fe3+, NTA-Fe3+ and citrate-Fe3+. For steady state kinetics with membrane fractions, we have obtained kinetic constants with EDTA-Fe3+, NTA- Fe3+, citrate-Fe3+ and goethite. The rates are expressed as per mole of MtrC. The hemeprotein MtrC, thought to be the terminal iron reductase, was quantified through Western-blot analysis using polyclonal antibodies. Second order rate constants from stop flow studies are then compared to kcat/Km values obtained from steady state kinetic studies. Similar studies were also performed with whole cells and again normalized to MtrC content. The kinetic constants scale up well for the soluble iron forms but not for the insoluble iron form (goethite). Mechanistic implications from these scale up studies will be

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

    PubMed Central

    Fennessey, Christine M.; Jones, Morris E.; Taillefert, Martial; DiChristina, Thomas J.

    2010-01-01

    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. PMID:20190086

  11. Elucidating the Molecular Basis and Regulation of Chromium(VI) Reduction by Shewanella oneidensis MR-1 and Resistance to Metal Toxicity Using Integrated Biochemical, Genomic and Proteomic Approaches

    SciTech Connect

    Dorothea K. Thompson; Robert Hettich

    2007-02-06

    Shewanella oneidensis MR-1 is a model environmental organism that possesses diverse respiratory capacities, including the ability to reduce soluble Cr(VI) to sparingly soluble, less toxic Cr(III). Chromate is a serious anthropogenic pollutant found in subsurface sediment and groundwater environments due to its widespread use in defense and industrial applications. Effective bioremediation of chromate-contaminated sites requires knowledge of the molecular mechanisms and regulation of heavy metal resistance and biotransformation by dissimilatory metal-reducing bacteria. Towards this goal, our ERSP-funded work was focused on the identification and functional analysis of genes/proteins comprising the response pathways for chromate detoxification and/or reduction. Our work utilized temporal transcriptomic profiling and whole-cell proteomic analyses to characterize the dynamic molecular response of MR-1 to an acute chromate shock (up to 90 min) as well as to a 24-h, low-dose exposure. In addition, we have examined the transcriptome of MR-1 cells actively engaged in chromate reduction. These studies implicated the involvement of a functionally undefined DNA-binding response regulator (SO2426) and a putative azoreductase (SO3585) in the chromate stress response of MR-1.

  12. 13C Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells

    PubMed Central

    Luo, Shuai; Guo, Weihua; H. Nealson, Kenneth; Feng, Xueyang; He, Zhen

    2016-01-01

    Microbial fuel cell (MFC) is a promising technology for direct electricity generation from organics by microorganisms. The type of electron donors fed into MFCs affects the electrical performance, and mechanistic understanding of such effects is important to optimize the MFC performance. In this study, we used a model organism in MFCs, Shewanella oneidensis MR-1, and 13C pathway analysis to investigate the role of formate in electricity generation and the related microbial metabolism. Our results indicated a synergistic effect of formate and lactate on electricity generation, and extra formate addition on the original lactate resulted in more electrical output than using formate or lactate as a sole electron donor. Based on the 13C tracer analysis, we discovered decoupled cell growth and electricity generation in S. oneidensis MR-1 during co-utilization of lactate and formate (i.e., while the lactate was mainly metabolized to support the cell growth, the formate was oxidized to release electrons for higher electricity generation). To our best knowledge, this is the first time that 13C tracer analysis was applied to study microbial metabolism in MFCs and it was demonstrated to be a valuable tool to understand the metabolic pathways affected by electron donors in the selected electrochemically-active microorganisms. PMID:26868848

  13. ¹³C Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells.

    PubMed

    Luo, Shuai; Guo, Weihua; Nealson, Kenneth H; Feng, Xueyang; He, Zhen

    2016-02-12

    Microbial fuel cell (MFC) is a promising technology for direct electricity generation from organics by microorganisms. The type of electron donors fed into MFCs affects the electrical performance, and mechanistic understanding of such effects is important to optimize the MFC performance. In this study, we used a model organism in MFCs, Shewanella oneidensis MR-1, and (13)C pathway analysis to investigate the role of formate in electricity generation and the related microbial metabolism. Our results indicated a synergistic effect of formate and lactate on electricity generation, and extra formate addition on the original lactate resulted in more electrical output than using formate or lactate as a sole electron donor. Based on the (13)C tracer analysis, we discovered decoupled cell growth and electricity generation in S. oneidensis MR-1 during co-utilization of lactate and formate (i.e., while the lactate was mainly metabolized to support the cell growth, the formate was oxidized to release electrons for higher electricity generation). To our best knowledge, this is the first time that (13)C tracer analysis was applied to study microbial metabolism in MFCs and it was demonstrated to be a valuable tool to understand the metabolic pathways affected by electron donors in the selected electrochemically-active microorganisms.

  14. 13C Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells

    NASA Astrophysics Data System (ADS)

    Luo, Shuai; Guo, Weihua; H. Nealson, Kenneth; Feng, Xueyang; He, Zhen

    2016-02-01

    Microbial fuel cell (MFC) is a promising technology for direct electricity generation from organics by microorganisms. The type of electron donors fed into MFCs affects the electrical performance, and mechanistic understanding of such effects is important to optimize the MFC performance. In this study, we used a model organism in MFCs, Shewanella oneidensis MR-1, and 13C pathway analysis to investigate the role of formate in electricity generation and the related microbial metabolism. Our results indicated a synergistic effect of formate and lactate on electricity generation, and extra formate addition on the original lactate resulted in more electrical output than using formate or lactate as a sole electron donor. Based on the 13C tracer analysis, we discovered decoupled cell growth and electricity generation in S. oneidensis MR-1 during co-utilization of lactate and formate (i.e., while the lactate was mainly metabolized to support the cell growth, the formate was oxidized to release electrons for higher electricity generation). To our best knowledge, this is the first time that 13C tracer analysis was applied to study microbial metabolism in MFCs and it was demonstrated to be a valuable tool to understand the metabolic pathways affected by electron donors in the selected electrochemically-active microorganisms.

  15. Comparative Temporal Proteomics of a Response Regulator (SO2426)-Deficient Strain and Wild-Type Shewanella oneidensis MR-1 During Chromate Transformation

    SciTech Connect

    Chourey, Karuna; Thompson, Melissa R; Shah, Manesh B; Zhang, Bing; Verberkmoes, Nathan C; Thompson, Dorothea K.; Hettich, Robert {Bob} L

    2009-01-01

    Predicted orphan response regulators encoded in the Shewanella oneidensis MR-1 genome are poorly understood from a cellular function perspective. Our previous transcriptomic and proteomic analyses demonstrated that an annotated DNA-binding response regulator, SO2426, was significantly up-regulated in wild-type S. oneidensis cells at both themRNAand protein levels in response to acute chromate [Cr(VI)] challenge, suggesting a potential regulatory role for this protein in metal stress pathways. To investigate the impact of SO2426 activity on chromate stress response at a genome-wide scale, we describe here comparative and temporal proteome characterizations using multidimensional HPLC-MS/MS and statistical analysis to identify differentially expressed proteins in biological replicates of wild-type S. oneidensis MR-1 and a so2426 deletion ( so2426) strain, which exhibited an impaired Cr(VI) transformation rate compared to that of the parental strain. Global protein profiles were examined at different time intervals (0, 1, 3, 4 h) following exogenous chromate challenge. Results indicated that deletion of the so2426 gene negatively affected expression of a small protein subset (27 proteins) including those with annotated functions in siderophore biosynthesis (SO3032), Fe uptake (SO4743), intracellular Fe storage (Bfr1), and other transport processes. Cr(VI) exposure and subsequent ransformation dramatically increased the number of differentially expressed proteins detected,with up-regulated bundance patterns observed largely for proteins involved in general stress protection and detoxification trategies, cell motility, and protein fate. In addition, the proteome data sets were mined for amino acids with otential post-translational modifications (PTMs) indicative of a level of gene expression regulation extending eyond the transcriptional control imposed by SO2426.

  16. Electrochemical interaction of Shewanella oneidensis MR-1 and its outer membrane cytochromes OmcA and MtrC with hematite electrodes

    SciTech Connect

    Meitl, Leisa A.; Eggleston, Carrick M.; Colberg, Patricia J.; Khare, Nidhi; Reardon, Catherine L.; Shi, Liang

    2009-09-15

    Bacterial metal reduction is an important biogeochemical process in anaerobic environments. An understanding of electron transfer pathways from dissimilatory metal reducing bacteria (DMRB) to solid phase metal (hydr)oxides is important for understanding metal redox cycling in soils and sediments, for utilizing DMRB in bioremedation, and for developing technologies such as microbial fuel cells. Here we hypothesize that the outer membrane cytochromes OmcA and MtrC from Shewanella oneidensis MR-1 are the only terminal reductases capable of direct electron transfer to a hematite working electrode. Cyclic voltammetry (CV) was used to study electron transfer between hematite electrodes and protein films, S. oneidensis MR-1 wild-type cell suspensions, and cytochrome deletion mutants. After controlling for hematite electrode dissolution at negative potential, the midpoint potentials of adsorbed OmcA and MtrC were measured (-201 mV and -163 mV vs. Ag/AgCl, respectively). Cell suspensions of wild-type MR-1, deletion mutants deficient in OmcA (ΔomcA), MtrC (ΔmtrC), and both OmcA and MtrC (ΔmtrC-ΔomcA) were also studied; voltammograms for ΔmtrC-ΔomcA were indistinguishable from the control. When the control was subtracted from the single deletion mutant voltammograms, redox peaks were consistent with the present cytochrome (i.e., ΔomcA consistent with MtrC and ΔmtrC consistent with OmcA). The results indicate that OmcA and MtrC are capable of direct electron exchange with hematite electrodes, consistent with a role as terminal reductases in the S. oneidensis MR-1 anaerobic respiratory pathway involving ferric minerals. There was no evidence for other terminal reductases operating under the conditions investigated. A Marcus-based approach to electron transfer kinetics indicated that the rate constant for electron transfer ket varies from 0.025 s-1 in the absence of a barrier to 63.5 s-1 with a 0.2 eV barrier.

  17. Shewanella Oneidensis MR-1 Msh Pilin Proteins are Involved in Extracellular Electron Transfer in Microbial Fuel Cells

    DTIC Science & Technology

    2011-01-01

    comparison of the 16 MR-1 Msh pilin complex proteins to Vibrio cholerae O1 biovar El Tor (tax id: 686) using the BLAST-search algorithm [21] for proteins... Vibrio cholerae mannose-sensitive hemagglutinin type 4 pilus gene locus. J Bacteriol 1999;181:1110–7. [23] Ringeisen BR, Henderson E, Wu PK...BLASTp) with default algorithm parameters. Sequence homology of MR-1 Msh proteins to the well-studied Msh pilin complex from V. cholera [22] allowed

  18. Crystallization and preliminary X-ray crystallographic studies of the outer membrane cytochrome OmcA from Shewanella oneidensis MR-1

    SciTech Connect

    Tomanicek, S.J.; Johs, A.; Sawhney, M.S.; Shi, L.; Liang, L.

    2012-05-24

    The outer membrane cytochrome OmcA functions as a terminal metal reductase in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. The ten-heme centers shuttle electrons from the transmembrane donor complex to extracellular electron acceptors. Here, the crystallization and preliminary crystallographic analysis of OmcA are reported. Crystals of OmcA were grown by the sitting-drop vapor-diffusion method using PEG 20 000 as a precipitant. The OmcA crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 93.0, b = 246.0, c = 136.6 {angstrom}, = 90, {beta} = 97.8, {gamma} = 90{sup o}. X-ray diffraction data were collected to a maximum resolution of 3.25 {angstrom}.

  19. Crystallization and preliminary X-ray crystallographic studies of the outer membrane cytochrome OmcA from Shewanella oneidensis MR-1

    SciTech Connect

    Tomanicek, S. J.; Johs, Alexander; Sawhney, M. S.; Shi, Liang; Liang, L.

    2012-01-01

    The outer membrane cytochrome OmcA functions as a terminal metal reductase in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. The ten-heme centers shuttle electrons from the transmembrane donor complex to extracellular electron acceptors. Here, the crystallization and preliminary crystallographic analysis of OmcA are reported. Crystals of OmcA were grown by the sitting-drop vapor-diffusion method using PEG 20 000 as a precipitant. The OmcA crystals belonged to space group P21, with unit-cell parameters a = 93.0, b = 246.0, c = 136.6 A ° , * = 90, * = 97.8, * = 90*. X-ray diffraction data were collected to a maximum resolution of 3.25 A ° .

  20. Identifying the role of cytochromes upon the attachment, growth and detachment of Shewanella oneidensis MR-1 on hematite during dissimilatory iron reduction under natural- flow conditions

    NASA Astrophysics Data System (ADS)

    Mitchell, A. C.; Geesey, G. G.

    2006-12-01

    Current understanding of bacterial respiration by dissimilatory iron (Fe) reduction is based primarily on studies of closed systems using soluble Fe(III). However, natural environments likely to support Fe reduction are typically open systems and contain Fe(III) primarily in the form of crystalline (hydr)oxides. Mechanisms by which electrons are transported between bacteria and mineral terminal electron acceptors (TEAs) under open system conditions are still poorly understood. However, a number of cytochromes have been identified as potentially playing a critical role in the electron transport system of some Fe reducing bacteria. Experiments were performed using (i) omcA, (ii) mtrC, or (iii) omcA and mtrC cytochrome deficient mutants of the Fe-reducing bacteria, Shewanella oneidensis MR-1, in transparent-window flow- reactors containing hematite as the only TEA. These were operated under defined hydrodynamic and anaerobic conditions. Cells expressed green fluorescent protein (gfp), allowing real time measurement of cells at the mineral surface by epifluorescence microscopy. Cytochromes which play a critical role in the anaerobic growth of S. Oneidensis by Fe reduction under open system natural-flow conditions could then be identified. Differences in the accumulation, maximum density, detachment and total production of surface-associated cells growing on hematite surfaces were apparent between the mutants, and between the mutants and the wild-type. Mutants deficient in cytochromes grew to a lower max density by up to 2 orders of magnitude than the wild-type, and exhibited no reduced Fe in the reactor effluent or at the surface of the hematite at the conclusion of the experiment, as revealed by X-Ray photoelectron spectroscopy (XPS). Therefore omcA and / or mtrC cytochromes appear critical for electron shuttling and anaerobic growth of S. Oneidensis on hematite under natural-flow conditions.

  1. Specific Bonds between an Iron Oxide Surface and Outer Membrane Cytochromes MtrC and OmcA from Shewanella oneidensis MR-1

    SciTech Connect

    Lower, Brian H.; Shi, Liang; Yongsunthon, Ruchirej; Droubay, Timothy C.; Mccready, David E.; Lower, Steven

    2007-07-31

    Shewanella oneidensis MR-1 is purported to express outer membrane cytochromes (e.g., MtrC and OmcA) that transfer electrons directly to Fe(III) in a mineral during anaerobic respiration.  A prerequisite for this type of reaction would be the formation of a stable bond between a cytochrome and an iron oxide surface.  Atomic force microscopy (AFM) was used to detect whether a specific bond forms between a hematite (Fe2O3) thin film, created with oxygen plasma assisted molecular beam epitaxy (MBE), and recombinant MtrC or OmcA molecules coupled to gold substrates.  Force spectra displayed a unique force signature indicative of a specific bond between each cytochrome and the hematite surface.  The strength of the OmcA-hematite bond was approximately twice as strong as the MtrC-hematite bond, but direct binding to hematite was twice as favorable for MtrC.  Reversible folding/unfolding reactions were observed for mechanically denatured MtrC molecules bound to hematite.  The force measurements for the hematite-cytochrome pairs were compared to spectra collected between an iron oxide and S. oneidensis under anaerobic conditions.  There is a strong correlation between the whole cell and pure protein force spectra suggesting that the unique binding attributes of each cytochrome complement one another and allow both MtrC and OmcA to play a prominent role in the transfer of electrons to Fe(III) in minerals.  Finally, by comparing the magnitude of binding force for the whole cell vs. pure protein data, we were able to estimate that a single bacterium of S. oneidensis (2 x 0.5 μm) expresses ~104 cytochromes on its outer surface. 

  2. Chromate/Nitrite Interactions in Shewanella Oneidensis MR-1: Evidence for Multiple Cr(VI) Reduction Mechanisms Dependent on Physiological Growth Conditions

    SciTech Connect

    Apel, William Arnold; Viamajala, S.; Peyton, Brent Michael; Petersen, J. N.

    2002-06-01

    Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was also observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple

  3. A gold-sputtered carbon paper as an anode for improved electricity generation from a microbial fuel cell inoculated with Shewanella oneidensis MR-1.

    PubMed

    Sun, Min; Zhang, Feng; Tong, Zhong-Hua; Sheng, Guo-Ping; Chen, Yong-Zhen; Zhao, Yue; Chen, You-Peng; Zhou, Shi-Yue; Liu, Gang; Tian, Yang-Chao; Yu, Han-Qing

    2010-10-15

    Gold is among the highly conductive and stable materials, which are ideal anodes for microbial fuel cells (MFCs). However, previous studies have shown that bare gold surface is recalcitrant for the colonization of some exoelectrogens, e.g., Shewanella putrefacians. In this work, the problem regarding the poor bio-compatibility of gold as an anode material was sorted out through coupling it with carbon paper. A new composite anode material was fabricated through sputtering gold layer homogeneously on carbon paper matrix. Results of cyclic voltammetry and electrochemical impedance spectroscopy in Fe(CN)6(3-/4-) solution demonstrated better electrochemical performance of the carbon paper-gold (C-Au) composite than either carbon paper or bare gold, when they were used in MFCs. With Shewanella oneidensis MR-1 as the inoculum, the C-Au anode-based MFC produced total electric charges higher than the carbon-paper-anode-based MFC by 47%. The cyclic voltammetry analysis and the scanning electron microscopy observation showed that the MR-1 biofilm growth was accelerated when the carbon paper surface was sputtered with gold. Utilization of such a carbon paper-gold composite significantly enhanced the MFC performance.

  4. Global Molecular and Morphological Effects of 24-Hour Chromium(VI)Exposure on Shewanella oneidensis MR-1

    SciTech Connect

    Chourey, Karuna; Thompson, Melissa R; Morrell-Falvey, Jennifer L; Verberkmoes, Nathan C; Brown, Steven D; Shah, Manesh B; Zhou, Jizhong; Doktycz, Mitchel John; Hettich, Robert {Bob} L; Thompson, Dorothea K

    2006-01-01

    The biological impact of 24-h ("chronic") chromium(VI) [Cr(VI) or chromate] exposure on ShewanellaoneidensisMR-1 was assessed by analyzing cellular morphology as well as genome-wide differential gene and protein expression profiles. Cells challenged aerobically with an initial chromate concentration of 0.3 mM in complex growth medium were compared to untreated control cells grown in the absence of chromate. At the 24-h time point at which cells were harvested for transcriptome and proteome analyses, no residual Cr(VI) was detected in the culture supernatant, thus suggesting the complete uptake and/or reduction of this metal by cells. In contrast to the untreated control cells, Cr(VI)-exposed cells formed apparently aseptate, nonmotile filaments that tended to aggregate. Transcriptome profiling and mass spectrometry-based proteomic charac terization revealed that the principal molecular response to 24-h Cr(VI) exposure was the induction of prophage-related genes and their encoded products as well as a number of functionally undefined hypothetical genes that were located within the integrated phage regions of the MR-1 genome. In addition, genes with annotated functions in DNA metabolism, cell division, biosynthesis and degradation of the murein (pepti doglycan) sacculus, membrane response, and general environmental stress protection were upregulated, while genes encoding chemotaxis, motility, and transport/binding proteins were largely repressed under conditions of 24-h chromate treatment.

  5. SO2907, A Putative TonB-dependent Receptor, Is Involved in Dissimilatory Iron Reduction by Shewanella oneidensis Strain MR-1

    SciTech Connect

    Qian, Yufeng; Shi, Liang; Tien, Ming

    2011-09-30

    Shewanella oneidensis strain MR-1 utilizes soluble and insoluble ferric ions as terminal electron acceptors during anaerobic respiration. The components of respiratory metabolism are localized in the membrane fractions which include the outer membrane and cytoplasmic membrane. Many of the biological components that interact with the various iron forms are proposed to be localized in these membrane fractions. To identify the iron-binding proteins acting either as an iron transporter or as a terminal iron reductase, we used metal-catalyzed oxidation reactions. This system catalyzed the oxidation of amino acids in close proximity to the iron binding site. The carbonyl groups formed from this oxidation can then be labeled with fluoresceinamine (FLNH2). The peptide harboring the FLNH2 can then be proteolytically digested, purified by HPLC and then identified by MALDI-TOF tandem MS. A predominant peptide was identified to be part of SO2907 that encodes a putative TonB-dependent receptor. Compared to wild type (wt), the so2097 gene deletion (ΔSO2907) mutant has impaired ability to reduce soluble Fe(III), but retains the same ability to respire oxygen or fumarate as the wt. The ΔSO2907 mutant was also impacted in reduction of insoluble iron. Iron binding assays using isothermal titration calorimetry and fluorescence tryptophan quenching demonstrated that a truncated form of heterologous-expressed SO2907 that contains the Fe(III) binding site, is capable of binding soluble Fe(III) forms with Kd of approximate 50 μM. To the best of our knowledge, this is the first report of the physiological role of SO2907 in Fe(III) reduction by MR-1.

  6. Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1

    NASA Technical Reports Server (NTRS)

    Tsapin, A. I.; Vandenberghe, I.; Nealson, K. H.; Scott, J. H.; Meyer, T. E.; Cusanovich, M. A.; Harada, E.; Kaizu, T.; Akutsu, H.; Leys, D.; Van Beeumen, J. J.

    2001-01-01

    Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

  7. A Method Adapting Microarray Technology for Signature-Tagged Mutagenesis of Desulfovibrio desulfuricans G20 and Shewanella oneidensis MR-1 in Anaerobic Sediment Survival Experiments†

    PubMed Central

    Groh, Jennifer L.; Luo, Qingwei; Ballard, Jimmy D.; Krumholz, Lee R.

    2005-01-01

    Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn10 transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized Desulfovibrio desulfuricans G20 in addition to Shewanella oneidensis MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats. PMID:16269742

  8. Enrichment of Functional Redox Reactive Proteins and Identification by Mass Spectrometry Results in Several Terminal Fe(III)-reducing Candidate Proteins in Shewanella oneidensis MR-1.

    SciTech Connect

    Elias, Dwayne A.; Yang, Feng; Mottaz, Heather M.; Beliaev, Alex S.; Lipton, Mary S.

    2007-02-01

    Identification of the proteins directly involved in microbial metal-reduction is important to understanding the biochemistry involved in heavy metal reduction/immobilization and the ultimate cleanup of DOE contaminated sites. Although previous strategies for the identification of these proteins have traditionally required laborious protein purification/characterization of metal-reducing capability, activity is often lost before the final purification step, thus creating a significant knowledge gap. In the current study, subcellular fractions of S. oneidensis MR-1 were enriched for Fe(III)-NTA reducing proteins in a single step using several orthogonal column matrices. The protein content of eluted fractions that demonstrated activity were determined by ultra high pressure liquid chromatography coupled with tandem mass spectrometry (LCMS/ MS). A comparison of the proteins identified from active fractions in all separations produced 30 proteins that may act as the terminal electron-accepting protein for Fe(III)-reduction. These include MtrA, MtrB, MtrC and OmcA as well as a number of other proteins not previously associated with Fe(III)-reduction. This is the first report of such an approach where the laborious procedures for protein purification are not required for identification of metal-reducing proteins. Such work provides the basis for a similar approach with other cultured organisms as well as analysis of sediment and groundwater samples from biostimulation efforts at contaminated sites.

  9. A method adapting microarray technology for signature tagged mutagenesis of Dusulfovibrio dusulfuricans G20 and Shewanella oneidensis MR-1 in anaerobic sediment survival experiments

    USGS Publications Warehouse

    Groh, Jennifer L.; Luo, Qingwei; Ballard , Jimmy D.; Krumholz, Lee R.

    2005-01-01

    Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn10 transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized Desulfovibrio desulfuricans G20 in addition to Shewanella oneidensis MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats.

  10. Crystallization of uridine phosphorylase from Shewanella oneidensis MR-1 in the laboratory and under microgravity and preliminary X-ray diffraction analysis.

    PubMed

    Safonova, Tatyana N; Mordkovich, Nadezhda N; Polyakov, Konstantin M; Manuvera, Valentin A; Veiko, Vladimir P; Popov, Vladimir O

    2012-11-01

    Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP from Shewanella oneidensis MR-1 was performed in the recipient strain Escherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296 K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9 Å from crystals of the free form grown on earth, 1.6 Å from crystals of the complex with uridine and 0.95 Å from crystals of the free form grown under microgravity. All crystals belong to the space group P2(1) and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data.

  11. Role of Outer-Membrane Cytochromes MtrC and OmcA in the Biomineralization of Ferrihydrite by Shewanella oneidensis MR-1.

    SciTech Connect

    Reardon, Catherine L.; Dohnalkova, Alice; Nachimuthu, Ponnusamy; Kennedy, David W.; Saffarini, Daad; Arey, Bruce W.; Shi, Liang; Wang, Zheming; Moore, Dean A.; Mclean, Jeffrey S.; Moyles, Dianne M.; Marshall, Matthew J.; Zachara, John M.; Fredrickson, Jim K.; Beliaev, Alex S.

    2010-01-01

    In an effort to improve the understanding of electron transfer mechanisms at the microbe-mineral interface, Shewanella oneidensis MR-1 mutants with in-frame deletions of outer membrane cytochrome genes mtrC, omcA, or both, were characterized for the ability to reduce metal oxides using a suite of microscopic, spectroscopic, and biochemicalr techniques. The results indicate that neither MtrC nor OmcA are essential for the reduction of soluble, complexed Fe(III)-citrate or Fe(III)-NTA; however, at least one of these outer membrane cytochromes is required for the reduction of Fe(III)- and Mn(III/IV)- oxides. In vitro analysis of purified, recombinant protein demonstrated that both cytochromes transfer electrons directly to metal-oxides; however, MtrC transfers electrons at a faster rate than OmcA. Immunolocalization of MtrC and OmcA reveal that both cytochromes are surface-exposed on the cell outer-membrane and co-localize with insoluble iron precipitates when respiring ferrihydrite or cultured aerobically with Fe(III)-citrate. Additionally, during prolonged incubation, wild-type cells promoted biotransformation of ferrihydrite to vivianite [Fe3(PO4)2•8H2O] while the double cytochrome mutant was unable to form any secondary mineral phases. Collectively, our results support a role for direct electron transfer from OMCs to metal oxides by establishing their in vitro electron transfer activities, confirming the requirement of either MtrC or OmcA for in vivo reductive biomineralization of ferrihydrite, and localizing the cytochromes to the cell exterior where they can directly contact mineral substrates.

  12. Effects of Incubation Conditions on Cr(VI) Reduction by c-type Cytochromes in Intact Shewanella oneidensis MR-1 Cells

    PubMed Central

    Han, Rui; Li, Fangbai; Liu, Tongxu; Li, Xiaomin; Wu, Yundang; Wang, Ying; Chen, Dandan

    2016-01-01

    It is widely recognized that the outer membrane c-type cytochromes (OM c-Cyts) of metal-reducing bacteria play a key role in microbial metal reduction processes. However, the in situ redox status of OM c-Cyts during microbial metal reduction processes remain poorly understood. In this study, diffuse-transmission UV/Vis spectroscopy is used to investigate the in situ spectral reaction of Cr(VI) reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions. The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time. The Cr(VI) reduction rates decreased with increasing initial Cr(VI) concentrations, and Cr(III) was identified as a reduced product. The presence of Cr(III) substantially inhibited Cr(VI) reduction and the recovery of reduced c-Cyts, indicating that Cr(III) might inhibit cell growth. Cr(VI) reduction rates increased with increasing cell density. The highest Cr(VI) reduction rate and fastest recovery of c-Cyts were obtained at pH 7.0 and 30°C, with sodium lactate serving as an electron donor. The presence of O2 strongly inhibited Cr(VI) reduction, suggesting that O2 might compete with Cr(VI) as an electron acceptor in cells. This study provides a case of directly examining in vivo reaction properties of an outer-membrane enzyme during microbial metal reduction processes under non-invasive physiological conditions. PMID:27242759

  13. Oxygen Tension and Riboflavin Gradients Cooperatively Regulate the Migration of Shewanella oneidensis MR-1 Revealed by a Hydrogel-Based Microfluidic Device

    PubMed Central

    Kim, Beum Jun; Chu, Injun; Jusuf, Sebastian; Kuo, Tiffany; TerAvest, Michaela A.; Angenent, Largus T.; Wu, Mingming

    2016-01-01

    Shewanella oneidensis is a model bacterial strain for studies of bioelectrochemical systems (BESs). It has two extracellular electron transfer pathways: (1) shuttling electrons via an excreted mediator riboflavin; and (2) direct contact between the c-type cytochromes at the cell membrane and the electrode. Despite the extensive use of S. oneidensis in BESs such as microbial fuel cells and biosensors, many basic microbiology questions about S. oneidensis in the context of BES remain unanswered. Here, we present studies of motility and chemotaxis of S. oneidensis under well controlled concentration gradients of two electron acceptors, oxygen and oxidized form of riboflavin (flavin+), using a newly developed microfluidic platform. Experimental results demonstrate that either oxygen or flavin+ is a chemoattractant to S. oneidensis. The chemotactic tendency of S. oneidensis in a flavin+ concentration gradient is significantly enhanced in an anaerobic in contrast to an aerobic condition. Furthermore, either a low oxygen tension or a high flavin+ concentration considerably enhances the speed of S. oneidensis. This work presents a robust microfluidic platform for generating oxygen and/or flavin+ gradients in an aqueous environment, and demonstrates that two important electron acceptors, oxygen and oxidized riboflavin, cooperatively regulate S. oneidensis migration patterns. The microfluidic tools presented as well as the knowledge gained in this work can be used to guide the future design of BESs for efficient electron production. PMID:27703448

  14. Transcriptional analysis of Shewanella oneidensis MR-1 with an electrode compared to Fe(III)citrate or oxygen as terminal electron acceptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background. Shewanella oneidensis is a target of extensive research efforts in the fields of bioelectrochemical systems and bioremediation because of its versatile metabolic capabilities, especially in regards to the respiration with extracellular electron acceptors. Here, we took a global approach ...

  15. A mechanistic study of TiO2 nanoparticle toxicity on Shewanella oneidensis MR-1 with UV-containing simulated solar irradiation: Bacterial growth, riboflavin secretion, and gene expression.

    PubMed

    Qiu, Tian A; Meyer, Ben M; Christenson, Ky G; Klaper, Rebecca D; Haynes, Christy L

    2017-02-01

    Toxicity of nanomaterials to ecological systems has recently emerged as an important field of research, and thus, many researchers are exploring the mechanisms of how nanoparticles impact organisms. Herein, we probe the mechanisms of bacteria-nanoparticle interaction by investigating how TiO2 nanoparticles impact a model organism, the metal-reducing bacterium Shewanella oneidensis MR-1. In addition to examining the effect of TiO2 exposure, the effect of synergistic simulated solar irradiation containing UV was explored in this study, as TiO2 nanoparticles are known photocatalysts. The data reveal that TiO2 nanoparticles cause an inhibition of S. oneidensis growth at high dosage without compromising cell viability, yet co-exposure of nanoparticles and illumination does not increase the adverse effects on bacterial growth relative to TiO2 alone. Measurements of intracellular reactive oxygen species and riboflavin secretion, on the same nanoparticle-exposed bacteria, reveal that TiO2 nanoparticles have no effect on these cell functions, but application of UV-containing illumination with TiO2 nanoparticles has an impact on the level of riboflavin outside bacterial cells. Finally, gene expression studies were employed to explore how cells respond to TiO2 nanoparticles and illumination, and these results were correlated with cell growth and cell function assessment. Together these data suggest a minimal impact of TiO2 NPs and simulated solar irradiation containing UV on S. oneidensis MR-1, and the minimal impact could be accounted for by the nutrient-rich medium used in this work. These measurements demonstrate a comprehensive scheme combining various analytical tools to enable a mechanistic understanding of nanoparticle-cell interactions and to evaluate the potential adverse effects of nanoparticles beyond viability/growth considerations.

  16. Kinetics of Reduction of Fe(III) Complexes by Outer Membrane Cytochromes MtrC and OmcA of Shewanella oneidensis MR-1

    SciTech Connect

    Wang, Zheming; Liu, Chongxuan; Wang, Xuelin; Marshall, Matthew J.; Zachara, John M.; Rosso, Kevin M.; Dupuis, Michel; Fredrickson, Jim K.; Heald, Steve M.; Shi, Liang

    2008-09-12

    Shewanella Oneidensis MR-1 possesses up to 42 c-type cytochromes with heme content varying between 1 to as many as 37. Among them, the outer-membrane cytochromes, particularly MtrC and OmcA, are suspected to function as terminal reductases and are responsible for its enzymatic catalysis capability. So far, the mechanisms of metal reduction by these outer-membrane cytochromes are unknown. In this work, we report the study of reduction kinetics of a series of Fe(III) complexes with citrate, NTA and EDTA by abiotically reduced MtrC and OmcA using a stopped-flow technique in combination with theoretical computation methods within the framework of the electron transfer theory of Marcus and speciation calculations based on the current thermodynamic database. Stopped-flow kinetic data showed that the reaction was very fast and appeared to proceed in two stages, a fast stage that completes in much less than a second and a slower stage afterwards. For a given complex, the reaction is faster by reduction with MtrC than OmcA, while for a given protein, the reaction completes in the decreasing order of Fe-EDTA > Fe-NTA > Fe-citrate. All the stopped-flow kinetic curves could be modeled by two parallel second-order bimolecular redox reactions with second-order rate constants ranging from 0.872 µM-1s-1 for the fast reaction between MtrC with Fe-EDTA complex to 0.012 µM-1s-1 for the slow reaction between OmcA and Fe-citrate complex. Speciation calculations indicated that at both metal:ligand ratios, 1:1.5 and 1:10, a single dominant ferric complex was responsible for the observed reaction for each ligand and, therefore, the observed dual-reaction pathways was attributed to the differences in the reduction behavior among various heme groups within each protein. The results of redox potential calculations with known thermodynamic data show only small differences on the scale of a few millivolts among the three complexes, suggested that

  17. Anaerobic Respiration of Elemental Sulfur and Thiosulfate by Shewanella oneidensis MR-1 Requires psrA, a Homolog of the phsA Gene of Salmonella enterica Serovar Typhimurium LT2▿ †

    PubMed Central

    Burns, Justin L.; DiChristina, Thomas J.

    2009-01-01

    Shewanella oneidensis MR-1, a facultatively anaerobic gammaproteobacterium, respires a variety of anaerobic terminal electron acceptors, including the inorganic sulfur compounds sulfite (SO32−), thiosulfate (S2O32−), tetrathionate (S4O62−), and elemental sulfur (S0). The molecular mechanism of anaerobic respiration of inorganic sulfur compounds by S. oneidensis, however, is poorly understood. In the present study, we identified a three-gene cluster in the S. oneidensis genome whose translated products displayed 59 to 73% amino acid similarity to the products of phsABC, a gene cluster required for S0 and S2O32− respiration by Salmonella enterica serovar Typhimurium LT2. Homologs of phsA (annotated as psrA) were identified in the genomes of Shewanella strains that reduce S0 and S2O32− yet were missing from the genomes of Shewanella strains unable to reduce these electron acceptors. A new suicide vector was constructed and used to generate a markerless, in-frame deletion of psrA, the gene encoding the putative thiosulfate reductase. The psrA deletion mutant (PSRA1) retained expression of downstream genes psrB and psrC but was unable to respire S0 or S2O32− as the terminal electron acceptor. Based on these results, we postulate that PsrA functions as the main subunit of the S. oneidensis S2O32− terminal reductase whose end products (sulfide [HS−] or SO32−) participate in an intraspecies sulfur cycle that drives S0 respiration. PMID:19542325

  18. Anaerobic respiration of elemental sulfur and thiosulfate by Shewanella oneidensis MR-1 requires psrA, a homolog of the phsA gene of Salmonella enterica serovar typhimurium LT2.

    PubMed

    Burns, Justin L; DiChristina, Thomas J

    2009-08-01

    Shewanella oneidensis MR-1, a facultatively anaerobic gammaproteobacterium, respires a variety of anaerobic terminal electron acceptors, including the inorganic sulfur compounds sulfite (SO3(2-)), thiosulfate (S2O3(2-)), tetrathionate (S4O6(2-)), and elemental sulfur (S(0)). The molecular mechanism of anaerobic respiration of inorganic sulfur compounds by S. oneidensis, however, is poorly understood. In the present study, we identified a three-gene cluster in the S. oneidensis genome whose translated products displayed 59 to 73% amino acid similarity to the products of phsABC, a gene cluster required for S(0) and S2O3(2-) respiration by Salmonella enterica serovar Typhimurium LT2. Homologs of phsA (annotated as psrA) were identified in the genomes of Shewanella strains that reduce S(0) and S2O3(2-) yet were missing from the genomes of Shewanella strains unable to reduce these electron acceptors. A new suicide vector was constructed and used to generate a markerless, in-frame deletion of psrA, the gene encoding the putative thiosulfate reductase. The psrA deletion mutant (PSRA1) retained expression of downstream genes psrB and psrC but was unable to respire S(0) or S2O3(2-) as the terminal electron acceptor. Based on these results, we postulate that PsrA functions as the main subunit of the S. oneidensis S2O3(2-) terminal reductase whose end products (sulfide [HS-] or SO3(2-)) participate in an intraspecies sulfur cycle that drives S(0) respiration.

  19. Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1.

    PubMed

    Shi, Liang; Chen, Baowei; Wang, Zheming; Elias, Dwayne A; Mayer, M Uljana; Gorby, Yuri A; Ni, Shuison; Lower, Brian H; Kennedy, David W; Wunschel, David S; Mottaz, Heather M; Marshall, Matthew J; Hill, Eric A; Beliaev, Alexander S; Zachara, John M; Fredrickson, James K; Squier, Thomas C

    2006-07-01

    Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., Delta omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the Delta omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.

  20. Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-Type Cytochromes of Shewanella oneidensis MR-1

    PubMed Central

    Shi, Liang; Chen, Baowei; Wang, Zheming; Elias, Dwayne A.; Mayer, M. Uljana; Gorby, Yuri A.; Ni, Shuison; Lower, Brian H.; Kennedy, David W.; Wunschel, David S.; Mottaz, Heather M.; Marshall, Matthew J.; Hill, Eric A.; Beliaev, Alexander S.; Zachara, John M.; Fredrickson, James K.; Squier, Thomas C.

    2006-01-01

    Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., ΔomcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the ΔomcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1. PMID:16788180

  1. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

    SciTech Connect

    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.

    2010-07-19

    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  2. In Vivo Identification of the Outer Membrane Protein OmcA-MtrC Interaction Network in Shewanella oneidensis MR-1 Cells Using Novel Hydrophobic Chemical Cross-Linkers

    SciTech Connect

    Zhang, Haizhen; Tang, Xiaoting; Munske, Gerhard R.; Zakharova, Natalia L.; Yang, Li; Zheng, Chunxiang; Wolff, Meagan A.; Tolic, Nikola; Anderson, Gordon A.; Shi, Liang; Marshall, Matthew J.; Fredrickson, Jim K.; Bruce, James E.

    2008-04-01

    Outer membrane (OM) cytochromes OmcA (SO1779) and MtrC (SO1778) are the integral components of electron transfer used by Shewanella oneidensis for anaerobic respiration of metal (hydr)oxides. Here the OmcA-MtrC interaction was identified in vivo using a novel hydrophobic chemical cross-linker (MRN) combined with immunoprecipitation techniques. In addition, identification of other OM proteins from the cross-linked complexes allows first visualization of the OmcA-MtrC interaction network. Further experiments on omcA and mtrC mutant cells showed OmcA plays a central role in the network interaction. For comparison, two commercial cross-linkers were also used in parallel and both resulted in fewer OM protein identifications, indicating the superior properties of MRN for identification of membrane protein interactions. Finally, comparison experiments of in vivo cross-linking and cell lysate cross-linking resulted in significantly different protein interaction data, demonstrating the importance of in vivo cross-linking for study of protein-protein interactions in cells.

  3. Role of outer membrane c-type cytochromes MtrC and OmcA in Shewanella oneidensis MR-1 cell production, accumulation, and detachment during respiration on hematite.

    PubMed

    Mitchell, A C; Peterson, L; Reardon, C L; Reed, S B; Culley, D E; Romine, M R; Geesey, G G

    2012-07-01

    The iron-reducing bacterium Shewanella oneidensis MR-1 has the capacity to contribute to iron cycling over the long term by respiring on crystalline iron oxides such as hematite when poorly crystalline phases are depleted. The ability of outer membrane cytochromes OmcA and MtrC of MR-1 to bind to and transfer electrons to hematite has led to the suggestion that they function as terminal reductases when this mineral is used as a respiratory substrate. Differences in their redox behavior and hematite-binding properties, however, indicate that they play different roles in the electron transfer reaction. Here, we investigated how these differences in cytochrome behavior with respect to hematite affected biofilm development when the mineral served as terminal electron acceptor (TEA). Upon attachment to hematite, cells of the wild-type (WT) strain as well as those of a ΔomcA mutant but not those of a ΔmtrC mutant replicated and accumulated on the mineral surface. The results indicate that MtrC but not OmcA is required for growth when this mineral serves as TEA. While an OmcA deficiency did not impede cell replication and accumulation on hematite prior to achievement of a maximum surface cell density comparable to that established by WT cells, OmcA was required for efficient electron transfer and cell attachment to hematite once maximum surface cell density was achieved. OmcA may therefore play a role in overcoming barriers to electron transfer and cell attachment to hematite imposed by reductive dissolution of the mineral surface from cell respiration associated with achievement of high surface cell densities.

  4. Contributions of the [NiFe]- and [FeFe]-hydrogenase to H2 production in Shewanella oneidensis MR-1 as revealed by isotope ratio analysis of evolved H2

    SciTech Connect

    Kreuzer, Helen W.; Hill, Eric A.; Moran, James J.; Bartholomew, Rachel A.; Hui, Yang; Hegg, Eric L.

    2014-03-01

    Shewanella oneidensis MR-1 encodes both a [NiFe]- and an [FeFe]-hydrogenase. While the output of these proteins has been characterized in mutant strains expressing only one of the enzymes, the contribution of each to H2 synthesis in the wild-type organism is not clear. Here we use stable isotope analysis of H2 in the culture headspace, along with transcription data and measurements of the concentrations of gases in the headspace, to characterize H2 production in the wild-type strain. After most of the O2 in the headspace had been consumed, H2 was produced and then consumed by the bidirectional [NiFe]-hydrogenase. Once the cultures were completely anaerobic, a new burst of H2 synthesis catalyzed by both enzymes took place. Our data is consistent with the hypothesis that at this point in the culture cycle, a pool of electrons is shunted toward both hydrogenases in the wild-type organism, but that in the absence of one of the hydrogenases, the flux is redirected to the available enzyme. To our knowledge, this is the first use of stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.

  5. Particle size effect and the mechanism of hematite reduction by the outer membrane cytochrome OmcA of Shewanella oneidensis MR-1

    NASA Astrophysics Data System (ADS)

    Liu, Juan; Pearce, Carolyn I.; Shi, Liang; Wang, Zheming; Shi, Zhi; Arenholz, Elke; Rosso, Kevin M.

    2016-11-01

    The cycling of iron at the Earth's near surface is profoundly influenced by dissimilatory metal reducing microorganisms, and many studies have focused on unraveling electron transfer mechanisms between these bacteria and Fe(III)-(oxyhydr)oxides. However, these efforts have been complicated by the fact that these minerals often occur in the micro- to nanosize regime, and in relevant natural environments as well as in the laboratory are subject to aggregation. The nature of the physical interface between the cellular envelope, the outer-membrane cytochromes responsible for facilitating the interfacial electron transfer step, and these complex mineral particulates is thus difficult to probe. Previous studies using whole cells have reported reduction rates that do not correlate with particle size. In the present study we isolate the interaction between the decaheme outer-membrane cytochrome OmcA of Shewanella oneidensis and nanoparticulate hematite, examining the reduction rate as a function of particle size and reaction products through detailed characterization of the electron balance and the structure and valence of iron at particle surfaces. By comparison with abiotic reduction via the smaller molecule ascorbic acid, we show that the reduction rate is systematically controlled by the sterically accessible interfacial contact area between OmcA and hematite in particle aggregates; rates increase once pore throat sizes in aggregates become as large as OmcA. Simultaneous measure of OmcA oxidation against Fe(II) release shows a ratio of 1:10, consistent with a cascade OmcA oxidation mechanism heme by heme. X-ray absorption spectroscopies reveal incipient magnetite on the reacted surfaces of the hematite nanoparticles after reaction. The collective findings establish the importance of accessibility of physical contact between the terminal reductases and iron oxide surfaces, and through apparent consistency of observations help reconcile behavior reported at the larger

  6. Role of Outer Membrane C-Type Cytochromes MtrC and OmcA in Shewanella Oneidensis MR-1 Cell Production, Accumulation, and Detachment During Respiration on Hematite

    SciTech Connect

    Mitchell, Andrew C.; Peterson, L.; Reardon, Catherine L.; Reed, Samantha B.; Culley, David E.; Romine, Margaret F.; Geesey, Gill G.

    2012-07-01

    Solid phase iron oxides are considered to be important terminal electron acceptors for microbial respiration in many anoxic environments. Besides the knowledge that cells attach to and reduce these substrates, other aspects of surface-associated cell behavior and the related cell surface components that influence cell-mineral interactions are not well understood. In the present study, wild-type cells of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 formed thin biofilms one-to-two cell layers in thickness when respiring on natural specular hematite under flow conditions similar to those which exist in aquatic sediments and subsurface environments. The distribution of cells within the biofilm indicated that direct contact was not required for electron transfer from cells to the mineral surface. Detached biomass in the form of single cells represented >99% of the surface-associated wild-type cell production from respiration on hematite over the biofilm life cycle. A mutant deficient in the outer membrane c35 type cytochrome OmcA, while still able to respire and replicate on hematite, established a lower steady-state cell density on the mineral surface than that of the wild-type strain. A mutant deficient in MtrC, another outer membrane c-type cytochrome, and a mutant deficient in both cytochromes were unable to reduce sufficient amounts of hematite to support detectable growth on the mineral surface. When considered in the context of previous work, the results support a growing body of evidence that the relative importance of OmcA and MtrC to cell respiration and replication depends on the form of iron oxide available as terminal electron acceptor.

  7. Methods for Imaging Shewanella Oneidensis MR-1 Nanofilaments

    DTIC Science & Technology

    2010-01-01

    has been made that, “Previous studies may have missed nanowires because bacteria in biofilms are Journal of Microbiological Methods 82 (2010) 187–191...j.mimet.2010.05.011 Contents lists available at ScienceDirect Journal of Microbiological Methods j ourna l homepage: www.e lsev ie r.com/ locate...188 R. Ray et al. / Journal of Microbiological Methods 82 (2010) 187–191 samples (Technique 3) until the specimens were coated in palladium

  8. The Influence of Acidity on Microbial Fuel Cells Containing Shewanella Oneidensis (PREPRINT)

    DTIC Science & Technology

    2008-09-01

    AFRL-RX-TY-TP-2008-4508 PREPRINT THE INFLUENCE OF ACIDITY ON MICROBIAL FUEL CELLS CONTAINING SHEWANELLA ONEIDENSIS (PREPRINT) Justin C...8. However, the effect of pH on the growth and electron transfer abilities of Shewanella oneidensis MR-1 (wild-type) and DSP10 (spontaneous mutant...2005; Dague et al., 2006; Gaboriaud et al., 2005; ao et al., 2004; Leaphart et al., 2006), but only an isolated report iscusses the effects of

  9. Influence of riboflavin on the reduction of radionuclides by Shewanella oneidenis MR-1.

    PubMed

    Cherkouk, Andrea; Law, Gareth T W; Rizoulis, Athanasios; Law, Katie; Renshaw, Joanna C; Morris, Katherine; Livens, Francis R; Lloyd, Jonathan R

    2016-03-28

    Uranium (as UO2(2+)), technetium (as TcO4(-)) and neptunium (as NpO2(+)) are highly mobile radionuclides that can be reduced enzymatically by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble species. The redox chemistry of Pu is more complicated, but the dominant oxidation state in most environments is highly insoluble Pu(IV), which can be reduced to Pu(III) which has a potentially increased solubility which could enhance migration of Pu in the environment. Recently it was shown that flavins (riboflavin and flavin mononucleotide (FMN)) secreted by Shewanella oneidensis MR-1 can act as electron shuttles, promoting anoxic growth coupled to the accelerated reduction of poorly-crystalline Fe(III) oxides. Here, we studied the role of riboflavin in mediating the reduction of radionuclides in cultures of Shewanella oneidensis MR-1. Our results demonstrate that the addition of 10 μM riboflavin enhances the reduction rate of Tc(VII) to Tc(IV), Pu(IV) to Pu(III) and to a lesser extent, Np(V) to Np(IV), but has no significant influence on the reduction rate of U(VI) by Shewanella oneidensis MR-1. Thus riboflavin can act as an extracellular electron shuttle to enhance rates of Tc(VII), Np(V) and Pu(IV) reduction, and may therefore play a role in controlling the oxidation state of key redox active actinides and fission products in natural and engineered environments. These results also suggest that the addition of riboflavin could be used to accelerate the bioremediation of radionuclide-contaminated environments.

  10. Global transcriptome analysis of the heat shock response ofshewanella oneidensis

    SciTech Connect

    Gao, Haichun; Wang, Sarah; Liu, Xueduan; Yan, Tinfeng; Wu, Liyou; Alm, Eric; Arkin, Adam P.; Thompson, Dorothea K.; Zhou, Jizhong

    2004-04-30

    Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. However, the genetic basis and regulatory mechanisms underlying the ability of S. oneidensis to survive and adapt to various environmentally relevant stresses is poorly understood. To define this organism's molecular response to elevated growth temperatures, temporal gene expression profiles were examined in cells subjected to heat stress using whole-genome DNA microarrays for S. oneidensis MR-1. Approximately 15 percent (711) of the predicted S. oneidensis genes represented on the microarray were significantly up- or down-regulated (P < 0.05) over a 25-min period following shift to the heat shock temperature (42 C). As expected, the majority of S. oneidensis genes exhibiting homology to known chaperones and heat shock proteins (Hsps) were highly and transiently induced. In addition, a number of predicted genes encoding enzymes in glycolys is and the pentose cycle, [NiFe] dehydrogenase, serine proteases, transcriptional regulators (MerR, LysR, and TetR families), histidine kinases, and hypothetical proteins were induced in response to heat stress. Genes encoding membrane proteins were differentially expressed, suggesting that cells possibly alter their membrane composition or structure in response to variations in growth temperature. A substantial number of the genes encoding ribosomal proteins displayed down-regulated co-expression patterns in response to heat stress, as did genes encoding prophage and flagellar proteins. Finally, based on computational comparative analysis of the upstream promoter regions of S.oneidensis heat-inducible genes, a putative regulatory motif, showing high conservation to the Escherichia coli sigma 32-binding consensus sequence, was identified.

  11. Programming the quorum sensing-based AND gate in Shewanella oneidensis for logic gated-microbial fuel cells.

    PubMed

    Hu, Yidan; Yang, Yun; Katz, Evgeny; Song, Hao

    2015-03-11

    An AND logic gate based on a synthetic quorum-sensing (QS) module was constructed in a Shewanella oneidensis MR-1 mtrA knockout mutant. The presence of two input signals activated the expression of a periplasmic decaheme cytochrome MtrA to regenerate the extracellular electron transfer conduit, enabling the construction of AND-gated microbial fuel cells.

  12. Flavin Electron Shuttles Dominate Extracellular Electron Transfer by Shewanella oneidensis

    PubMed Central

    Kotloski, Nicholas J.; Gralnick, Jeffrey A.

    2013-01-01

    ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δbfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. PMID:23322638

  13. Identification and analysis of the Shewanella oneidensis major oxygen-independent coproporphyrinogen III oxidase gene.

    PubMed

    Al-Sheboul, Suhaila; Saffarini, Daad

    2011-12-01

    Shewanella oneidenesis MR-1 is a facultative anaerobe that can use a large number of electron acceptors including metal oxides. During anaerobic respiration, S. oneidensis MR-1 synthesizes a large number of c cytochromes that give the organism its characteristic orange color. Using a modified mariner transposon, a number of S. oneidensis mutants deficient in anaerobic respiration were generated. One mutant, BG163, exhibited reduced pigmentation and was deficient in c cytochromes normally synthesized under anaerobic condition. The deficiencies in BG163 were due to insertional inactivation of hemN1, which exhibits a high degree of similarity to genes encoding anaerobic coproporphyrinogen III oxidases that are involved in heme biosynthesis. The ability of BG163 to synthesize c cytochromes under anaerobic conditions, and to grow anaerobically with different electron acceptors was restored by the introduction of hemN1 on a plasmid. Complementation of the mutant was also achieved by the addition of hemin to the growth medium. The genome sequence of S. oneidensis contains three putative anaerobic coproporphyrinogen III oxidase genes. The protein encoded by hemN1 appears to be the major enzyme that is involved in anaerobic heme synthesis of S. oneidensis. The other two putative anaerobic coproporphyrinogen III oxidase genes may play a minor role in this process.

  14. The Role of Shewanella oneidensis MR-1 Outer Surface Structures in Extracellular Electron Transfer

    DTIC Science & Technology

    2010-01-01

    less current than the wild type in an MFC and was unable to reduce Fe(III). These results indicated that although nanofilaments and soluble mediators...mutant that lacks the outer membrane c cytochromes, MtrC and OmcA. This mutant generated significantly less current than the wild type in an MFC and...published previously [34]. The anodes were single-sided carbon-coated titanium flags and the cathode system was graphite paper in 50 mM potassium

  15. Final Summary of "Interdisciplinary Study of Shewanella oneidensis MR-1's Metabolism & Metal Reduction"

    SciTech Connect

    Kolker, Eugene

    2007-06-26

    Our project focused primarily on analysis of different types of data produced by global high-throughput technologies, data integration of gene annotation, and gene and protein expression information, as well as on getting a better functional annotation of Shewanella genes. Specifically, four of our numerous major activities and achievements include the development of: statistical models for identification and expression proteomics, superior to currently available approaches (including our own earlier ones); approaches to improve gene annotations on the whole-organism scale; standards for annotation, transcriptomics and proteomics approaches; and generalized approaches for data integration of gene annotation, gene and protein expression information.

  16. Formate Metabolism in Shewanella oneidensis Generates Proton Motive Force and Prevents Growth without an Electron Acceptor

    PubMed Central

    Kane, Aunica L.; Brutinel, Evan D.; Joo, Heena; Maysonet, Rebecca; VanDrisse, Chelsey M.; Kotloski, Nicholas J.

    2016-01-01

    ABSTRACT Shewanella oneidensis strain MR-1 is a facultative anaerobe that thrives in redox-stratified environments due to its ability to utilize a wide array of terminal electron acceptors. Conversely, the electron donors utilized by S. oneidensis are more limited and include products of primary fermentation such as lactate, pyruvate, formate, and hydrogen. Lactate, pyruvate, and hydrogen metabolisms in S. oneidensis have been described previously, but little is known about the role of formate oxidation in the ecophysiology of these bacteria. Formate is produced by S. oneidensis through pyruvate formate lyase during anaerobic growth on carbon sources that enter metabolism at or above the level of pyruvate, and the genome contains three gene clusters predicted to encode three complete formate dehydrogenase complexes. To determine the contribution of each complex to formate metabolism, strains lacking one, two, or all three annotated formate dehydrogenase gene clusters were generated and examined for growth rates and yields on a variety of carbon sources. Here, we report that formate oxidation contributes to both the growth rate and yield of S. oneidensis through the generation of proton motive force. Exogenous formate also greatly accelerated growth on N-acetylglucosamine, a carbon source normally utilized very slowly by S. oneidensis under anaerobic conditions. Surprisingly, deletion of all three formate dehydrogenase gene clusters enabled growth of S. oneidensis using pyruvate in the absence of a terminal electron acceptor, a mode of growth never before observed in these bacteria. Our results demonstrate that formate oxidation is a fundamental strategy under anaerobic conditions for energy conservation in S. oneidensis. IMPORTANCE Shewanella species have garnered interest in biotechnology applications for their ability to respire extracellular terminal electron acceptors, such as insoluble iron oxides and electrodes. While much effort has gone into studying the

  17. Cellular Response of Shewanella oneidensis to StrontiumStress

    SciTech Connect

    Brown, Steven D.; Martin, Madhavi; Deshpande, Sameer; Seal,Sudipta; Huang, Katherine; Alm, Eric; Yang, Yunfeng; Wu, Liyou; Yan,Tengfen; Liu, Xueduan; Arkin, Adam; Chourey, Karuna; Zhou, Jizhong; Thompson, Dorothea K.

    2007-03-30

    The physiology and transcriptome dynamics of the metalion-reducing bacterium Shewanella oneidensis strain MR-1 in response tononradioactive strontium (Sr) exposure were investigated. Studiesindicated that MR-1 was able to grow aerobically in complex medium in thepresence of 180 mMSrCl2 but showed severe growth inhibition at levelsabove that concentration. Temporal gene expression profiles weregenerated from aerobically grown, mid-exponential-phase MR-1 cellsshocked with 180 mM SrCl2 and analyzed for significant differences inmRNA abundance with reference to data for nonstressed MR-1 cells. Geneswith annotated functions in siderophore biosynthesis and iron transportwere among the most highly induced (>100-fold [P<0.05]) openreading frames in response to acute Sr stress, and a mutant(SO3032::pKNOCK) defective in siderophore production was found to behypersensitive to SrCl2 exposure, compared to parental and wild-typestrains. Transcripts encoding multidrug and heavy metal efflux pumps,proteins involved in osmotic adaptation, sulfate ABC transporters, andassimilative sulfur metabolism enzymes also were differentially expressedfollowing Sr exposure but at levels that were several orders of magnitudelower than those for iron transport genes. Precipitate formation wasobserved during aerobic growth of MR-1 in broth cultures amended with 50,100, or 150 mM SrCl2 but not in cultures of the SO3032::pKNOCK mutant orin the abiotic control. Chemical analysis of this precipitate usinglaser-induced breakdown spectroscopy and static secondary ion massspectrometry indicated extracellular solid-phase sequestration of Sr,with at least a portion of the heavy metal associated with carbonatephases.

  18. Analysis of Shewanella oneidensis Membrane Protein Expression in Response to Electron Acceptor Availability

    SciTech Connect

    Giometti, Carol S.; Khare, Tripti; Verberkmoes, Nathan; O'Loughlin, Ed; Lindberg, Carl; Thompson, Melissa; Hettich, Robert

    2006-04-05

    Shewanella oneidensis MR-1, a gram negative metal-reducing bacterium, can utilize a large number of electron acceptors. In the natural environment, S. oneidensis utilizes insoluble metal oxides as well as soluble terminal electron acceptors. The purpose of this ERSP project is to identify differentially expressed proteins associated with the membranes of S. oneidensis MR-1 cells grown with different electron acceptors, including insoluble metal oxides. We hypothesize that through the use of surface labeling, subcellular fractionation, and a combination of proteome analysis tools, proteins involved in the reduction of different terminal electron acceptors will be elucidated. We are comparing the protein profiles from cells grown with the soluble electron acceptors oxygen and fumarate and with those from cells grown with the insoluble iron oxides goethite, ferrihydrite and lepidocrocite. Comparison of the cell surface proteins isolated from cells grown with oxygen or anaerobically with fumarate revealed an increase in the abundance of over 25 proteins in anaerobic cells, including agglutination protein and flagellin proteins along with the several hypothetical proteins. In addition, the surface protein composition of cells grown with the insoluble iron oxides varies considerably from the protein composition observed with either soluble electron acceptor as well as between the different insoluble acceptors.

  19. Shewanella oneidensis Hfq promotes exponential phase growth, stationary phase culture density, and cell survival

    PubMed Central

    2013-01-01

    Background Hfq is an RNA chaperone protein that has been broadly implicated in sRNA function in bacteria. Here we describe the construction and characterization of a null allele of the gene that encodes the RNA chaperone Hfq in Shewanella oneidensis strain MR-1, a dissimilatory metal reducing bacterium. Results Loss of hfq in S. oneidensis results in a variety of mutant phenotypes, all of which are fully complemented by addition of a plasmid-borne copy of the wild type hfq gene. Aerobic cultures of the hfq∆ mutant grow more slowly through exponential phase than wild type cultures, and hfq∆ cultures reach a terminal cell density in stationary phase that is ~2/3 of that observed in wild type cultures. We have observed a similar growth phenotype when the hfq∆ mutant is cultured under anaerobic conditions with fumarate as the terminal electron acceptor, and we have found that the hfq∆ mutant is defective in Cr(VI) reduction. Finally, the hfq∆ mutant exhibits a striking loss of colony forming units in extended stationary phase and is highly sensitive to oxidative stress induced by H2O2 or methyl viologen (paraquat). Conclusions The hfq mutant in S. oneidensis exhibits pleiotropic phenotypes, including a defect in metal reduction. Our results also suggest that hfq mutant phenotypes in S. oneidensis may be at least partially due to increased sensitivity to oxidative stress. PMID:23394078

  20. Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

    PubMed

    Brennan, Christopher M; Mazzucca, Nicholas Q; Mezoian, Taylor; Hunt, Taylor M; Keane, Meaghan L; Leonard, Jessica N; Scola, Shelby E; Beer, Emma N; Perdue, Sarah; Pellock, Brett J

    2014-01-01

    The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

  1. Reduced Heme Levels Underlie the Exponential Growth Defect of the Shewanella oneidensis hfq Mutant

    PubMed Central

    Mezoian, Taylor; Hunt, Taylor M.; Keane, Meaghan L.; Leonard, Jessica N.; Scola, Shelby E.; Beer, Emma N.; Perdue, Sarah; Pellock, Brett J.

    2014-01-01

    The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step. PMID:25356668

  2. Physiological and Transcriptomic Analyses to Characterize the Function of Fur and Iron Response in Shewanella oneidensis

    SciTech Connect

    Yang, Yunfeng; Harris, Daniel P; Luo, Feng; Wu, Liyou; Parsons, Andrea; Palumbo, Anthony Vito; Zhou, Jizhong

    2008-01-01

    Maintaining iron homeostasis is a key metabolic challenge for most organisms. In many bacterial species, regulation of iron homeostasis is carried out by the global transcriptional regulator Fur. Physiological examination showed that Shewanella oneidensis harboring a fur deletion mutation had deficiencies in both growth and acid tolerance response. However, the fur mutant better tolerated iron-limited environments than the wild-type strain MR-1. Transcriptomic studies comparing the fur mutant and MR-1 confirmed previous findings that iron acquisition systems were highly induced by Fur inactivation. In addition, the temporal gene expression profiling of the fur mutant in response to iron depletion and repletion suggested that a number of genes involved in energy transport were iron-responsive but Fur-independent. Further identification of Fur-independent genes was obtained by generating a gene co-expression network from temporal gene expression profiles. A group of genes is involved in heat shock and has an rpoH-binding site at their promoters, and genes related to anaerobic energy transport has a highly conserved Crp binding site at the promoters. Together, this work provides useful information for the characterization of the function of Fur and the iron response in S. oneidensis.

  3. Genome-scale metabolic network validation of Shewanella oneidensis using transposon insertion frequency analysis.

    PubMed

    Yang, Hong; Krumholz, Elias W; Brutinel, Evan D; Palani, Nagendra P; Sadowsky, Michael J; Odlyzko, Andrew M; Gralnick, Jeffrey A; Libourel, Igor G L

    2014-09-01

    Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA). TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1) previous genome-wide direct gene-essentiality assignments; and, 2) flux balance analysis (FBA) predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.

  4. Genome-Scale Metabolic Network Validation of Shewanella oneidensis Using Transposon Insertion Frequency Analysis

    PubMed Central

    Yang, Hong; Krumholz, Elias W.; Brutinel, Evan D.; Palani, Nagendra P.; Sadowsky, Michael J.; Odlyzko, Andrew M.; Gralnick, Jeffrey A.; Libourel, Igor G. L.

    2014-01-01

    Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA). TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1) previous genome-wide direct gene-essentiality assignments; and, 2) flux balance analysis (FBA) predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions. PMID:25233219

  5. Impacts of Shewanella oneidensis c-type cytochromes on aerobic and anaerobic respiration

    SciTech Connect

    Gao, Haichun; Barua, Soumitra; Liang, Yili; Wu, Lianming; Dong, Yangyang; Reed, Samantha B.; Chen, Jingrong; Culley, David E.; Kennedy, David W.; Yang, Yunfeng; He, Zhili; Nealson, Kenneth H.; Fredrickson, Jim K.; Tiedje, James M.; Romine, Margaret F.; Zhou, Jizhong

    2010-06-24

    Shewanella are renowned for their ability to utilize a wide range of electron acceptors (EA) for respiration, which has been partially accredited to the presence of a large number of the c-type cytochromes. To investigate the involvement of c-type cytochrome proteins in aerobic and anaerobic respiration of Shewanella oneidensis Mr -1, 36 in-frame deletion mutants, among possible 41 predicted, c-type cytochrome genes were obtained. The potential involvement of each individual c-type cytochrome in the reduction of a variety of EAs was assessed individually as well as in competition experiments. While results on the wellstudied c-type cytochromes CymA(SO4591) and MtrC(SO1778) were consistent with previous findings, collective observations were very interesting: the responses of S. oneidensis Mr -1 to low and highly toxic metals appeared to be significantly different; CcoO, CcoP and PetC, proteins involved in aerobic respiration in various organisms, played critical roles in both aerobic and anaerobic respiration with highly toxic metals as EA. In addition, these studies also suggested that an uncharacterized c-type cytochrome (SO4047) may be important to both aerobiosis and anaerobiosis.

  6. Impacts of Shewanella oneidensis c-type cytochromes on aerobic and anaerobic respiration.

    PubMed

    Gao, Haichun; Barua, Soumitra; Liang, Yili; Wu, Lin; Dong, Yangyang; Reed, Samantha; Chen, Jingrong; Culley, Dave; Kennedy, David; Yang, Yunfeng; He, Zhili; Nealson, Kenneth H; Fredrickson, James K; Tiedje, James M; Romine, Margaret; Zhou, Jizhong

    2010-07-01

    Shewanella are renowned for their ability to utilize a wide range of electron acceptors (EA) for respiration, which has been partially accredited to the presence of a large number of the c-type cytochromes. To investigate the involvement of c-type cytochrome proteins in aerobic and anaerobic respiration of Shewanella oneidensis Mr -1, 36 in-frame deletion mutants, among possible 41 predicted, c-type cytochrome genes were obtained. The potential involvement of each individual c-type cytochrome in the reduction of a variety of EAs was assessed individually as well as in competition experiments. While results on the well-studied c-type cytochromes CymA(SO4591) and MtrC(SO1778) were consistent with previous findings, collective observations were very interesting: the responses of S. oneidensis Mr -1 to low and highly toxic metals appeared to be significantly different; CcoO, CcoP and PetC, proteins involved in aerobic respiration in various organisms, played critical roles in both aerobic and anaerobic respiration with highly toxic metals as EA. In addition, these studies also suggested that an uncharacterized c-type cytochrome (SO4047) may be important to both aerobiosis and anaerobiosis.

  7. Fluxomics of the Eastern Oyster for Environmental Stress Studies

    PubMed Central

    Tikunov, Andrey P.; Stoskopf, Michael K.; Macdonald, Jeffrey M.

    2014-01-01

    The metabolism of 2-13C/15N-glycine and U-13C-glucose was determined in four tissue blocks (adductor muscle, stomach and digestive gland, mantle, and gills) of the Eastern oyster (Crassostrea virginica) using proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR) spectroscopy. The oysters were treated in aerated seawater with three treatments (5.5 mM U-13C-glucose, 2.7 mM 2-13C/15N-glycine, and 5.5 mM U-13C-glucose plus 2.7 mM 2-13C/15N-glycine) and the relative mass balance and 13C fractional enrichments were determined in the four tissue blocks. In all tissues, glycine was metabolized by the glycine cycle forming serine exclusively in the mitochondria by the glycine cleavage system forming 2,3-13C-serine. In muscle, a minor amount of serine-derived pyruvate entered the Krebs cycle as substantiated by detection of a trace of 2,3-13C-aspartate. In all tissues, U-13C-glucose formed glycogen by glycogen synthesis, alanine by glycolysis, and glutamate and aspartate through the Krebs cycle. Alanine was formed exclusively from glucose via alanine transaminase and not glycine via alanine-glyoxylate transaminase. Based on isotopomer analysis, pyruvate carboxylase and pyruvate dehydrogenase appeared to be equal points for pyruvate entry into the Krebs cycle. In the 5.5 mM U-13C-glucose plus 2.7 mM 2-13C/15N-glycine emergence treatment used to simulate 12 h of “low tide”, oysters accumulated more 13C-labeled metabolites, including both anaerobic glycolytic and aerobic Krebs cycle intermediates. The aerobic metabolites could be the biochemical result of the gaping behavior of mollusks during emergence. The change in tissue distribution and mass balance of 13C-labeled nutrients (U-13C-glucose and 2-13C/15N-glycine) provides the basis for a new quantitative fluxomic method for elucidating sub-lethal environmental effects in marine organisms called whole body mass balance phenotyping (WoMBaP). PMID:24958387

  8. Use of an Electrochemical Split Cell Technique to Evaluate the Influence of Shewanella oneidensis Activities on Corrosion of Carbon Steel

    PubMed Central

    Miller, Robert Bertram; Sadek, Anwar; Rodriguez, Alvaro; Iannuzzi, Mariano; Giai, Carla; Senko, John M.; Monty, Chelsea N.

    2016-01-01

    Microbially induced corrosion (MIC) is a complex problem that affects various industries. Several techniques have been developed to monitor corrosion and elucidate corrosion mechanisms, including microbiological processes that induce metal deterioration. We used zero resistance ammetry (ZRA) in a split chamber configuration to evaluate the effects of the facultatively anaerobic Fe(III) reducing bacterium Shewanella oneidensis MR-1 on the corrosion of UNS G10180 carbon steel. We show that activities of S. oneidensis inhibit corrosion of steel with which that organism has direct contact. However, when a carbon steel coupon in contact with S. oneidensis was electrically connected to a second coupon that was free of biofilm (in separate chambers of the split chamber assembly), ZRA-based measurements indicated that current moved from the S. oneidensis-containing chamber to the cell-free chamber. This electron transfer enhanced the O2 reduction reaction on the coupon deployed in the cell free chamber, and consequently, enhanced oxidation and corrosion of that electrode. Our results illustrate a novel mechanism for MIC in cases where metal surfaces are heterogeneously covered by biofilms. PMID:26824529

  9. Use of an Electrochemical Split Cell Technique to Evaluate the Influence of Shewanella oneidensis Activities on Corrosion of Carbon Steel.

    PubMed

    Miller, Robert Bertram; Sadek, Anwar; Rodriguez, Alvaro; Iannuzzi, Mariano; Giai, Carla; Senko, John M; Monty, Chelsea N

    2016-01-01

    Microbially induced corrosion (MIC) is a complex problem that affects various industries. Several techniques have been developed to monitor corrosion and elucidate corrosion mechanisms, including microbiological processes that induce metal deterioration. We used zero resistance ammetry (ZRA) in a split chamber configuration to evaluate the effects of the facultatively anaerobic Fe(III) reducing bacterium Shewanella oneidensis MR-1 on the corrosion of UNS G10180 carbon steel. We show that activities of S. oneidensis inhibit corrosion of steel with which that organism has direct contact. However, when a carbon steel coupon in contact with S. oneidensis was electrically connected to a second coupon that was free of biofilm (in separate chambers of the split chamber assembly), ZRA-based measurements indicated that current moved from the S. oneidensis-containing chamber to the cell-free chamber. This electron transfer enhanced the O2 reduction reaction on the coupon deployed in the cell free chamber, and consequently, enhanced oxidation and corrosion of that electrode. Our results illustrate a novel mechanism for MIC in cases where metal surfaces are heterogeneously covered by biofilms.

  10. Dynamic Modeling of Aerobic Growth of Shewanella oneidensis. Predicting Triauxic Growth, Flux Distributions and Energy Requirement for Growth

    SciTech Connect

    Song, Hyun-Seob; Ramkrishna, Doraiswami; Pinchuk, Grigoriy E.; Beliaev, Alex S.; Konopka, Allan; Fredrickson, Jim K.

    2013-01-01

    A model-based analysis is conducted to investigate metabolism of Shewanella oneidensis MR-1 strain in aerobic batch culture, which exhibits an intriguing growth pattern by sequentially consuming substrate (i.e., lactate) and by-products (i.e., pyruvate and acetate). A general protocol is presented for developing a detailed network-based dynamic model for S. oneidensis based on the Lumped Hybrid Cybernetic Model (LHCM) framework. The L-HCM, although developed from only limited data, is shown to accurately reproduce exacting dynamic metabolic shifts, and provide reasonable estimates of energy requirement for growth. Flux distributions in S. oneidensis predicted by the L-HCM compare very favorably with 13C-metabolic flux analysis results reported in the literature. Predictive accuracy is enhanced by incorporating measurements of only a few intracellular fluxes, in addition to extracellular metabolites. The L-HCM developed here for S. oneidensis is consequently a promising tool for the analysis of intracellular flux distribution and metabolic engineering.

  11. Rapid Prediction of Bacterial Heterotrophic Fluxomics Using Machine Learning and Constraint Programming.

    PubMed

    Wu, Stephen Gang; Wang, Yuxuan; Jiang, Wu; Oyetunde, Tolutola; Yao, Ruilian; Zhang, Xuehong; Shimizu, Kazuyuki; Tang, Yinjie J; Bao, Forrest Sheng

    2016-04-01

    13C metabolic flux analysis (13C-MFA) has been widely used to measure in vivo enzyme reaction rates (i.e., metabolic flux) in microorganisms. Mining the relationship between environmental and genetic factors and metabolic fluxes hidden in existing fluxomic data will lead to predictive models that can significantly accelerate flux quantification. In this paper, we present a web-based platform MFlux (http://mflux.org) that predicts the bacterial central metabolism via machine learning, leveraging data from approximately 100 13C-MFA papers on heterotrophic bacterial metabolisms. Three machine learning methods, namely Support Vector Machine (SVM), k-Nearest Neighbors (k-NN), and Decision Tree, were employed to study the sophisticated relationship between influential factors and metabolic fluxes. We performed a grid search of the best parameter set for each algorithm and verified their performance through 10-fold cross validations. SVM yields the highest accuracy among all three algorithms. Further, we employed quadratic programming to adjust flux profiles to satisfy stoichiometric constraints. Multiple case studies have shown that MFlux can reasonably predict fluxomes as a function of bacterial species, substrate types, growth rate, oxygen conditions, and cultivation methods. Due to the interest of studying model organism under particular carbon sources, bias of fluxome in the dataset may limit the applicability of machine learning models. This problem can be resolved after more papers on 13C-MFA are published for non-model species.

  12. Rapid Prediction of Bacterial Heterotrophic Fluxomics Using Machine Learning and Constraint Programming

    PubMed Central

    Wu, Stephen Gang; Wang, Yuxuan; Jiang, Wu; Oyetunde, Tolutola; Yao, Ruilian; Zhang, Xuehong; Shimizu, Kazuyuki; Tang, Yinjie J.; Bao, Forrest Sheng

    2016-01-01

    13C metabolic flux analysis (13C-MFA) has been widely used to measure in vivo enzyme reaction rates (i.e., metabolic flux) in microorganisms. Mining the relationship between environmental and genetic factors and metabolic fluxes hidden in existing fluxomic data will lead to predictive models that can significantly accelerate flux quantification. In this paper, we present a web-based platform MFlux (http://mflux.org) that predicts the bacterial central metabolism via machine learning, leveraging data from approximately 100 13C-MFA papers on heterotrophic bacterial metabolisms. Three machine learning methods, namely Support Vector Machine (SVM), k-Nearest Neighbors (k-NN), and Decision Tree, were employed to study the sophisticated relationship between influential factors and metabolic fluxes. We performed a grid search of the best parameter set for each algorithm and verified their performance through 10-fold cross validations. SVM yields the highest accuracy among all three algorithms. Further, we employed quadratic programming to adjust flux profiles to satisfy stoichiometric constraints. Multiple case studies have shown that MFlux can reasonably predict fluxomes as a function of bacterial species, substrate types, growth rate, oxygen conditions, and cultivation methods. Due to the interest of studying model organism under particular carbon sources, bias of fluxome in the dataset may limit the applicability of machine learning models. This problem can be resolved after more papers on 13C-MFA are published for non-model species. PMID:27092947

  13. Natural isotope correction of MS/MS measurements for metabolomics and (13)C fluxomics.

    PubMed

    Niedenführ, Sebastian; ten Pierick, Angela; van Dam, Patricia T N; Suarez-Mendez, Camilo A; Nöh, Katharina; Wahl, S Aljoscha

    2016-05-01

    Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of (13)C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS-type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC-MS(/MS) requires derivatization for the usually non-volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full use of LC- and GC-MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; (13)C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation.

  14. Rapid Precipitation of Amorphous Silica in Experimental Systems with Nontronite (NAu-1) and Shewanella oneidensis MR-1

    DTIC Science & Technology

    2007-01-15

    response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and... reviewing the collection of Information. Send comments regarding this burden estimate or any other aspect of this collection of information, Including...suspensions of organoclay complexes for TEM into fresh LB media and cultured under continuous ultrathin sectioning (Perret et al., 1991; Wilkinson et al

  15. Disruption of Putrescine Biosynthesis in Shewanella oneidensis Enhances Biofilm Cohesiveness and Performance in Cr(VI) Immobilization

    PubMed Central

    Ding, Yuanzhao; Peng, Ni; Du, Yonghua; Ji, Lianghui

    2014-01-01

    Although biofilm-based bioprocesses have been increasingly used in various applications, the long-term robust and efficient biofilm performance remains one of the main bottlenecks. In this study, we demonstrated that biofilm cohesiveness and performance of Shewanella oneidensis can be enhanced through disrupting putrescine biosynthesis. Through random transposon mutagenesis library screening, one hyperadherent mutant strain, CP2-1-S1, exhibiting an enhanced capability in biofilm formation, was obtained. Comparative analysis of the performance of biofilms formed by S. oneidensis MR-1 wild type (WT) and CP2-1-S1 in removing dichromate (Cr2O72−), i.e., Cr(VI), from the aqueous phase showed that, compared with the WT biofilms, CP2-1-S1 biofilms displayed a substantially lower rate of cell detachment upon exposure to Cr(VI), suggesting a higher cohesiveness of the mutant biofilms. In addition, the amount of Cr(III) immobilized by CP2-1-S1 biofilms was much larger, indicating an enhanced performance in Cr(VI) bioremediation. We further showed that speF, a putrescine biosynthesis gene, was disrupted in CP2-1-S1 and that the biofilm phenotypes could be restored by both genetic and chemical complementations. Our results also demonstrated an important role of putrescine in mediating matrix disassembly in S. oneidensis biofilms. PMID:24362428

  16. Disruption of putrescine biosynthesis in Shewanella oneidensis enhances biofilm cohesiveness and performance in Cr(VI) immobilization.

    PubMed

    Ding, Yuanzhao; Peng, Ni; Du, Yonghua; Ji, Lianghui; Cao, Bin

    2014-02-01

    Although biofilm-based bioprocesses have been increasingly used in various applications, the long-term robust and efficient biofilm performance remains one of the main bottlenecks. In this study, we demonstrated that biofilm cohesiveness and performance of Shewanella oneidensis can be enhanced through disrupting putrescine biosynthesis. Through random transposon mutagenesis library screening, one hyperadherent mutant strain, CP2-1-S1, exhibiting an enhanced capability in biofilm formation, was obtained. Comparative analysis of the performance of biofilms formed by S. oneidensis MR-1 wild type (WT) and CP2-1-S1 in removing dichromate (Cr2O7(2-)), i.e., Cr(VI), from the aqueous phase showed that, compared with the WT biofilms, CP2-1-S1 biofilms displayed a substantially lower rate of cell detachment upon exposure to Cr(VI), suggesting a higher cohesiveness of the mutant biofilms. In addition, the amount of Cr(III) immobilized by CP2-1-S1 biofilms was much larger, indicating an enhanced performance in Cr(VI) bioremediation. We further showed that speF, a putrescine biosynthesis gene, was disrupted in CP2-1-S1 and that the biofilm phenotypes could be restored by both genetic and chemical complementations. Our results also demonstrated an important role of putrescine in mediating matrix disassembly in S. oneidensis biofilms.

  17. Transcriptome and Proteome Dynamics of the Cellular Response of Shewanella oneidensis to Chromium Stress

    SciTech Connect

    Thompson, D.K.

    2005-04-18

    The overall goal of this DOE NABIR project is to characterize the molecular basis and regulation of hexavalent chromium [Cr(VI)] stress response and reduction by Shewanella oneidensis strain MR-1. Temporal genomic profiling and mass spectrometry-based proteomic analysis were employed to characterize the dynamic molecular response of S. oneidensis MR-1 to both acute and chronic Cr(VI) exposure. The acute stress response of aerobic, mid-exponential phase cells shocked to a final concentration of 1 mM potassium chromate (K2CrO4) was examined at post-exposure time intervals of 5, 30, 60, and 90 min relative to untreated cells. The transcriptome of mid-exponential cultures was also analyzed 30 min after shock doses of 0.3, 0.5, or 1 mM K{sub 2}CrO{sub 4}. The tonB1-exbB1-exbD1 genes comprising the TonB1 iron transport system were some of the most highly induced coding sequences (CDSs) after 90 min (up to {approx}240 fold), followed by other genes involved in heme transport, sulfate transport, and sulfur assimilation pathways. In addition, transcript levels for CDSs with annotated functions in DNA repair (dinP, recX, recA, recN) and detoxification processes (so3585, so3586) were substantially increased in Cr(VI)-exposed cells compared to untreated cells. By contrast, genes predicted to encode hydrogenases (HydA, HydB), oxidoreductases (SO0902-03-04, SO1911), iron-sulfur cluster binding proteins (SO4404), decaheme cytochrome c proteins (MtrA, OmcA, OmcB), and a number of LysR or TetR family transcriptional regulators were some of the most highly repressed CDSs following the 90-min shock period. Transcriptome profiles generated from MR-1 cells adapted to 0.3 mM Cr(VI) differed significantly from those characterizing cells exposed to acute Cr(VI) stress without adaptation. Parallel proteomic characterization of soluble protein and membrane protein fractions extracted from Cr(VI)-shocked and Cr(VI)-adapted MR-1 cells was performed using multidimensional HPLC-ESI-MS/MS (both

  18. Transcriptome Profiling of Shewanella oneidensis Gene Expressionfollowing Exposure to Acidic and Alkaline pH

    SciTech Connect

    Leaphart, Adam B.; Thompson, Dorothea K.; Huang, Katherine; Alm,Eric; Wan, Xiu-Feng; Arkin, Adam P.; Brown, Steven D.; Wu, Liyou; Yan,Tingfen; Liu, Xueduan; Wickham, Gene S.; Zhou, Jizhong

    2007-04-02

    The molecular response of Shewanella oneidensis MR-1 tovariations in extracellular pH was investigated based on genomewide geneexpression profiling. Microarray analysis revealed that cells elicitedboth general and specific transcriptome responses when challenged withenvironmental acid (pH 4) or base (pH 10) conditions over a 60-minperiod. Global responses included the differential expression of genesfunctionally linked to amino acid metabolism, transcriptional regulationand signal transduction, transport, cell membrane structure, andoxidative stress protection. Response to acid stress included theelevated expression of genes encoding glycogen biosynthetic enzymes,phosphate transporters, and the RNA polymerase sigma-38 factor (rpoS),whereas the molecular response to alkaline pH was characterized byupregulation of nhaA and nhaR, which are predicted to encode an Na+/H+antiporter and transcriptional activator, respectively, as well assulfate transport and sulfur metabolism genes. Collectively, theseresults suggest that S. oneidensis modulates multiple transporters, cellenvelope components, and pathways of amino acid consumption and centralintermediary metabolism as part of its transcriptome response to changingexternal pH conditions.

  19. Biofabrication of discrete spherical gold nanoparticles using the metal-reducing bacterium, Shewanella oneidensis

    SciTech Connect

    Suresh, Anil K; Pelletier, Dale A; Wang, Wei; Broich, Michael L; Moon, Ji Won; Gu, Baohua; Allison, David P; Joy, David Charles; Phelps, Tommy Joe; Doktycz, Mitchel John

    2011-01-01

    Nanocrystallites have garnered substantial interest due to their varying applications including catalysis. Consequently important aspects related to control of shape/size and syntheses through economical and non-hazardous means are desirable. Highly efficient bioreduction based natural fabrication approaches that utilize microbes and or -plant extracts are poised to meet these needs. Here we show that the gamma- proteobacterium, Shewanella oneidensis MR-1, can reduce tetrachloro aurate (III) ions, producing discrete extracellular spherical gold nanocrystallites. The particles were homogeneous with multiple size distributions and produced under ambient conditions at high yield, 88% of theoretical maximum. Further characterization revealed that the particles consist of spheres in the size range of 2-50 nm, with an average of 12 5 nm. The nanoparticles were hydrophilic, biocompatible, and resisted aggregation even after several months. The particles are likely capped by a detachable protein/peptide coat. UV-vis and Fourier transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectra and transmission electron microscopy measurements confirmed the formation as well the crystalline nature of the nanoparticles. The antibacterial activity of these gold nanoparticles was assessed using Gram-negative (E. coli and S. oneidensis) and Gram-positive (B. subtilis) bacteria. Toxicity assessments divulged that the particles were neither toxic nor inhibitory to any of these bacteria.

  20. Differential label-free quantitative proteomic analysis of Shewanella oneidensis cultured under aerobic and suboxic conditions by accurate mass and time tag approach.

    PubMed

    Fang, Ruihua; Elias, Dwayne A; Monroe, Matthew E; Shen, Yufeng; McIntosh, Martin; Wang, Pei; Goddard, Carrie D; Callister, Stephen J; Moore, Ronald J; Gorby, Yuri A; Adkins, Joshua N; Fredrickson, Jim K; Lipton, Mary S; Smith, Richard D

    2006-04-01

    We describe the application of LC-MS without the use of stable isotope labeling for differential quantitative proteomic analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and suboxic conditions. LC-MS/MS was used to initially identify peptide sequences, and LC-FTICR was used to confirm these identifications as well as measure relative peptide abundances. 2343 peptides covering 668 proteins were identified with high confidence and quantified. Among these proteins, a subset of 56 changed significantly using statistical approaches such as statistical analysis of microarrays, whereas another subset of 56 that were annotated as performing housekeeping functions remained essentially unchanged in relative abundance. Numerous proteins involved in anaerobic energy metabolism exhibited up to a 10-fold increase in relative abundance when S. oneidensis was transitioned from aerobic to suboxic conditions.

  1. Differential Label-free Quantitative Proteomic Analysis of Shewanella oneidensis Cultured under Aerobic and Suboxic Conditions by Accurate Mass and Time Tag Approach

    SciTech Connect

    Fang, Ruihua; Elias, Dwayne A.; Monroe, Matthew E.; Shen, Yufeng; McIntosh, Martin; Wang, Pei; Goddard, Carrie D.; Callister, Stephen J.; Moore, Ronald J.; Gorby, Yuri A.; Adkins, Joshua N.; Fredrickson, Jim K.; Lipton, Mary S.; Smith, Richard D.

    2006-04-01

    We describe the application of liquid chromatography coupled to mass spectrometry (LC/MS) without the use of stable isotope labeling for differential quantitative proteomics analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and sub-oxic conditions. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to initially identify peptide sequences, and LC coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) was used to confirm these identifications, as well as measure relative peptide abundances. 2343 peptides, covering 668 proteins were identified with high confidence and quantified. Among these proteins, a subset of 56 changed significantly using statistical approaches such as SAM, while another subset of 56 that were annotated as performing housekeeping functions remained essentially unchanged in relative abundance. Numerous proteins involved in anaerobic energy metabolism exhibited up to a 10-fold increase in relative abundance when S. oneidensis is transitioned from aerobic to sub-oxic conditions.

  2. Cell surface expression of MR1B, a splice variant of the MHC class I-related molecule MR1, revealed with antibodies.

    PubMed

    Yamaguchi, Hisateru; Tsukamoto, Kentaro; Hashimoto, Keiichiro

    2014-01-10

    The major histocompatibility complex (MHC) class I-related molecule, MR1, is highly conserved in mammals and can present bacteria-derived vitamin B metabolites to mucosal-associated invariant T (MAIT) cells, possibly having important defense function in the microbial infection. MR1B is a splice variant of MR1 and possesses an intriguing domain structure with only two extracellular domains resembling some NKG2D ligand molecules. Thus far, cell surface expression of MR1B could not be analyzed with flow cytometry due to a lack of appropriate antibodies reactive with MR1B. Here we clarified the expression of MR1B recombinant protein on the cell surface of the transfected cells by flow cytometry analyses using the antiserum against MR1. Consistently, MR1B tagged with FLAG peptide at the N-terminus also could be detected with anti-FLAG monoclonal antibodies. Our result showed that MR1B can be recognized on the cell surface by macromolecules such as antibodies, indicating its potential of interaction with certain receptor(s). We discuss possibility of interaction of MR1B and/or the full-length MR1 with some receptor(s) other than αβ T cell receptor (TCR) of MAIT cells based on the highly conserved characteristic residues of the ligand-binding domains of MR1 and its MAIT cells αβTCR footprints.

  3. Strategies for Extending Metabolomics Studies with Stable Isotope Labelling and Fluxomics

    PubMed Central

    Srivastava, Anubhav; Kowalski, Greg M.; Callahan, Damien L.; Meikle, Peter J.; Creek, Darren J.

    2016-01-01

    This is a perspective from the peer session on stable isotope labelling and fluxomics at the Australian & New Zealand Metabolomics Conference (ANZMET) held from 30 March to 1 April 2016 at La Trobe University, Melbourne, Australia. This report summarizes the key points raised in the peer session which focused on the advantages of using stable isotopes in modern metabolomics and the challenges in conducting flux analyses. The session highlighted the utility of stable isotope labelling in generating reference standards for metabolite identification, absolute quantification, and in the measurement of the dynamic activity of metabolic pathways. The advantages and disadvantages of different approaches of fluxomics analyses including flux balance analysis, metabolic flux analysis and kinetic flux profiling were also discussed along with the use of stable isotope labelling in in vivo dynamic metabolomics. A number of crucial technical considerations for designing experiments and analyzing data with stable isotope labelling were discussed which included replication, instrumentation, methods of labelling, tracer dilution and data analysis. This report reflects the current viewpoint on the use of stable isotope labelling in metabolomics experiments, identifying it as a great tool with the potential to improve biological interpretation of metabolomics data in a number of ways. PMID:27706078

  4. From Metabolomics to Fluxomics: A Computational Procedure to Translate Metabolite Profiles into Metabolic Fluxes

    PubMed Central

    Cortassa, Sonia; Caceres, Viviane; Bell, Lauren N.; O’Rourke, Brian; Paolocci, Nazareno; Aon, Miguel A.

    2015-01-01

    We describe a believed-novel procedure for translating metabolite profiles (metabolome) into the set of metabolic fluxes (fluxome) from which they originated. Methodologically, computational modeling is integrated with an analytical platform comprising linear optimization, continuation and dynamic analyses, and metabolic control. The procedure was tested with metabolite profiles obtained from ex vivo mice Langendorff-heart preparations perfused with glucose. The metabolic profiles were analyzed using a detailed kinetic model of the glucose catabolic pathways including glycolysis, pentose phosphate (PP), glycogenolysis, and polyols to translate the glucose metabolome of the heart into the fluxome. After optimization, the ability of the model to simulate the initial metabolite profile was confirmed, and metabolic fluxes as well as the structure of control and regulation of the glucose catabolic network could be calculated. We show that the step catalyzed by phosphofructokinase together with ATP demand and glycogenolysis exert the highest control on the glycolytic flux. The negative flux control exerted by phosphofructokinase on the PP and polyol pathways revealed that the extent of glycolytic flux directly affects flux redirection through these pathways, i.e., the higher the glycolytic flux the lower the PP and polyols. This believed-novel methodological approach represents a step forward that may help in designing therapeutic strategies targeted to diagnose, prevent, and treat metabolic diseases. PMID:25564863

  5. Electrochemical selection and characterization of a high current-generating Shewanella oneidensis mutant with altered cell-surface morphology and biofilm-related gene expression

    PubMed Central

    2014-01-01

    Background Shewanella oneidensis MR-1 exhibits extracellular electron transfer (EET) activity that is influenced by various cellular components, including outer-membrane cytochromes, cell-surface polysaccharides (CPS), and regulatory proteins. Here, a random transposon-insertion mutant library of S. oneidensis MR-1 was screened after extended cultivation in electrochemical cells (ECs) with a working electrode poised at +0.2 V (vs. Ag/AgCl) to isolate mutants that adapted to electrode-respiring conditions and identify as-yet-unknown EET-related factors. Results Several mutants isolated from the enrichment culture exhibited rough morphology and extraordinarily large colonies on agar plates compared to wild-type MR-1. One of the isolated mutants, designated strain EC-2, produced 90% higher electric current than wild-type MR-1 in ECs and was found to have a transposon inserted in the SO_1860 (uvrY) gene, which encodes a DNA-binding response regulator of the BarA/UvrY two-component regulatory system. However, an in-frame deletion mutant of SO_1860 (∆SO_1860) did not exhibit a similar level of current generation as that of EC-2, suggesting that the enhanced current-generating capability of EC-2 was not simply due to the disruption of SO_1860. In both EC-2 and ∆SO_1860, the transcription of genes related to CPS synthesis was decreased compared to wild-type MR-1, suggesting that CPS negatively affects current generation. In addition, transcriptome analyses revealed that a number of genes, including those involved in biofilm formation, were differentially expressed in EC-2 compared to those in ∆SO_1860. Conclusions The present results indicate that the altered expression of the genes related to CPS biosynthesis and biofilm formation is associated with the distinct morphotype and high current-generating capability of strain EC-2, suggesting an important role of these genes in determining the EET activity of S. oneidensis. PMID:25028134

  6. A novel platform for automated high-throughput fluxome profiling of metabolic variants.

    PubMed

    Heux, Stéphanie; Poinot, Juliette; Massou, Stéphane; Sokol, Serguei; Portais, Jean-Charles

    2014-09-01

    Advances in metabolic engineering are enabling the creation of a large number of cell factories. However, high-throughput platforms do not yet exist for rapidly analyzing the metabolic network of the engineered cells. To fill the gap, we developed an integrated solution for fluxome profiling of large sets of biological systems and conditions. This platform combines a robotic system for (13)C-labelling experiments and sampling of labelled material with NMR-based isotopic fingerprinting and automated data interpretation. As a proof-of-concept, this workflow was applied to discriminate between Escherichia coli mutants with gradual expression of the glucose-6-phosphate dehydrogenase. Metabolic variants were clearly discriminated while pathways that support metabolic flexibility towards modulation of a single enzyme were elucidating. By directly connecting the data flow between cell cultivation and flux quantification, considerable advances in throughput, robustness, release of resources and screening capacity were achieved. This will undoubtedly facilitate the development of efficient cell factories.

  7. [Cloning of mMR-1 gene and expression in Pichia pastoris systems].

    PubMed

    Li, Tian-Bo; Hu, Yang; Wang, Yi-Guang; Xia, Huan-Zhang

    2005-01-01

    hMR-1 (Homo Myofibrillogenesis Regulator 1, AF417001) is a novel homo gene, which was firstly cloned in our laboratory. The former studies revealed that hMR-1 is a transmembrane protein which shows protein interaction with sarcomeric proteins like myomesin I, myosin regulatory light chain, alpha-enolase and some cell regulator proteins such as eukaryotic translation initiation factor3 subunit 5 (eIF3S5) and etc. In this work, we focused on cloning the homologous gene of hMR-1 from mouse C57BL/6J and exploring its expression using Pichia pastoris yeast system. Two pairs of primers were synthesized according to the hMR-1 gene homologous sequence on mouse genome chromosome 1. The mouse MR-1 gene (mMR-1) was cloned by PCR following the first round RT-PCR from mouse C57BL/6J spleen total RNA. Sequence analysis verified that mMR-1 gene and amino acids sequence showed 90.4% and 90.1% identity with hMR-1, respectively. The prediction of hydrophobic transmembrane structure of mMR-1 suggested it is also a transmembrane protein. The mMR-1 Pichia pastoris expression vector pPIC9-mMR-1 was constructed by fusion of the flanking mMR-1 ORF in the pPIC9 plasmid. After linearization of pPIC9-mMR-1 with Sal I, the 8.5kb DNA fragment was transformed into Pichia pastoris GS115 strain by electroporation. GS115/Mut+ pPIC9-mMR-1 transformants were selected on minimal methanol medium. Integration of mMR-1 gene into the yeast genome in the recombinants was verified by PCR from the transformants total DNA. The mMR-1 protein was expressed by induction under the concentration of 0.5 % methanol. The specific induced protein of 25 kD molecular mass in SDS-PAGE was confirmed to be the mMR-1 protein by Western blot rsing hMR-1 polyclonal antibody. The expression level of this recombinant mMR-1 protein was about 50 mg/L. The successful expression of mMR-1 in the Pichia pastoris GS115 will facilitate the further functional analysis of the novel gene MR-1 in animal model.

  8. Expression of terminal oxidases under nutrient-starved conditions in Shewanella oneidensis: detection of the A-type cytochrome c oxidase

    PubMed Central

    Le Laz, Sébastien; kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2016-01-01

    Shewanella species are facultative anaerobic bacteria that colonize redox-stratified habitats where O2 and nutrient concentrations fluctuate. The model species Shewanella oneidensis MR-1 possesses genes coding for three terminal oxidases that can perform O2 respiration: a bd-type quinol oxidase and cytochrome c oxidases of the cbb3-type and the A-type. Whereas the bd- and cbb3-type oxidases are routinely detected, evidence for the expression of the A-type enzyme has so far been lacking. Here, we investigated the effect of nutrient starvation on the expression of these terminal oxidases under different O2 tensions. Our results reveal that the bd-type oxidase plays a significant role under nutrient starvation in aerobic conditions. The expression of the cbb3-type oxidase is also modulated by the nutrient composition of the medium and increases especially under iron-deficiency in exponentially growing cells. Most importantly, under conditions of carbon depletion, high O2 and stationary-growth, we report for the first time the expression of the A-type oxidase in S. oneidensis, indicating that this terminal oxidase is not functionally lost. The physiological role of the A-type oxidase in energy conservation and in the adaptation of S. oneidensis to redox-stratified environments is discussed. PMID:26815910

  9. Evolution of Cell Size Homeostasis and Growth Rate Diversity during Initial Surface Colonization of Shewanella oneidensis.

    PubMed

    Lee, Calvin K; Kim, Alexander J; Santos, Giancarlo S; Lai, Peter Y; Lee, Stella Y; Qiao, David F; Anda, Jaime De; Young, Thomas D; Chen, Yujie; Rowe, Annette R; Nealson, Kenneth H; Weiss, Paul S; Wong, Gerard C L

    2016-09-06

    Cell size control and homeostasis are fundamental features of bacterial metabolism. Recent work suggests that cells add a constant size between birth and division ("adder" model). However, it is not known how cell size homeostasis is influenced by the existence of heterogeneous microenvironments, such as those during biofilm formation. Shewanella oneidensis MR-1 can use diverse energy sources on a range of surfaces via extracellular electron transport (EET), which can impact growth, metabolism, and size diversity. Here, we track bacterial surface communities at single-cell resolution to show that not only do bacterial motility appendages influence the transition from two- to three-dimensional biofilm growth and control postdivisional cell fates, they strongly impact cell size homeostasis. For every generation, we find that the average growth rate for cells that stay on the surface and continue to divide (nondetaching population) and that for cells that detach before their next division (detaching population) are roughly constant. However, the growth rate distribution is narrow for the nondetaching population, but broad for the detaching population in each generation. Interestingly, the appendage deletion mutants (ΔpilA, ΔmshA-D, Δflg) have significantly broader growth rate distributions than that of the wild type for both detaching and nondetaching populations, which suggests that Shewanella appendages are important for sensing and integrating environmental inputs that contribute to size homeostasis. Moreover, our results suggest multiplexing of appendages for sensing and motility functions contributes to cell size dysregulation. These results can potentially provide a framework for generating metabolic diversity in S. oneidensis populations to optimize EET in heterogeneous environments.

  10. The tetraheme cytochrome CymA is required for anaerobic respiration with dimethyl sulfoxide and nitrite in Shewanella oneidensis.

    PubMed

    Schwalb, Carsten; Chapman, Stephen K; Reid, Graeme A

    2003-08-12

    The tetraheme c-type cytochrome, CymA, from Shewanella oneidensis MR-1 has previously been shown to be required for respiration with Fe(III), nitrate, and fumarate [Myers, C. R., and Myers, J. M. (1997) J. Bacteriol. 179, 1143-1152]. It is located in the cytoplasmic membrane where the bulk of the protein is exposed to the periplasm, enabling it to transfer electrons to a series of redox partners. We have expressed and purified a soluble derivative of CymA (CymA(sol)) that lacks the N-terminal membrane anchor. We show here, by direct measurements of electron transfer between the purified proteins, that CymA(sol) efficiently reduces S. oneidensis fumarate reductase. This indicates that no further proteins are required for electron transfer between the quinone pool and fumarate if we assume direct reduction of CymA by quinols. By expressing CymA(sol) in a mutant lacking CymA, we have shown that this soluble form of the protein can complement the defect in fumarate respiration. We also demonstrate that CymA is essential for growth with DMSO (dimethyl sulfoxide) and for reduction of nitrite, implicating CymA in at least five different electron transfer pathways in Shewanella.

  11. Spatiometabolic stratification of Shewanella oneidensis biofilms.

    PubMed

    Teal, Tracy K; Lies, Douglas P; Wold, Barbara J; Newman, Dianne K

    2006-11-01

    Biofilms, or surface-attached microbial communities, are both ubiquitous and resilient in the environment. Although much is known about how biofilms form, develop, and detach, very little is understood about how these events are related to metabolism and its dynamics. It is commonly thought that large subpopulations of cells within biofilms are not actively producing proteins or generating energy and are therefore dead. An alternative hypothesis is that within the growth-inactive domains of biofilms, significant populations of living cells persist and retain the capacity to dynamically regulate their metabolism. To test this, we employed unstable fluorescent reporters to measure growth activity and protein synthesis in vivo over the course of biofilm development and created a quantitative routine to compare domains of activity in independently grown biofilms. Here we report that Shewanella oneidensis biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of size. Within domains of growth-inactive cells, genes typically upregulated under anaerobic conditions are expressed well after growth has ceased. These findings reveal that, far from being dead, the majority of cells in mature S. oneidensis biofilms have actively turned-on metabolic programs appropriate to their local microenvironment and developmental stage.

  12. A study on the polymorphism of human MHC class I-related MR1 gene and identification of an MR1-like pseudogene.

    PubMed

    Parra-Cuadrado, J F; Navarro, P; Mirones, I; Setién, F; Oteo, M; Martínez-Naves, E

    2000-08-01

    Human MR1 is a recently discovered, ubiquitously transcribed gene very similar to the HLA class I loci and of unknown function. Mouse and rat MR1 sequences have also been described showing high similarity with the human gene. The goal of this work was to investigate if human MR1 was polymorphic. We have found that DNA sequences of MR1-specific polymerase chain reaction (PCR) products obtained from samples of diverse ethnic origin were invariant except in one case in which two silent mutations were detected. We also found an MR1-like sequence displaying significant differences with the previously described, the most remarkable of which is a STOP codon in the alpha2 domain indicating that is a pseudogene.

  13. c-Type Cytochrome-Dependent Formation of U(IV) Nanoparticles by Shewanella oneidensis

    SciTech Connect

    Marshall, Matthew J.; Beliaev, Alex S.; Dohnalkova, Alice; Kennedy, David W.; Shi, Liang; Wang, Zheming; Boyanov, Maxim I.; Lai, Barry; Kemner, Kenneth M.; Mclean, Jeffrey S.; Reed, Samantha B.; Culley, David E.; Bailey, Vanessa L.; Simonson, Cody J.; Saffarini, Daad; Romine, Margaret F.; Zachara, John M.; Fredrickson, Jim K.

    2006-08-08

    Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal reducing bacterium, Shewanella oneidensis MR-1 are essential for the reduction of U(VI) and formation of extracelluar UO2 nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC, previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild type MR-1. Similar to the wild type, the mutants accumulated UO2 nanoparticles extracellularly to high densities in association with an exopolymeric substance (EPS). In wild type cells, this UO2-EPS matrix exhibited glycocalyx-like properties, contained multiple elements of the OM, polysaccharide, and heme containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO2 nanoparticles with MtrC and OmcA. This is the first study to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO2 nanoparticles. In the environment, such association of UO2 nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O2 or transport in soils and sediments.

  14. Surface-Enhanced Raman Imaging of Intracellular Bioreduction of Chromate in Shewanella oneidensis

    PubMed Central

    Ravindranath, Sandeep P.; Henne, Kristene L.; Thompson, Dorothea K.; Irudayaraj, Joseph

    2011-01-01

    This proposed research aims to use novel nanoparticle sensors and spectroscopic tools constituting surface-enhanced Raman spectroscopy (SERS) and Fluorescence Lifetime imaging (FLIM) to study intracellular chemical activities within single bioremediating microorganism. The grand challenge is to develop a mechanistic understanding of chromate reduction and localization by the remediating bacterium Shewanella oneidensis MR-1 by chemical and lifetime imaging. MR-1 has attracted wide interest from the research community because of its potential in reducing multiple chemical and metallic electron acceptors. While several biomolecular approaches to decode microbial reduction mechanisms exist, there is a considerable gap in the availability of sensor platforms to advance research from population-based studies to the single cell level. This study is one of the first attempts to incorporate SERS imaging to address this gap. First, we demonstrate that chromate-decorated nanoparticles can be taken up by cells using TEM and Fluorescence Lifetime imaging to confirm the internalization of gold nanoprobes. Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells. Distinctive differences in Raman signatures of Cr(VI) and Cr(III) enabled their spatial identification within single cells from the Raman images. A comprehensive evaluation of toxicity and cellular interference experiments conducted revealed the inert nature of these probes and that they are non-toxic. Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported. While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be

  15. Impact of silver(I) on the metabolism of Shewanella oneidensis.

    PubMed

    Wang, Hui; Law, Nicholas; Pearson, Geraldine; van Dongen, Bart E; Jarvis, Roger M; Goodacre, Royston; Lloyd, Jonathan R

    2010-02-01

    Anaerobic cultures of Shewanella oneidensis MR-1 reduced toxic Ag(I), forming nanoparticles of elemental Ag(0), as confirmed by X-ray diffraction analyses. The addition of 1 to 50 microM Ag(I) had a limited impact on growth, while 100 microM Ag(I) reduced both the doubling time and cell yields. At this higher Ag(I) concentration transmission electron microscopy showed the accumulation of elemental silver particles within the cell, while at lower concentrations the metal was exclusively reduced and precipitated outside the cell wall. Whole organism metabolite fingerprinting, using the method of Fourier transform infrared spectroscopy analysis of cells grown in a range of silver concentrations, confirmed that there were significant physiological changes at 100 microM silver. Principal component-discriminant function analysis scores and loading plots highlighted changes in certain functional groups, notably, lipids, amides I and II, and nucleic acids, as being discriminatory. Molecular analyses confirmed a dramatic drop in cellular yields of both the phospholipid fatty acids and their precursor molecules at high concentrations of silver, suggesting that the structural integrity of the cellular membrane was compromised at high silver concentrations, which was a result of intracellular accumulation of the toxic metal.

  16. The Influence of Cultivation Methods on Shewanella oneidensis Physiology and Proteome Expression

    SciTech Connect

    Elias, Dwayne A.; Tollaksen, Sandra L.; Kennedy, David W.; Mottaz, Heather M.; Giometti, Carol S.; Mclean, Jeffrey S.; Hill, Eric A.; Pinchuk, Grigoriy E.; Lipton, Mary S.; Fredrickson, Jim K.; Gorby, Yuri A.

    2008-04-01

    High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O2 deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell metabolic variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.

  17. Molecular Underpinnings of Nitrite Effect on CymA-Dependent Respiration in Shewanella oneidensis

    PubMed Central

    Jin, Miao; Fu, Huihui; Yin, Jianhua; Yuan, Jie; Gao, Haichun

    2016-01-01

    Shewanella exhibit a remarkable versatility of respiration, with a diverse array of electron acceptors (EAs). In environments where these bacteria thrive, multiple EAs are usually present. However, we know little about strategies by which these EAs and their interaction affect ecophysiology of Shewanella. In this study, we demonstrate in the model strain, Shewanella oneidensis MR-1, that nitrite, not through nitric oxide to which it may convert, inhibits respiration of fumarate, and probably many other EAs whose reduction depends on quinol dehydrogenase CymA. This is achieved via the repression of cyclic adenosine monophosphate (cAMP) production, a second messenger required for activation of cAMP-receptor protein (Crp) which plays a primary role in regulation of respiration. If nitrite is not promptly removed, intracellular cAMP levels drop, and this impairs Crp activity. As a result, the production of nitrite reductase NrfA, CymA, and fumarate reductase FccA is substantially reduced. In contrast, nitrite can be simultaneously respired with trimethylamine N-oxide, resulting in enhanced biomass. PMID:27493647

  18. Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku

    PubMed Central

    Baym, Michael; Shaket, Lev; Anzai, Isao A.; Adesina, Oluwakemi; Barstow, Buz

    2016-01-01

    Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction. PMID:27830751

  19. Core Fluxome and Metafluxome of Lactic Acid Bacteria under Simulated Cocoa Pulp Fermentation Conditions

    PubMed Central

    Adler, Philipp; Bolten, Christoph Josef; Dohnt, Katrin; Hansen, Carl Erik

    2013-01-01

    In the present work, simulated cocoa fermentation was investigated at the level of metabolic pathway fluxes (fluxome) of lactic acid bacteria (LAB), which are typically found in the microbial consortium known to convert nutrients from the cocoa pulp into organic acids. A comprehensive 13C labeling approach allowed to quantify carbon fluxes during simulated cocoa fermentation by (i) parallel 13C studies with [13C6]glucose, [1,2-13C2]glucose, and [13C6]fructose, respectively, (ii) gas chromatography-mass spectrometry (GC/MS) analysis of secreted acetate and lactate, (iii) stoichiometric profiling, and (iv) isotopomer modeling for flux calculation. The study of several strains of L. fermentum and L. plantarum revealed major differences in their fluxes. The L. fermentum strains channeled only a small amount (4 to 6%) of fructose into central metabolism, i.e., the phosphoketolase pathway, whereas only L. fermentum NCC 575 used fructose to form mannitol. In contrast, L. plantarum strains exhibited a high glycolytic flux. All strains differed in acetate flux, which originated from fractions of citrate (25 to 80%) and corresponding amounts of glucose and fructose. Subsequent, metafluxome studies with consortia of different L. fermentum and L. plantarum strains indicated a dominant (96%) contribution of L. fermentum NCC 575 to the overall flux in the microbial community, a scenario that was not observed for the other strains. This highlights the idea that individual LAB strains vary in their metabolic contribution to the overall fermentation process and opens up new routes toward streamlined starter cultures. L. fermentum NCC 575 might be one candidate due to its superior performance in flux activity. PMID:23851099

  20. Reconstruction of Gene Networks of Iron Response in Shewanella oneidensis

    SciTech Connect

    Yang, Yunfeng; Harris, Daniel P; Luo, Feng; Joachimiak, Marcin; Wu, Liyou; Dehal, Paramvir; Jacobsen, Janet; Yang, Zamin Koo; Gao, Haichun; Arkin, Adam; Palumbo, Anthony Vito; Zhou, Jizhong

    2009-01-01

    It is of great interest to study the iron response of the -proteobacterium Shewanella oneidensis since it possesses a high content of iron and is capable of utilizing iron for anaerobic respiration. We report here that the iron response in S. oneidensis is a rapid process. To gain more insights into the bacterial response to iron, temporal gene expression profiles were examined for iron depletion and repletion, resulting in identification of iron-responsive biological pathways in a gene co-expression network. Iron acquisition systems, including genes unique to S. oneidensis, were rapidly and strongly induced by iron depletion, and repressed by iron repletion. Some were required for iron depletion, as exemplified by the mutational analysis of the putative siderophore biosynthesis protein SO3032. Unexpectedly, a number of genes related to anaerobic energy metabolism were repressed by iron depletion and induced by repletion, which might be due to the iron storage potential of their protein products. Other iron-responsive biological pathways include protein degradation, aerobic energy metabolism and protein synthesis. Furthermore, sequence motifs enriched in gene clusters as well as their corresponding DNA-binding proteins (Fur, CRP and RpoH) were identified, resulting in a regulatory network of iron response in S. oneidensis. Together, this work provides an overview of iron response and reveals novel features in S. oneidensis, including Shewanella-specific iron acquisition systems, and suggests the intimate relationship between anaerobic energy metabolism and iron response.

  1. Single-Molecule Methods for the Large-Scale Characterization of Expression Levels and Protein-Protein Interactions in Shewanella Oneidensis MR-1

    SciTech Connect

    Weiss, Shimon; Michalet, Xavier

    2008-10-01

    This project has demonstrated a new approach to localize binding sites of proteins regulating gene expression (also known as transcription factors) on the genome of bacteria. Knowledge of the precise binding site(s) of a specific transcription factor helps determining its role in the cell cycle and by extension provides further understanding of the mechanisms at play in the organism. The approach entails labeling transcription factors (or any other DNA-binding protein of interest) with quantum dots, a new class of very bright fluorescent probes, which allow detection of individual molecules with a simple microscope. Detection is then followed with very accurate localization of the probe (with nanometer resolution) with respect to specific parts of the DNA or other proteins bound to the DNA. We have confirmed the precision of our measurement using another technique based on atomic force microscopy, which provides a nanometer-resolution topographic picture of a sample. Quantum dots and DNA are readily observable (and distinguishable) in the atomic force microscope, and can be simultaneously observed by fluorescence microscopy, allowing a direct comparison of the two methods. Precise nanometer-localization of protein binding sites using fluorescent quantum dots is thus a direct and visual method for physical mapping of transcription factor binding sites on whole genomes.

  2. Molecular Dynamics of the Shewanella oneidensis Response toChromate Stress

    SciTech Connect

    Brown, S.D.; Thompson, M.R.; VerBerkmoes, N.C.; Chourey, K.; Shah, M.; Zhou, J.-Z.; Hettich, R.L.; Thompson, D.K.

    2007-09-21

    Temporal genomic profiling and whole-cell proteomic analyseswere performed to characterize the dynamic molecular response of themetal-reducing bacterium Shewanella oneidensis MR-1 to an acute chromateshock. The complex dynamics of cellular processes demand the integrationof methodologies that describe biological systems at the levels ofregulation, gene and protein expression, and metabolite production.Genomic microarray analysis of the transcriptome dynamics ofmidexponential phase cells subjected to 1 mM potassium chromate (K2CrO4)at exposure time intervals of 5, 30, 60, and 90 min revealed 910 genesthat were differentially expressed at one or more time points. Stronglyinduced genes included those encoding components of a TonB1 irontransport system (tonB1-exbB1-exbD1), hemin ATP-binding cassettetransporters (hmuTUV), TonB-dependent receptors as well as sulfatetransporters (cysP, cysW-2, and cysA-2), and enzymes involved inassimilative sulfur metabolism (cysC, cysN, cysD, cysH, cysI, and cysJ).Transcript levels for genes with annotated functions in DNA repair (lexA,recX, recA, recN, dinP, and umuD), cellular detoxification (so1756,so3585, and so3586), and two-component signal transduction systems(so2426) were also significantly up-regulated (p<0.05) inCr(VI)-exposed cells relative to untreated cells. By contrast, genes withfunctions linked to energy metabolism, particularly electron transport(e.g. so0902-03-04, mtrA, omcA, and omcB), showed dramatic temporalalterations in expression with the majority exhibiting repression.Differential proteomics based on multidimensional HPLC-MS/MS was used tocomplement the transcriptome data, resulting in comparable induction andrepression patterns for a subset of corresponding proteins. In total,expression of 2,370 proteins were confidently verified with 624 (26percent) of these annotated as hypothetical or conserved hypotheticalproteins. The initial response of S. oneidensis to chromate shock appearsto require a combination of

  3. Effects of Bio-Au Nanoparticles on Electrochemical Activity of Shewanella oneidensis Wild Type and ΔomcA/mtrC Mutant

    PubMed Central

    Wu, Ranran; Cui, Li; Chen, Lixiang; Wang, Chao; Cao, Changli; Sheng, Guoping; Yu, Hanqing; Zhao, Feng

    2013-01-01

    Both Shewanella oneidensis MR-1 wild type and its mutant ΔomcA/mtrC are capable of transforming AuIII into Au nanoparticles (AuNPs). Cyclic voltammetry reveals a decrease in redox current after the wild type is exposed to AuIII but an increase in oxidation current for the mutant. The peak current of the wild type is much higher than that of the mutant before the exposure of AuIII, but lower than that of the mutant after the formation of AuNPs. This suggests that damage to the electron transfer chain in the mutant could be repaired by AuNPs to a certain extent. Spectroscopy and SDS-PAGE analysis indicate a decrease in cell protein content after the formation of AuNPs, which provides a convenient way to detect intracellular information on cells. PMID:24264440

  4. Systematic Assessment of the Benefits and Caveats in Mining Microbial Post-Translational Modifications from Shotgun Proteomic Data; Response of Shewanella oneidensis to Chromate Exposure

    SciTech Connect

    Thompson, Melissa R; Thompson, Dorothea K; Hettich, Robert {Bob} L

    2008-01-01

    Microbes are known to regulate both gene expression and protein activity through the use of post-translational modifications (PTMs). Common PTMs involved in cellular signaling and gene control include methylations, acetylations, and phosphorylations; whereas oxidations have been implicated as an indicator for stress. Shewanella oneidensis MR-1 is a gram-negative bacterium that demonstrates both respiratory versatility and the ability to sense and adapt to diverse environmental conditions. The dataset used in this study consisted of tandem mass spectra derived from mid-log phase aerobic cultures of S. oneidensis shocked either with or without 1 mM chromate [Cr(VI)]. In this study, three algorithms (DBDigger, Sequest, and InsPecT) were evaluated for their ability to scrutinize shotgun proteomic data for evidence of PTMs. The use of conservative scoring filters for peptides or proteins versus creating a sub-database first from a non-modification search was evaluated with DBDigger. The use of higher scoring filters for peptide identifications was found to result in optimal identifications of PTM peptides with a 2% false discovery rate (FDR) for the total dataset using the DBDigger algorithm. However, the FDR climbs to about 50% when considering PTM peptides only. Sequest was evaluated as a method for confirming PTM peptides putatively identified using DBDigger; however, there was a low identification rate (~25%) for the searched spectra. InsPecT was found to have a lower FDR (~9%) than DBDigger for PTM peptides. Comparisons between InsPecT and DBDigger were made with respect to both the FDR and PTM peptide identifications. As a demonstration of this approach, a number of S. oneidensis chemotaxis proteins as well as low-abundance signal transduction proteins were identified as being post-translationally modified in response to chromate challenge.

  5. Silencing MR-1 attenuates inflammatory damage in mice heart induced by AngII

    SciTech Connect

    Dai, Wenjian; Chen, Haiyang; Jiang, Jiandong; Kong, Weijia; Wang, Yiguang

    2010-01-15

    Myofibrillogenesis regulator-1(MR-1) can aggravate cardiac hypertrophy induced by angiotensin(Ang) II in mice through activation of NF-{kappa}B signaling pathway, and nuclear transcription factor (NF)-{kappa}B and activator protein-1(AP-1) regulate inflammatory and immune responses by increasing the expression of specific inflammatory genes in various tissues including heart. Whether inhibition of MR-1 expression will attenuate AngII-induced inflammatory injury in mice heart has not been explored. Herein, we monitored the activation of NF-{kappa}B and AP-1, together with expression of pro-inflammatory of interleukin(IL)-6, tumor necrosis factor(TNF)-{alpha}, vascular-cell adhesion molecule (VCAM)-1, platelet endothelial cell adhesion molecule (PECAM), and inflammatory cell infiltration in heart of mice which are induced firstly by AngII (PBS),then received MR-1-siRNA or control-siRNA injecting. We found that the activation of NF-{kappa}B and AP-1 was inhibited significantly, together with the decreased expression of IL-6, TNF-{alpha}, VCAM-1, and PECAM in AngII-induced mice myocardium in MR-1-siRNA injection groups compared with control-siRNA injecting groups. However, the expression level of MR-1 was not an apparent change in PBS-infused groups than in unoperation groups, and MR-1-siRNA do not affect the expression of MR-1 in PBS-infused mice. Our findings suggest that silencing MR-1 protected mice myocardium against inflammatory injury induced by AngII by suppression of pro-inflammatory transcription factors NF-{kappa}B and AP-1 signaling pathway.

  6. Genetic and Molecular Characterization of Flagellar Assembly in Shewanella oneidensis

    PubMed Central

    Wu, Lin; Wang, Jixuan; Tang, Peng; Chen, Haijiang; Gao, Haichun

    2011-01-01

    Shewanella oneidensis is a highly motile organism by virtue of a polar flagellum. Unlike most flagellated bacteria, it contains only one major chromosome segment encoding the components of the flagellum with the exception of the motor proteins. In this region, three genes encode flagellinsaccording to the original genome annotation. However, we find that only flaA and flaB encode functional filament subunits. Although these two genesare under the control of different promoters, they are actively transcribed and subsequently translated, producing a considerable number of flagellin proteins. Additionally, both flagellins are able to interact with their chaperon FliS and are subjected to feedback regulation. Furthermore, FlaA and FlaB are glycosylated by a pathwayinvolving a major glycosylating enzyme,PseB, in spite of the lack of the majority of theconsensus glycosylation sites. In conclusion, flagellar assembly in S. oneidensis has novel features despite the conservation of homologous genes across taxa. PMID:21731763

  7. High expression of CD26 accurately identifies human bacteria-reactive MR1-restricted MAIT cells

    PubMed Central

    Sharma, Prabhat K; Wong, Emily B; Napier, Ruth J; Bishai, William R; Ndung'u, Thumbi; Kasprowicz, Victoria O; Lewinsohn, Deborah A; Lewinsohn, David M; Gold, Marielle C

    2015-01-01

    Mucosa-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor TRAV1–2 and detect a range of bacteria and fungi through the MHC-like molecule MR1. However, knowledge of the function and phenotype of bacteria-reactive MR1-restricted TRAV1–2+ MAIT cells from human blood is limited. We broadly characterized the function of MR1-restricted MAIT cells in response to bacteria-infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria-reactive MR1-restricted T cells shared effector functions of cytolytic effector CD8+ T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8+ subsets we demonstrated that high expression of CD26 on CD8+ TRAV1–2+ cells identified with high specificity and sensitivity, bacteria-reactive MR1-restricted T cells from human blood. CD161hi was also specific for but lacked sensitivity in identifying all bacteria-reactive MR1-restricted T cells, some of which were CD161dim. Using cell surface expression of CD8, TRAV1–2, and CD26hi in the absence of stimulation we confirm that bacteria-reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis. PMID:25752900

  8. High expression of CD26 accurately identifies human bacteria-reactive MR1-restricted MAIT cells.

    PubMed

    Sharma, Prabhat K; Wong, Emily B; Napier, Ruth J; Bishai, William R; Ndung'u, Thumbi; Kasprowicz, Victoria O; Lewinsohn, Deborah A; Lewinsohn, David M; Gold, Marielle C

    2015-07-01

    Mucosa-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor TRAV1-2 and detect a range of bacteria and fungi through the MHC-like molecule MR1. However, knowledge of the function and phenotype of bacteria-reactive MR1-restricted TRAV1-2(+) MAIT cells from human blood is limited. We broadly characterized the function of MR1-restricted MAIT cells in response to bacteria-infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria-reactive MR1-restricted T cells shared effector functions of cytolytic effector CD8(+) T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8(+) subsets we demonstrated that high expression of CD26 on CD8(+)  TRAV1-2(+) cells identified with high specificity and sensitivity, bacteria-reactive MR1-restricted T cells from human blood. CD161(hi) was also specific for but lacked sensitivity in identifying all bacteria-reactive MR1-restricted T cells, some of which were CD161(dim) . Using cell surface expression of CD8, TRAV1-2, and CD26(hi) in the absence of stimulation we confirm that bacteria-reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis.

  9. From position-specific isotope labeling towards soil fluxomics: a novel toolbox to assess the microbial impact on biogeochemical cycles

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Dippold, M. A.; Kuzyakov, Y.

    2015-12-01

    Understanding the microbial impact on C and nutrient cycles is one of the most important challenges in terrestrial biogeochemistry. Transformation of low molecular weight organic substances (LMWOS) is a key step in all biogeochemical cycles because 1) all high molecular substances pass the LMWOS pool during their degradation and 2) only LMWOS can be taken up by microorganisms intact. Thus, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the microbial metabolic network and its control mechanism. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools but studies were nearly exclusively based on uniformly labeled substances. However, such tracers do not allow the differentiation of the intact use of the initial substances from its transformation to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of basic metabolites and quantification of isotope incorporation in CO2 and bulk soil enabled following the basic metabolic pathways of microorganisms. However, the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites like phospholipid fatty acids (PLFA) or amino sugars revealed new insights into the soil fluxome: First, it enables tracing specific anabolic pathways in diverse microbial communities in soils e.g. carbon starvation pathways versus pathways reflecting microbial growth. Second, it allows identification of specific pathways of individual functional microbial groups in soils in situ. Tracing metabolic pathways and understanding their regulating factors are crucial for soil C fluxomics i.e. the unravaling of the complex network of C transformations

  10. Aerated Shewanella oneidensis in Continuously-fed Bioelectrochemical Systems for Power and Hydrogen Production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the effects of aeration of Shewanella oneidensis on potentiostatic current production, iron(III) reduction, hydrogen production in a microbial electrolysis cell, and electric power generation in a microbial fuel cell. The potentiostatic performance of aerated S. oneidensis was considerab...

  11. TLR signaling in human antigen‐presenting cells regulates MR1‐dependent activation of MAIT cells

    PubMed Central

    van Wilgenburg, Bonnie; Hannaway, Rachel F.; Ruustal, Kerstin; Phalora, Prabhjeet; Kurioka, Ayako; Hansen, Ted H.; Willberg, Christian B.; Phillips, Rodney E.; Klenerman, Paul

    2016-01-01

    Mucosal‐associated invariant T (MAIT) cells are an abundant innate‐like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1‐mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1‐mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1‐mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B‐cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B‐cell lines, suggesting differential regulation in different cell types. APC activation and NF‐κB signaling were critical for MR1‐mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1‐mediated MAIT cell activation. Overall, MR1‐mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs. PMID:27105778

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

    PubMed Central

    Sekar, Ramanan; Shin, Hyun Dong

    2016-01-01

    ABSTRACT 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

  13. Engineering of Isogenic Cells Deficient for MR1 with a CRISPR/Cas9 Lentiviral System: Tools To Study Microbial Antigen Processing and Presentation to Human MR1-Restricted T Cells

    PubMed Central

    Lloyd, Angharad; Meermeier, Erin W.; Crowther, Michael D.; Connor, Thomas R.; Dolton, Garry; Miles, John J.; Burrows, Scott R.; Gold, Marielle C.; Lewinsohn, David M.

    2016-01-01

    The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1−/− clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1−/− clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells. PMID:27307560

  14. Draft Genome Sequence of Burkholderia sp. MR1, a Methylarsenate-Reducing Bacterial Isolate from Florida Golf Course Soil

    PubMed Central

    Pawitwar, Shashank S.; Utturkar, Sagar M.; Brown, Steven D.; Yoshinaga, Masafumi

    2015-01-01

    To elucidate the environmental organoarsenical biocycle, we isolated a soil organism, Burkholderia sp. MR1, which reduces relatively nontoxic pentavalent methylarsenate to the more toxic trivalent methylarsenite, with the goal of identifying the gene for the reductase. Here, we report the draft genome sequence of Burkholderia sp. MR1. PMID:26044439

  15. Biochemical and Physiological Characterization: Development & Apply Optical Methods for Charaterizing Biochemical Protein-Protein Interactions in MR-1

    SciTech Connect

    Weiss, Shimon

    2006-08-30

    The objectives of this report are to: Develop novel site-specific protein labeling chemistries for assaying protein-protein interactions in MR-1; and development of a novel optical acquisition and data analysis method for characterizing protein-protein interactions in MR-1 model systems. Our work on analyzing protein-protein interactions in MR-1 is divided in four areas: (1) expression and labeling of MR-1 proteins; (2) general scheme for site-specific fluorescent labeling of expressed proteins; (3) methodology development for monitoring protein-protein interactions; and (4) study of protein-protein interactions in MR-1. In this final report, we give an account for our advances in all areas.

  16. MR1-restricted mucosal associated invariant T (MAIT) cells in the immune response to Mycobacterium tuberculosis

    PubMed Central

    Gold, Marielle C.; Napier, Ruth J.; Lewinsohn, David M.

    2014-01-01

    Summary The intracellular pathogen Mycobacterium tuberculosis (Mtb) and its human host have long co-evolved. Although the host cellular immune response is critical to the control of the bacterium information on the specific contribution of different immune cell subsets in humans is incomplete. Mucosal associated invariant T (MAIT) cells are a prevalent and unique T-cell population in humans with the capacity to detect intracellular infection with bacteria including Mtb. MAIT cells detect bacterially derived metabolites presented by the evolutionarily conserved major histocompatibility complex-like molecule MR1. Here we review recent advances in our understanding of this T-cell subset and address the potential roles for MR1-restricted T cells in the control, diagnosis, and therapy of tuberculosis. PMID:25703558

  17. Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino

  18. Posttranslational Modification of Flagellin FlaB in Shewanella oneidensis

    PubMed Central

    Sun, Linlin; Jin, Miao; Ding, Wen; Yuan, Jie

    2013-01-01

    Shewanella oneidensis is a highly motile organism by virtue of a polar, glycosylated flagellum composed of flagellins FlaA and FlaB. In this study, the functional flagellin FlaB was isolated and analyzed with nano-liquid chromatography-mass spectrometry (MS) and tandem MS. In combination with the mutational analysis, we propose that the FlaB flagellin protein from S. oneidensis is modified at five serine residues with a series of novel O-linked posttranslational modifications (PTMs) that differ from each other by 14 Da. These PTMs are composed in part of a 274-Da sugar residue that bears a resemblance to the nonulosonic acids. The remainder appears to be composed of a second residue whose mass varies by 14 Da depending on the PTM. Further investigation revealed that synthesis of the glycans initiates with PseB and PseC, the first two enzymes of the Pse pathway. In addition, a number of lysine residues are found to be methylated by SO4160, an analogue of the lysine methyltransferase of Salmonella enterica serovar Typhimurium. PMID:23543712

  19. Validating Annotations for Uncharacterized Proteins in Shewanella oneidensis

    PubMed Central

    Louie, Brenton; Tarczy-Hornoch, Peter; Higdon, Roger

    2008-01-01

    Abstract Proteins of unknown function are a barrier to our understanding of molecular biology. Assigning function to these “uncharacterized” proteins is imperative, but challenging. The usual approach is similarity searches using annotation databases, which are useful for predicting function. However, since the performance of these databases on uncharacterized proteins is basically unknown, the accuracy of their predictions is suspect, making annotation difficult. To address this challenge, we developed a benchmark annotation dataset of 30 proteins in Shewanella oneidensis. The proteins in the dataset were originally uncharacterized after the initial annotation of the S. oneidensis proteome in 2002. In the intervening 5 years, the accumulation of new experimental evidence has enabled specific functions to be predicted. We utilized this benchmark dataset to evaluate several commonly utilized annotation databases. According to our criteria, six annotation databases accurately predicted functions for at least 60% of proteins in our dataset. Two of these six even had a “conditional accuracy” of 90%. Conditional accuracy is another evaluation metric we developed which excludes results from databases where no function was predicted. Also, 27 of the 30 proteins' functions were correctly predicted by at least one database. These represent one of the first performance evaluations of annotation databases on uncharacterized proteins. Our evaluation indicates that these databases readily incorporate new information and are accurate in predicting functions for uncharacterized proteins, provided that experimental function evidence exists. PMID:18687039

  20. Biochemical Analyses of Dissimilatory Iron Reduction by Shewanella oneidensis

    NASA Astrophysics Data System (ADS)

    Ruebush, S. S.; Tien, M.; Icopini, G. A.; Brantley, S. L.

    2002-12-01

    Shewanella oneidensis demonstrates respiratory flexibility by the transfer of electrons to Fe (III) and Mn (IV) oxides under anaerobic conditions. Researchers postulate that the bacterium utilizes surface proteins to facilitate the respiratory mechanism for dissimilatory iron(III) reduction. Previous genetic and biochemical studies has shown that iron reduction is associated with the outer membrane of the cell. The identity of the terminal reductase is not yet known. S. oneidensis has been shown to use soluble extra-cellular compounds to facilitate iron(III) reduction as well as expression of novel proteins on the cell surface when interacting with iron(III) oxides. Our results show that the outer membrane fraction possess enzymatic activity for converting Fe(III) to Fe(II) as measured by ferrozine complexation. AQDS, extra-cellular organic extracts, and iron(III) both soluble and solid have been assayed for activity with outer membrane fractions. Zymograms of the membrane fractions separated by isoelectric focusing and native PAGE electrophoresis stained using ferrozine have implicated proteins that are directly involved in the Fe(III) reduction process. A proteomics analysis of outer membrane proteins has also been implemented to identify different expression patterns under Fe(III) reducing conditions. Proteins that are unique to Fe(III) reduction have been isolated and identified using N-terminal sequence analysis. We will also attempt to examine the effect of enzymatic iron(III) reduction on isotopic partitioning from in vitro assays.

  1. humMR1, a highly specific humanized single chain antibody for targeting EGFRvIII.

    PubMed

    Safdari, Yaghoub; Farajnia, Safar; Asgharzadeh, Mohammad; Omidfar, Kobra; Khalili, Masoumeh

    2014-02-01

    Production of an efficient humanized single chain antibody is reported here to specifically target EGFRvIII, a truncated receptor expressed in a wide variety of human cancers. CDR loops of MR1, a phage display-derived murine single chain antibody developed against this mutant receptor, were grafted on human frameworks that had been selected based on similarity to MR1 in terms of two distinct parameters, variable domain protein sequence and CDR canonical structures. Moreover, two point mutations were introduced in CDR-H2 and CDR-H3 loops of the humanized antibody to destroy its cross-reactivity to wild-type EGFR. The resultant antibody, referred to as humMR1, was found by MTT assay, ELISA and western blot techniques to be highly specific for EGFRvIII. The affinity of this antibody for EGFRvIII-specific 14-amino acid synthetic peptide and HC2 cells were measured to be 1.87 × 10(10) and 2.17 × 10(10)/M respectively. This humanized antibody leads to 78.5% inhibition in proliferation of EGFRvIII-overexpressing cells.

  2. An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

    SciTech Connect

    Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan; Damron, Fredrick; Zhou, Jizhong; Qiu, Dongru

    2015-01-01

    Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.

  3. An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

    DOE PAGES

    Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan; ...

    2015-01-01

    Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essentialmore » for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.« less

  4. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

    PubMed Central

    Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

    2016-01-01

    Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

  5. Effect of biofilm coatings at metal-oxide/water interfaces I: Pb(II) and Zn(II) partitioning and speciation at Shewanella oneidensis/metal-oxide/water interfaces

    NASA Astrophysics Data System (ADS)

    Wang, Yingge; Gélabert, Alexandre; Michel, F. Marc; Choi, Yongseong; Gescher, Johannes; Ona-Nguema, Georges; Eng, Peter J.; Bargar, John R.; Farges, Francois; Spormann, Alfred M.; Brown, Gordon E.

    2016-09-01

    Microbial biofilms are often present as coatings on metal-oxide surfaces in natural and industrial environments and may induce significant changes in the partitioning behavior and speciation of aqueous metal ions, which in turn can impact their transport and fate. In this study, long-period X-ray standing wave-fluorescence yield (LP-XSW-FY) spectroscopy was used to measure under in situ conditions the partitioning of aqueous Pb(II) and Zn(II) between multilayer Shewanella oneidensis MR-1 biofilms and highly polished, oriented single-crystal surfaces of α-Al2O3 and α-Fe2O3 as a function of metal-ion concentration and time at pH 6.0. We show that after 3-h exposure time, Pb(II) binds preferentially to the α-Al2O3 (1-102) and α-Fe2O3 (0 0 0 1) surfaces at low Pb concentration ([Pb] = 10-7 M) and then increasingly partitions into the biofilm coatings at higher concentrations (10-6 to 10-4 M). In contrast, Zn(II) partitions preferentially into the biofilm coating for both surfaces at all Zn concentrations studied (10-7 to 10-4 M). In comparison, the α-Al2O3 (0 0 0 1) surface has a low affinity for both Pb(II) and Zn(II), and the biofilm coatings are the dominant sink for both ions. These findings suggest that in the presence of S. oneidensis biofilm coatings, α-Al2O3 (0 0 0 1) is the least reactive surface for Pb(II) and Zn(II) compared to α-Al2O3 (1-102) and α-Fe2O3 (0 0 0 1). They also show that Zn(II) has a lower affinity than Pb(II) for reactive sites on α-Al2O3 (1-102) and α-Fe2O3 (0 0 0 1) at [Me(II)] of 10-7 M; at 10-5 M, the bulk of the metal ions partition into the biofilm coatings. At longer exposure times (20-24 h), both Pb(II) and Zn(II) increasingly partition to the metal-oxide surfaces at [Me(II)] = 10-5 M and pH 6.0, indicating possible reaction/diffusion-controlled sorption processes. Pb LIII-edge and Zn K-edge grazing-incidence extended X-ray absorption fine structure (GI-EXAFS) measurements suggest that both Pb(II) and Zn(II) ions may be

  6. Effect of biofilm coatings at metal-oxide/water interfaces I: Pb(II) and Zn(II) partitioning and speciation at Shewanella oneidensis/metal-oxide/water interfaces

    DOE PAGES

    Wang, Yingge; Gelabert, Alexandre; Michel, F. Marc; ...

    2016-05-30

    Microbial biofilms are often present as coatings on metal-oxide surfaces in natural and industrial environments and may induce significant changes in the partitioning behavior and speciation of aqueous metal ions, which in turn can impact their transport and fate. In this study, long-period X-ray standing wave-fluorescence yield (LP-XSW-FY) spectroscopy was used to measure under in situ conditions the partitioning of aqueous Pb(II) and Zn(II) between multilayer Shewanella oneidensis MR-1 biofilms and highly polished, oriented single-crystal surfaces of α-Al2O3 and α-Fe2O3 as a function of metal-ion concentration and time at pH 6.0. We show that after 3-h exposure time, Pb(II) bindsmore » preferentially to the alpha-Al2O3 (1-102) and α-Fe2O3 (0001) surfaces at low Pb concentration ([Pb] = 10–7 M) and then increasingly partitions into the biofilm coatings at higher concentrations (10–6 to 10–4 M). In contrast, Zn(II) partitions preferentially into the biofilm coating for both surfaces at all Zn concentrations studied (10–7 to 10–4 M). In comparison, the α-Al2O3 (0001) surface has a low affinity for both Pb(II) and Zn(II), and the biofilm coatings are the dominant sink for both ions. These findings suggest that in the presence of S. oneidensis biofilm coatings, α-Al2O3 (0001) is the least reactive surface for Pb(II) and Zn(II) compared to α-Al2O3 (1-102) and α-Fe2O3 (0001). They also show that Zn(II) has a lower affinity than Pb(II) for reactive sites on α-Al2O3 (1-102) and α-Fe2O3 (0001) at [Me(II)] of 10–7 M; at 10–5 M, the bulk of the metal ions partition into the biofilm coatings. At longer exposure times (20-24 h), both Pb(II) and Zn(II) increasingly partition to the metal-oxide surfaces at [Me(II)] = 10–5 M and pH 6.0, indicating possible reaction/diffusion-controlled sorption processes. Pb L-III-edge and Zn K-edge grazing-incidence extended X-ray absorption fine structure (GI-EXAFS) measurements suggest that both Pb(II) and Zn

  7. Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

    SciTech Connect

    Hartshorne, Robert S.; Jepson, Brian N.; Clarke, Thomas A.; Field, Sarah J.; Fredrickson, Jim K.; Zachara, John M.; Shi, Liang; Butt, Julea N.; Richardson, David

    2007-09-04

    Abstract MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV–vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately *500 mV (vs. the standard hydrogen electrode). Across this potential window the UV– vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled lowspin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate.

  8. CD1d- and MR1-Restricted T Cells in Sepsis

    PubMed Central

    Szabo, Peter A.; Anantha, Ram V.; Shaler, Christopher R.; McCormick, John K.; Haeryfar, S.M. Mansour

    2015-01-01

    Dysregulated immune responses to infection, such as those encountered in sepsis, can be catastrophic. Sepsis is typically triggered by an overwhelming systemic response to an infectious agent(s) and is associated with high morbidity and mortality even under optimal critical care. Recent studies have implicated unconventional, innate-like T lymphocytes, including CD1d- and MR1-restricted T cells as effectors and/or regulators of inflammatory responses during sepsis. These cell types are typified by invariant natural killer T (iNKT) cells, variant NKT (vNKT) cells, and mucosa-associated invariant T (MAIT) cells. iNKT and vNKT cells are CD1d-restricted, lipid-reactive cells with remarkable immunoregulatory properties. MAIT cells participate in antimicrobial defense, and are restricted by major histocompatibility complex-related protein 1 (MR1), which displays microbe-derived vitamin B metabolites. Importantly, NKT and MAIT cells are rapid and potent producers of immunomodulatory cytokines. Therefore, they may be considered attractive targets during the early hyperinflammatory phase of sepsis when immediate interventions are urgently needed, and also in later phases when adjuvant immunotherapies could potentially reverse the dangerous state of immunosuppression. We will highlight recent findings that point to the significance or the therapeutic potentials of NKT and MAIT cells in sepsis and will also discuss what lies ahead in research in this area. PMID:26322041

  9. Cold adaptation regulated by cryptic prophage excision in Shewanella oneidensis.

    PubMed

    Zeng, Zhenshun; Liu, Xiaoxiao; Yao, Jianyun; Guo, Yunxue; Li, Baiyuan; Li, Yangmei; Jiao, Nianzhi; Wang, Xiaoxue

    2016-12-01

    Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation.

  10. Cold adaptation regulated by cryptic prophage excision in Shewanella oneidensis

    PubMed Central

    Zeng, Zhenshun; Liu, Xiaoxiao; Yao, Jianyun; Guo, Yunxue; Li, Baiyuan; Li, Yangmei; Jiao, Nianzhi; Wang, Xiaoxue

    2016-01-01

    Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation. PMID:27482926

  11. Highly efficient in vitro biosynthesis of silver nanoparticles using Lysinibacillus sphaericus MR-1 and their characterization

    PubMed Central

    Gou, Yujun; Zhou, Rongying; Ye, Xiujuan; Gao, Shanshan; Li, Xiangqian

    2015-01-01

    Silver nanoparticles (AgNPs) have been widely used in diverse fields due to their superior properties. Currently the biosynthesis of AgNPs is in the limelight of modern nanotechnology because of its green properties. However, relatively low yield and inefficiency diminish the prospect of applying these biosynthesized AgNPs. In this work, a rapid mass AgNP biosynthesis method using the cell-free extract of a novel bacterial strain, Lysinibacillus sphaericus MR-1, which has been isolated from a chemical fertilizer plant, is reported. In addition, the optimum synthesis conditions of AgNPs were investigated. The optimum pH, temperature, dosage, and reaction time were 12, 70 °C, 20 mM AgNO3, and 75 min, respectively. Finally, AgNPs were characterized by optical absorption spectroscopy, zeta potential and size distribution analysis, x-ray diffraction, electron microscopy, and energy-dispersive x-ray spectroscopy. The results revealed that these biosynthesized AgNPs were bimolecular covered, stable, well-dispersed face centered cubic (fcc) spherical crystalline particles with diameters in the range 5–20 nm. The advantages of this approach are its simplicity, high efficiency, and eco-friendly and cost-effective features. PMID:27877754

  12. Aerated Shewanella oneidensis in continuously fed bioelectrochemical systems for power and hydrogen production.

    PubMed

    Rosenbaum, Miriam; Cotta, Michael A; Angenent, Largus T

    2010-04-01

    We studied the effects of aeration of Shewanella oneidensis on potentiostatic current production, hydrogen production in a microbial electrolysis cell, and electric power generation in a microbial fuel cell (MFC). The potentiostatic performance of aerated S. oneidensis was considerably enhanced to a maximum current density of 0.45 A/m(2) or 80.3 A/m(3) (mean: 0.34 A/m(2), 57.2 A/m(3)) compared to anaerobically grown cultures. Biocatalyzed hydrogen production rates with aerated S. oneidensis were studied within the applied potential range of 0.3-0.9 V and were highest at 0.9 V with 0.3 m(3) H(2)/m(3) day, which has been reported for mixed cultures, but is approximately 10 times higher than reported for an anaerobic culture of S. oneidensis. Aerated MFC experiments produced a maximum power density of 3.56 W/m(3) at a 200-Omega external resistor. The main reasons for enhanced electrochemical performance are higher levels of active biomass and more efficient substrate utilization under aerobic conditions. Coulombic efficiencies, however, were greatly reduced due to losses of reducing equivalents to aerobic respiration in the anode chamber. The next challenge will be to optimize the aeration rate of the bacterial culture to balance between maximization of bacterial activation and minimization of aerobic respiration in the culture.

  13. The tricarboxylic acid cycle in Shewanella oneidensis is independent of Fur and RyhB control

    SciTech Connect

    Yang, Yunfeng; McCue, Lee Ann; Parsons, Andrea; Feng, Sheng; Zhou, Jizhong

    2010-01-01

    Background: It is well established in E. coli and Vibrio cholerae that strains harboring mutations in the ferric uptake regulator gene (fur) are unable to utilize tricarboxylic acid (TCA) compounds, due to the down-regulation of key TCA cycle enzymes, such as AcnA and SdhABCD. This down-regulation is mediated by a Fur-regulated small regulatory RNA named RyhB. It is unclear in the g-proteobacterium S. oneidensis whether TCA is also regulated by Fur and RyhB. Results: In the present study, we showed that a fur deletion mutant of S. oneidensis could utilize TCA compounds. Consistently, expression of the TCA cycle genes acnA and sdhA was not down-regulated in the mutant. To explore this observation further, we identified a ryhB gene in Shewanella species and experimentally demonstrated the gene expression. Further experiments suggested that RyhB was up-regulated in fur mutant, but that AcnA and SdhA were not controlled by RyhB. Conclusions: These cumulative results delineate an important difference of the Fur-RyhB regulatory cycle between S. oneidensis and other g-proteobacteria. This work represents a step forward for understanding the unique regulation in S. oneidensis.

  14. Unexpected chemoreceptors mediate energy taxis towards electron acceptors in Shewanella oneidensis.

    PubMed

    Baraquet, Claudine; Théraulaz, Laurence; Iobbi-Nivol, Chantal; Méjean, Vincent; Jourlin-Castelli, Cécile

    2009-07-01

    Shewanella oneidensis uses a wide range of terminal electron acceptors for respiration. In this study, we show that the chemotactic response of S. oneidensis to anaerobic electron acceptors requires functional electron transport systems. Deletion of the genes encoding dimethyl sulphoxide and trimethylamine N-oxide reductases, or inactivation of these molybdoenzymes as well as nitrate reductase by addition of tungstate, abolished electron acceptor taxis. Moreover, addition of nigericin prevented taxis towards trimethylamine N-oxide, dimethyl sulphoxide, nitrite, nitrate and fumarate, showing that this process depends on the DeltapH component of the proton motive force. These data, together with those concerning response to metals (Bencharit and Ward, 2005), support the idea that, in S. oneidensis, taxis towards electron acceptors is governed by an energy taxis mechanism. Surprisingly, energy taxis in S. oneidensis is not mediated by the PAS-containing chemoreceptors but rather by a chemoreceptor (SO2240) containing a Cache domain. Four other chemoreceptors also play a minor role in this process. These results indicate that energy taxis can be mediated by new types of chemoreceptors.

  15. Domain Analysis of ArcS, the Hybrid Sensor Kinase of the Shewanella oneidensis MR-1 Arc Two-Component System, Reveals Functional Differentiation of Its Two Receiver Domains

    PubMed Central

    Bubendorfer, Sebastian

    2013-01-01

    In all species of the genus Shewanella, the redox-sensing Arc two-component system consists of the response regulator ArcA, the sensor kinase ArcS, and the separate phosphotransfer protein HptA. Compared to its counterpart ArcB in Escherichia coli, ArcS has a significantly different domain structure. Resequencing and reannotation revealed that in the N-terminal part, ArcS possesses a periplasmic CaChe-sensing domain bracketed by two transmembrane domains and, moreover, that ArcS has two cytoplasmic PAS-sensing domains and two receiver domains, compared to a single one of each in ArcB. Here, we used a combination of in vitro phosphotransfer studies on purified proteins and phenotypic in vivo mutant analysis to determine the roles of the different domains in ArcS function. The analysis revealed that phosphotransfer occurs from and toward the response regulator ArcA and involves mainly the C-terminal RecII domain. However, RecI also can receive a phosphate from HptA. In addition, the PAS-II domain, located upstream of the histidine kinase domain, is crucial for function. The results support a model in which phosphorylation of RecI stimulates histidine kinase activity of ArcS in order to maintain an appropriate level of phosphorylated ArcA according to environmental conditions. In addition, the study reveals some fundamental mechanistic differences between ArcS/HptA and ArcB with respect to signal perception and phosphotransfer despite functional conservation of the Arc system in Shewanella and E. coli. PMID:23161031

  16. Silver nanocrystallites: Facile biofabrication using Shewanella oneidensis, and an evaluation of their comparative toxicity on Gram-negative and Gram-positive bacteria

    SciTech Connect

    Suresh, Anil K; Wang, Wei; Pelletier, Dale A; Moon, Ji Won; Gu, Baohua; Mortensen, Ninell P; Allison, David P; Joy, David Charles; Phelps, Tommy Joe; Doktycz, Mitchel John

    2010-01-01

    Microorganisms have long been known to develop resistance to metal ions either by sequestering metals inside the cell or by effluxing them into the extracellular media. Here we report the biosynthesis of extracellular silver based single nanocrystallites of well-defined composition and homogeneous morphology utilizing the -proteobacterium, Shewanella oneidensis strain MR-1, upon incubation with an aqueous solution of silver nitrate. Further characterization of these particles revealed that the crystals consist of small, reasonably monodispersed spheres in the size range 2 11 nm (with an average of 4 1.5 nm). The bactericidal effect of these biologically synthesized silver nanoparticles (biogenic-Ag) are compared to similar chemically synthesized nanoparticles (colloidal silver [colloidal-Ag] and oleate capped silver [oleate-Ag]). The determination of the bactericidal effect of these different silver nanoparticles was assessed using both Gram-negative (E. coli) and Gram-positive (B. subtilis) bacteria and based on the diameter of the inhibition zone in disc diffusion tests, minimum inhibitory concentrations, Live/Dead staining assays, and atomic force microscopy. From a toxicity perspective, a clear synthesis procedure, and a surface coat- and strain-dependent inhibition were observed for silver nanoparticles. Biogenic-Ag was found to be of higher toxicity when compared to colloidal-Ag for both E. coli and B. subtilis. E. coli was found to be more resistant to either of these nanoparticles than B. subtilis. In contrast, Oleate-Ag was not toxic to either of the bacteria. These findings have important implications for the potential uses of Ag nanomaterials and for their fate in biological and environmental systems.

  17. Direct Involvement of Type II Secretion System in Extracellular Translocation of Shewanella Oneidensis Outer Membrane Cytochromes MtrC and OmcA

    SciTech Connect

    Shi, Liang; Deng, Shuang; Marshall, Matthew J.; Wang, Zheming; Kennedy, David W.; Dohnalkova, Alice; Mottaz, Heather M.; Hill, Eric A.; Gorby, Yuri A.; Beliaev, Alex S.; Richardson, David J.; Zachara, John M.; Fredrickson, Jim K.

    2008-08-01

    Outer membrane decaheme c-type cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 are extracellular lipoproteins important for dissimilatory reduction of solid metal (hydr)oxides during anaerobic respiration. To investigate the roles of type II secretion system (T2S) in translocation of MtrC and OmcA across outer membrane, we measured the effects of deleting two T2S genes, gspD and gspG, on the secretion of MtrC and OmcA when cells were grown under anaerobic conditions. Deletion of gspD or gspG resulted in slightly yellowish supernatants, different from the pink supernatant of wild type (wt). Comparative proteomic analyses revealed that, although MtrC, OmcA and NrfA, a periplasmic nitrite reductase, were present the supernatants of wt and ΔgspD mutant, their peptides counts were much lower in ΔgspD than in wt. Subsequent analyses with heme-staining and Western blot not only confirmed that deletion of gspD or gspG reduced the abundances of MtrC and OmcA in the supernatants, but also revealed that the deletions consequently increased their abundances inside the cells. Complementation of ΔgspG mutant with functional GspG could reverse the effects of deleting gspG on the colors of the supernatants and the abundances of MtrC and OmcA. In contrast, Western results showed that the abundance of NrfA was reduced in the supernatant and the cells of ΔgspD mutant, suggesting that reduced NrfA in the periplasm, where MtrC and OmcA were accumulated, contributed to its reduction in the supernatant. Thus, our results demonstrate at the first time that T2S facilitates translocation of MtrC and OmcA across outer membrane.

  18. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage.

    PubMed

    Gold, Marielle C; McLaren, James E; Reistetter, Joseph A; Smyk-Pearson, Sue; Ladell, Kristin; Swarbrick, Gwendolyn M; Yu, Yik Y L; Hansen, Ted H; Lund, Ole; Nielsen, Morten; Gerritsen, Bram; Kesmir, Can; Miles, John J; Lewinsohn, Deborah A; Price, David A; Lewinsohn, David M

    2014-07-28

    Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire as a whole, especially for TCRβ chain sequences. Moreover, different pathogen-specific responses were characterized by distinct TCR usage, both between and within individuals, suggesting that MAIT cell adaptation was a direct consequence of exposure to various exogenous MR1-restricted epitopes. In line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped by microbial exposure.

  19. Microbial-enzymatic-hybrid biological fuel cell with optimized growth conditions for Shewanella oneidensis DSP-10.

    PubMed

    Roy, Jared N; Luckarift, Heather R; Sizemore, Susan R; Farrington, Karen E; Lau, Carolin; Johnson, Glenn R; Atanassov, Plamen

    2013-07-10

    In this work we present a biological fuel cell fabricated by combining a Shewanella oneidensis microbial anode and a laccase-modified air-breathing cathode. This concept is devised as an extension to traditional biochemical methods by incorporating diverse biological catalysts with the aim of powering small devices. In preparing the biological fuel cell anode, novel hierarchical-structured architectures and biofilm configurations were investigated. A method for creating an artificial biofilm based on encapsulating microorganisms in a porous, thin film of silica was compared with S. oneidensis biofilms that were allowed to colonize naturally. Results indicate comparable current and power densities for artificial and natural biofilm formations, based on growth characteristics. As a result, this work describes methods for creating controllable and reproducible bio-anodes and demonstrates the versatility of hybrid biological fuel cells.

  20. A Crp-Dependent Two-Component System Regulates Nitrate and Nitrite Respiration in Shewanella oneidensis

    PubMed Central

    Dong, Yangyang; Wang, Jixuan; Fu, Huihui; Zhou, Guangqi; Shi, Miaomiao; Gao, Haichun

    2012-01-01

    We have previously illustrated the nitrate/nitrite respiratory pathway of Shewanella oneidensis, which is renowned for its remarkable versatility in respiration. Here we investigated the systems regulating the pathway with a reliable approach which enables characterization of mutants impaired in nitrate/nitrite respiration by guaranteeing biomass. The S. oneidensis genome encodes an Escherichia coli NarQ/NarX homolog SO3981 and two E. coli NarP/NarL homologs SO1860 and SO3982. Results of physiological characterization and mutational analyses demonstrated that S. oneidensis possesses a single two-component system (TCS) for regulation of nitrate/nitrite respiration, consisting of the sensor kinase SO3981(NarQ) and the response regulator SO3982(NarP). The TCS directly controls the transcription of nap and nrfA (genes encoding nitrate and nitrite reductases, respectively) but regulates the former less tightly than the latter. Additionally, phosphorylation at residue 57 of SO3982 is essential for its DNA-binding capacity. At the global control level, Crp is found to regulate expression of narQP as well as nap and nrfA. In contrast to NarP-NarQ, Crp is more essential for nap rather than nrfA. PMID:23240049

  1. Metabolic Characteristics of a Glucose-Utilizing Shewanella oneidensis Strain Grown under Electrode-Respiring Conditions

    PubMed Central

    Nakagawa, Gen; Kouzuma, Atsushi; Hirose, Atsumi; Kasai, Takuya; Yoshida, Gen; Watanabe, Kazuya

    2015-01-01

    In bioelectrochemical systems, the electrode potential is an important parameter affecting the electron flow between electrodes and microbes and microbial metabolic activities. Here, we investigated the metabolic characteristics of a glucose-utilizing strain of engineered Shewanella oneidensis under electrode-respiring conditions in electrochemical reactors for gaining insight into how metabolic pathways in electrochemically active bacteria are affected by the electrode potential. When an electrochemical reactor was operated with its working electrode poised at +0.4 V (vs. an Ag/AgCl reference electrode), the engineered S. oneidensis strain, carrying a plasmid encoding a sugar permease and glucose kinase of Escherichia coli, generated current by oxidizing glucose to acetate and produced D-lactate as an intermediate metabolite. However, D-lactate accumulation was not observed when the engineered strain was grown with a working electrode poised at 0 V. We also found that transcription of genes involved in pyruvate and D-lactate metabolisms was upregulated at a high electrode potential compared with their transcription at a low electrode potential. These results suggest that the carbon catabolic pathway of S. oneidensis can be modified by controlling the potential of a working electrode in an electrochemical bioreactor. PMID:26394222

  2. Metabolic Characteristics of a Glucose-Utilizing Shewanella oneidensis Strain Grown under Electrode-Respiring Conditions.

    PubMed

    Nakagawa, Gen; Kouzuma, Atsushi; Hirose, Atsumi; Kasai, Takuya; Yoshida, Gen; Watanabe, Kazuya

    2015-01-01

    In bioelectrochemical systems, the electrode potential is an important parameter affecting the electron flow between electrodes and microbes and microbial metabolic activities. Here, we investigated the metabolic characteristics of a glucose-utilizing strain of engineered Shewanella oneidensis under electrode-respiring conditions in electrochemical reactors for gaining insight into how metabolic pathways in electrochemically active bacteria are affected by the electrode potential. When an electrochemical reactor was operated with its working electrode poised at +0.4 V (vs. an Ag/AgCl reference electrode), the engineered S. oneidensis strain, carrying a plasmid encoding a sugar permease and glucose kinase of Escherichia coli, generated current by oxidizing glucose to acetate and produced D-lactate as an intermediate metabolite. However, D-lactate accumulation was not observed when the engineered strain was grown with a working electrode poised at 0 V. We also found that transcription of genes involved in pyruvate and D-lactate metabolisms was upregulated at a high electrode potential compared with their transcription at a low electrode potential. These results suggest that the carbon catabolic pathway of S. oneidensis can be modified by controlling the potential of a working electrode in an electrochemical bioreactor.

  3. The role of host cell physiology in the productivity of the baculovirus-insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines.

    PubMed

    Monteiro, Francisca; Bernal, Vicente; Alves, Paula M

    2017-03-01

    The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is broadly used for the production of recombinant proteins and vaccine manufacture, yet the host physiological aspects that contribute to productivity are to be disclosed. This work provides the first quantitative analysis of the metabolic fluxes of High Five cells. This analysis was conducted in comparison with Sf9 cells, another major host for biologicals production via BEVS. Moreover, herein is presented, for the first time, quantitative data of the relative contribution of sugars and amino acids catabolism to the activity of the TCA cycle in Sf9 and High Five cells. High Five cells metabolic activity was markedly influenced by the amino acids concentration in culture medium, which determine the rates of amino acid catabolism, carbon overflow and by-product formation. This characteristic of High Five cells was reflected in the activities of anaplerotic metabolism and the TCA cycle, which may not work as a true cycle as a function of medium composition. This was not the case for Sf9 cells, in which the glucose carbon incorporation in the TCA cycle was significantly higher and lactate production minor. Following infection, the decrease in by-product accumulation rates was accompanied by an increase in net ATP synthesis in Sf9 and High Five cells, although through distinct mechanisms cell-line dependent. The impact of baculovirus infection on cellular metabolic status highlights the capacity of this virus to re-direct the cellular fluxome toward ATP production to support replication and progeny generation. These results pave the way to deepen our knowledge on the relationship between a host cell and the virus, contributing to disclosing the metabolic determinants that contribute to productivity. Biotechnol. Bioeng. 2017;114: 674-684. © 2016 Wiley Periodicals, Inc.

  4. Effect of biofilm coatings at metal-oxide/water interfaces II: Competitive sorption between Pb(II) and Zn(II) at Shewanella oneidensis/metal-oxide/water interfaces

    NASA Astrophysics Data System (ADS)

    Wang, Yingge; Gélabert, Alexandre; Michel, F. Marc; Choi, Yongseong; Eng, Peter J.; Spormann, Alfred M.; Brown, Gordon E.

    2016-09-01

    Competitive sorption of Pb(II) and Zn(II) on Shewanella oneidensis MR-1 biofilm-coated single-crystal α-Al2O3 (1 -1 0 2) and α-Fe2O3 (0 0 0 1) surfaces was investigated using long-period X-ray standing wave-florescence yield (LP-XSW-FY) spectroscopy. In situ partitioning of aqueous Pb(II) and Zn(II) between the biofilms and underlying metal-oxide substrates was probed following exposure of these complex interfaces to equi-molar Pb and Zn solutions (0.01 M NaNO3 as background electrolyte, pH = 6.0, and 3-h equilibration time). At higher Pb and Zn concentrations (⩾10-5 M), more than 99% of these ions partitioned into the biofilms at S. oneidensis/α-Al2O3 (1 -1 0 2)/water interfaces, which is consistent with the partitioning behavior of both Pb(II) or Zn(II) in single-metal-ion experiments. Thus, no apparent competitive effects were found in this system at these relatively high metal-ion concentrations. However, at lower equi-molar concentrations (⩽10-6 M), Pb(II) and Zn(II) partitioning in the same system changed significantly compared to the single-metal-ion systems. The presence of Zn(II) decreased Pb(II) partitioning onto α-Al2O3 (1 -1 0 2) substantially (∼52% to ∼13% at 10-7 M, and ∼23% to ∼5% at 10-6 M), whereas the presence of Pb(II) caused more Zn(II) to partition onto α-Al2O3 (1 -1 0 2) surfaces (∼15% to ∼28% at 10-7 M, and ∼1% to ∼7% at 10-6 M). The higher observed partitioning of Zn(II) (∼28%) at the α-Al2O3 (1 -1 0 2) surfaces compared to Pb(II) (∼13%) in the mixed-metal-ion systems at the lowest concentration (10-7 M) suggests that Zn(II) is slightly favored over Pb(II) for sorption sites on α-Al2O3 (1 -1 0 2) surfaces under our experimental conditions. Competitive sorption of Pb(II) and Zn(II) at S. oneidensis/α-Fe2O3 (0 0 0 1)/water interfaces at equi-molar metal-ion concentrations of ⩽10-6 M showed that the presence of Pb(II) ions decreased Zn(II) partitioning onto α-Fe2O3 (0 0 0 1) significantly (∼45% to <1% at 10

  5. Impact of TiO2 Nanoparticles on Growth, Biofilm Formation, and Flavin Secretion in Shewanella oneidensis

    PubMed Central

    Maurer-Jones, Melissa A.; Gunsolus, Ian L.; Meyer, Ben M.; Christenson, Cole J.; Haynes, Christy L.

    2013-01-01

    Understanding of nanoparticle impacts on critical bacteria functions allows us to gain a mechanistic understanding of toxicity and guides us towards design rules for creating safe nanomaterials. Herein and using analytical techniques, biofilm formation, a general bacteria function, and riboflavin secretion, a species-specific function, were monitored in Shewanella oneidensis, a metal reducing bacterium, following exposure to a variety of TiO2 nanoparticle types (synthesized, Aeroxide P25, and T-Eco). TEM images show that dosed nanoparticles are in close proximity to the bacteria but they are not internalized. Using quartz crystal microbalance (QCM), it was revealed that S. oneidensis biofilm formation is slowed in the presence of nanoparticles. Though S. oneidensis grows more slowly in the presence of TiO2 nanoparticles, riboflavin secretion, a function related to the S. oneidensis metal reducing capacity, was increased significantly in a nanoparticle dose-dependent manner. Both changes in biofilm formation and riboflavin secretion are supported by changes in gene expression in nanoparticle-exposed S. oneidensis. This broad study of bacterial nanotoxicity, including use of sensitive analytical tools for functional assessments of biofilm formation, riboflavin secretion, and gene expression has implications for total ecosystem health as the use of engineered nanoparticles grows. PMID:23701037

  6. Effect of biofilm coatings at metal-oxide/water interfaces I: Pb(II) and Zn(II) partitioning and speciation at Shewanella oneidensis/metal-oxide/water interfaces

    SciTech Connect

    Wang, Yingge; Gelabert, Alexandre; Michel, F. Marc; Choi, Yongseong; Gescher, Johannes; Ona-Nguema, Georges; Eng, Peter J.; Bargar, John R.; Farges, Francois; Spormann, Alfred M.; Brown, Jr., Gordon E.

    2016-05-30

    Microbial biofilms are often present as coatings on metal-oxide surfaces in natural and industrial environments and may induce significant changes in the partitioning behavior and speciation of aqueous metal ions, which in turn can impact their transport and fate. In this study, long-period X-ray standing wave-fluorescence yield (LP-XSW-FY) spectroscopy was used to measure under in situ conditions the partitioning of aqueous Pb(II) and Zn(II) between multilayer Shewanella oneidensis MR-1 biofilms and highly polished, oriented single-crystal surfaces of α-Al2O3 and α-Fe2O3 as a function of metal-ion concentration and time at pH 6.0. We show that after 3-h exposure time, Pb(II) binds preferentially to the alpha-Al2O3 (1-102) and α-Fe2O3 (0001) surfaces at low Pb concentration ([Pb] = 10–7 M) and then increasingly partitions into the biofilm coatings at higher concentrations (10–6 to 10–4 M). In contrast, Zn(II) partitions preferentially into the biofilm coating for both surfaces at all Zn concentrations studied (10–7 to 10–4 M). In comparison, the α-Al2O3 (0001) surface has a low affinity for both Pb(II) and Zn(II), and the biofilm coatings are the dominant sink for both ions. These findings suggest that in the presence of S. oneidensis biofilm coatings, α-Al2O3 (0001) is the least reactive surface for Pb(II) and Zn(II) compared to α-Al2O3 (1-102) and α-Fe2O3 (0001). They also show that Zn(II) has a lower affinity than Pb(II) for reactive sites on α-Al2O3 (1-102) and α-Fe2O3 (0001) at [Me(II)] of 10–7 M; at 10–5 M, the bulk of the metal ions partition into the biofilm coatings. At longer exposure times (20-24 h), both Pb(II) and Zn

  7. Snapshot of iron response in Shewanella oneidensis by gene network reconstruction

    SciTech Connect

    Yang, Yunfeng; Harris, Daniel P.; Luo, Feng; Xiong, Wenlu; Joachimiak, Marcin; Wu, Liyou; Dehal, Paramvir; Jacobsen, Janet; Yang, Zamin; Palumbo, Anthony V.; Arkin, Adam P.; Zhou, Jizhong

    2008-10-09

    Background: Iron homeostasis of Shewanella oneidensis, a gamma-proteobacterium possessing high iron content, is regulated by a global transcription factor Fur. However, knowledge is incomplete about other biological pathways that respond to changes in iron concentration, as well as details of the responses. In this work, we integrate physiological, transcriptomics and genetic approaches to delineate the iron response of S. oneidensis. Results: We show that the iron response in S. oneidensis is a rapid process. Temporal gene expression profiles were examined for iron depletion and repletion, and a gene co-expression network was reconstructed. Modules of iron acquisition systems, anaerobic energy metabolism and protein degradation were the most noteworthy in the gene network. Bioinformatics analyses suggested that genes in each of the modules might be regulated by DNA-binding proteins Fur, CRP and RpoH, respectively. Closer inspection of these modules revealed a transcriptional regulator (SO2426) involved in iron acquisition and ten transcriptional factors involved in anaerobic energy metabolism. Selected genes in the network were analyzed by genetic studies. Disruption of genes encoding a putative alcaligin biosynthesis protein (SO3032) and a gene previously implicated in protein degradation (SO2017) led to severe growth deficiency under iron depletion conditions. Disruption of a novel transcriptional factor (SO1415) caused deficiency in both anaerobic iron reduction and growth with thiosulfate or TMAO as an electronic acceptor, suggesting that SO1415 is required for specific branches of anaerobic energy metabolism pathways. Conclusions: Using a reconstructed gene network, we identified major biological pathways that were differentially expressed during iron depletion and repletion. Genetic studies not only demonstrated the importance of iron acquisition and protein degradation for iron depletion, but also characterized a novel transcriptional factor (SO1415) with a

  8. Effect of biofilm coatings at metal-oxide/water interfaces II: Competitive sorption between Pb(II) and Zn(II) at Shewanella oneidensis/metal-oxide/water interfaces

    DOE PAGES

    Wang, Yingge; Gelabert, Alexandre; Michel, F. Marc; ...

    2016-05-07

    Competitive sorption of Pb(II) and Zn(II) on Shewanella oneidensis MR-1 biofilm-coated single-crystal α-Al2O3 (1 –1 0 2) and α-Fe2O3 (0 0 0 1) surfaces was investigated using long-period X-ray standing wave-florescence yield (LP-XSW-FY) spectroscopy. In situ partitioning of aqueous Pb(II) and Zn(II) between the biofilms and underlying metal-oxide substrates was probed following exposure of these complex interfaces to equi-molar Pb and Zn solutions (0.01 M NaNO3 as background electrolyte, pH = 6.0, and 3-h equilibration time). At higher Pb and Zn concentrations (≥10–5 M), more than 99% of these ions partitioned into the biofilms at S. oneidensis/α-Al2O3 (1 –1 0more » 2)/water interfaces, which is consistent with the partitioning behavior of both Pb(II) or Zn(II) in single-metal-ion experiments. Furthermore, no apparent competitive effects were found in this system at these relatively high metal-ion concentrations. However, at lower equi-molar concentrations (≤10–6 M), Pb(II) and Zn(II) partitioning in the same system changed significantly compared to the single-metal-ion systems. The presence of Zn(II) decreased Pb(II) partitioning onto α-Al2O3 (1 –1 0 2) substantially (~52% to ~13% at 10–7 M, and ~23% to ~5% at 10–6 M), whereas the presence of Pb(II) caused more Zn(II) to partition onto α-Al2O3 (1 –1 0 2) surfaces (~15% to ~28% at 10–7 M, and ~1% to ~7% at 10–6 M) .The higher observed partitioning of Zn(II) (~28%) at the α-Al2O3 (1 –1 0 2) surfaces compared to Pb(II) (~13%) in the mixed-metal-ion systems at the lowest concentration (10–7 M) suggests that Zn(II) is slightly favored over Pb(II) for sorption sites on α-Al2O3 (1 –1 0 2) surfaces under our experimental conditions.« less

  9. MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase.

    PubMed

    Vidoni, Sara; Harbour, Michael E; Guerrero-Castillo, Sergio; Signes, Alba; Ding, Shujing; Fearnley, Ian M; Taylor, Robert W; Tiranti, Valeria; Arnold, Susanne; Fernandez-Vizarra, Erika; Zeviani, Massimo

    2017-02-14

    The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes.

  10. An essential role for UshA in processing of extracellular flavin electron shuttles by Shewanella oneidensis.

    PubMed

    Covington, Elizabeth D; Gelbmann, Christopher B; Kotloski, Nicholas J; Gralnick, Jeffrey A

    2010-10-01

    The facultative anaerobe Shewanella oneidensis can reduce a number of insoluble extracellular metals. Direct adsorption of cells to the metal surface is not necessary, and it has been shown that S. oneidensis releases low concentrations flavins, including riboflavin and flavin mononucleotide (FMN), into the surrounding medium to act as extracellular electron shuttles. However, the mechanism of flavin release by Shewanella remains unknown. We have conducted a transposon mutagenesis screen to identify mutants deficient in extracellular flavin accumulation. Mutations in ushA, encoding a predicted 5'-nucleotidase, resulted in accumulation of flavin adenine dinucleotide (FAD) in culture supernatants, with a corresponding decrease in FMN and riboflavin. Cellular extracts of S. oneidensis convert FAD to FMN, whereas extracts of ushA mutants do not, and fractionation experiments show that UshA activity is periplasmic. We hypothesize that S. oneidensis secretes FAD into the periplasmic space, where it is hydrolysed by UshA to FMN and adenosine monophosphate (AMP). FMN diffuses through outer membrane porins where it accelerates extracellular electron transfer, and AMP is dephosphorylated by UshA and reassimilated by the cell. We predict that transport of FAD into the periplasm also satisfies the cofactor requirement of the unusual periplasmic fumarate reductase found in Shewanella.

  11. Identification and characterization of a HEPN-MNT family type II toxin–antitoxin in Shewanella oneidensis

    PubMed Central

    Yao, Jianyun; Guo, Yunxue; Zeng, Zhenshun; Liu, Xiaoxiao; Shi, Fei; Wang, Xiaoxue

    2015-01-01

    Toxin–antitoxin (TA) systems are prevalent in bacteria and archaea. However, related studies in the ecologically and bioelectrochemically important strain Shewanella oneidensis are limited. Here, we show that SO_3166, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibited cell growth in S. oneidensis and Escherichia coli. SO_3165, a putative minimal nucleotidyltransferase (MNT), neutralized the toxicity of SO_3166. Gene SO_3165 lies upstream of SO_3166, and they are co-transcribed. Moreover, the SO_3165 and SO_3166 proteins interact with each other directly in vivo, and antitoxin SO_3165 bound to the promoter of the TA operon and repressed its activity. Finally, the conserved Rx4-6H domain in HEPN family was identified in SO_3166. Mutating either the R or H abolished SO_3166 toxicity, confirming that Rx4-6H domain is critical for SO_3166 activity. Taken together, these results demonstrate that SO_3166 and SO_3165 in S. oneidensis form a typical type II TA pair. This TA pair plays a critical role in regulating bacterial functions because its disruption led to impaired cell motility in S. oneidensis. Thus, we demonstrated for the first time that HEPN-MNT can function as a TA system, thereby providing important insights into the understanding of the function and regulation of HEPNs and MNTs in prokaryotes. PMID:26112399

  12. The tricarboxylic acid cycle in Shewanella oneidensis is independent of Fur and RyhB control

    SciTech Connect

    Yang, Yunfeng; McCue, Lee Ann; Parsons, Andrea B.; Feng, Sheng; Zhou, Jizhong

    2010-10-26

    It is well established in E. coli and Vibrio cholerae that strains harboring mutations in the ferric uptake regulator gene (fur) are unable to utilize tricarboxylic acid (TCA) compounds, due to the down-regulation of key TCA cycle enzymes, such as AcnA and SdhABCD. This down-regulation is mediated by a Fur-regulated small regulatory RNA named RyhB. In this study, we showed that a fur deletion mutant of the γ-proteobacterium S. oneidensis could utilize TCA compounds. In addition, expression of the TCA cycle genes acnA and sdhA was not down-regulated in the mutant. To explore this observation further, we identified a ryhB gene in Shewanella species and demonstrated its expression experimentally. Further experiments suggested that RyhB was up-regulated in fur mutant, but that AcnA and SdhA were not controlled by RyhB. This work delineates an important difference of the Fur-RyhB regulatory cycle between S. oneidensis and other γ-proteobacteria.

  13. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov.

    PubMed

    Venkateswaran, K; Moser, D P; Dollhopf, M E; Lies, D P; Saffarini, D A; MacGregor, B J; Ringelberg, D B; White, D C; Nishijima, M; Sano, H; Burghardt, J; Stackebrandt, E; Nealson, K H

    1999-04-01

    The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.

  14. Exoprotein Production Correlates with Morphotype Changes of Nonmotile Shewanella oneidensis Mutants

    PubMed Central

    Shi, Miaomiao; Wu, Lin; Xia, Yu; Chen, Haijiang; Luo, Qixia; Sun, Linlin

    2013-01-01

    We report a previously undescribed mechanism for the rugose morphotype in Shewanella oneidensis, a research model for investigating redox transformations of environmental contaminants. Bacteria may form smooth or rugose colonies on agar plates. In general, conversion from the smooth to rugose colony morphotype is attributed to increased production of exopolysaccharide (EPS). In this work, we discovered that aflagellate S. oneidensis mutants grew into rugose colonies, whereas those with nonfunctional flagellar filaments remained smooth. EPS production was not altered in either case, but mutants with the rugose morphotype showed significantly reduced exoprotein secretion. The idea that exoproteins at a reduced level correlate with rugosity gained support from smooth suppressor strains of an aflagellate rugose fliD (encoding the capping protein) mutant, which restored the exoprotein level to the levels of the wild-type and mutant strains with a smooth morphotype. Further analyses revealed that SO1072 (a putative GlcNAc-binding protein) was one of the highly upregulated exoproteins in these suppressor strains. Most intriguingly, this study identified a compensatory mechanism of SO1072 to flagellins possibly mediated by bis-(3′-5′)-cyclic dimeric GMP. PMID:23335418

  15. Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

    NASA Astrophysics Data System (ADS)

    Wen, Junlin; Zhou, Shungui; Chen, Junhua

    2014-06-01

    Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3', 5, 5'-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 103 and 5.0 × 106 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms.

  16. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov

    NASA Technical Reports Server (NTRS)

    Venkateswaran, K.; Moser, D. P.; Dollhopf, M. E.; Lies, D. P.; Saffarini, D. A.; MacGregor, B. J.; Ringelberg, D. B.; White, D. C.; Nishijima, M.; Sano, H.; Burghardt, J.; Stackebrandt, E.; Nealson, K. H.

    1999-01-01

    The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.

  17. Structure of 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase from Shewanella oneidensis at 1.6 A: identification of farnesyl pyrophosphate trapped in a hydrophobic cavity.

    PubMed

    Ni, Shuisong; Robinson, Howard; Marsing, Gregory C; Bussiere, Dirksen E; Kennedy, Michael A

    2004-11-01

    Isopentenyl pyrophosphate (IPP) is a universal building block for the ubiquitous isoprenoids that are essential to all organisms. The enzymes of the non-mevalonate pathway for IPP synthesis, which is unique to many pathogenic bacteria, have recently been explored as targets for antibiotic development. Several crystal structures of 2C-methyl-D-erythritol-2,4-cyclophosphate (MECDP) synthase, the fifth of seven enzymes involved in the non-mevalonate pathway for synthesis of IPP, have been reported; however, the composition of metal ions in the active site and the presence of a hydrophobic cavity along the non-crystallographic threefold symmetry axis has varied between the reported structures. Here, the structure of MEDCP from Shewanella oneidensis MR1 (SO3437) was determined to 1.6 A resolution in the absence of substrate. The presence of a zinc ion in the active-site cleft, tetrahedrally coordinated by two histidine side chains, an aspartic acid side chain and an ambiguous fourth ligand, was confirmed by zinc anomalous diffraction. Based on analysis of anomalous diffraction data and typical metal-to-ligand bond lengths, it was concluded that an octahedral sodium ion was 3.94 A from the zinc ion. A hydrophobic cavity was observed along the threefold non-crystallographic symmetry axis, filled by a well defined non-protein electron density that could be modeled as farnesyl pyrophosphate (FPP), a downstream product of IPP, suggesting a possible feedback mechanism for enzyme regulation. The high-resolution data clarified the FPP-binding mode compared with previously reported structures. Multiple sequence alignment indicated that the residues critical to the formation of the hydrophobic cavity and for coordinating the pyrophosphate group of FPP are present in the majority of MEDCP synthase enzymes, supporting the idea of a specialized biological function related to FPP binding in a subfamily of MEDCP synthase homologs.

  18. Sequence and Genetic Characterization of etrA, an fnr Analog that Regulates Anaerobic Respiration in Shewanella putrefaciens MR-1

    NASA Technical Reports Server (NTRS)

    Saffarini, Daad A.; Nelson, Kenneth H.

    1993-01-01

    An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-l. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Further, S.putrefaciens etrA is able to complement an fnr mutant of E.coli. In contrast to fnr, there is no recognizable Fnr box upstream of the etrA sequence. Gene replacement etr.A mutants of MR-1 were deficient in growth on nitrite, thiosulfate, sulfite, trimethylamine-N-oxide, dimethyl sulfoxide, Fe(III), and fumarate, suggesting that EtrA is involved in the regulation of the corresponding reductase genes. However, the mutants were all positive for reduction of and growth on nitrate and Mn(IV), indicating that EtrA is not involved in the regulation of these two systems. Southern blots of S.putrefaciens DNA with use of etrA as a probe revealed the expected etrA bands and a second set of hybridization signals whose genetic and functional properties remain to be determined.

  19. Loss of OxyR reduces efficacy of oxygen respiration in Shewanella oneidensis

    PubMed Central

    Wan, Fen; Shi, Miaomiao; Gao, Haichun

    2017-01-01

    In many bacteria, OxyR is the major regulator controlling cellular response to H2O2. A common phenotype resulting from OxyR loss is reduced growth rate, but the underlying mechanism is unknown. We demonstrated in Shewanella oneidensis, an important research model for applied and environmental microbes, that the defect is primarily due to an electron shortage to major terminal oxidase cytochrome cbb3. The loss of OxyR leads to enhanced production of electron carriers that compete for electrons against cytochrome cbb3, cytochrome bd in particular. We further showed that the oxyR mutation also results in increased production of menaquinone, an additional means to lessen electrons to cytochrome cbb3. Although regulation of OxyR on these biological processes appears to be indirect, these data indicate that the regulator plays a previously underappreciated role in mediating respiration. PMID:28195212

  20. Monodispersed biocompatible silver sulfide nanoparticles: Facile extracellular biosynthesis using the gamma-proteobacterium, Shewanella oneidensis

    SciTech Connect

    Doktycz, Mitchel John; Moon, Ji Won; Meyer III, Harry M; Hensley, Dale K; Phelps, Tommy Joe; Pelletier, Dale A

    2011-01-01

    Interest in engineered metal and semiconductor nanocrystallites continues to grow due to their unique size- and shape-dependent optoelectronic, physicochemical and biological properties. Therefore identifying novel non-hazardous nanoparticle synthesis routes that address hydrophilicity, size and shape control and production costs has become a priority. In the present article we report for the first time on the efficient generation of extracellular silver sulfide (Ag{sub 2}S) nanoparticles by the metal-reducing bacterium Shewanella oneidensis. The particles are reasonably monodispersed and homogeneously shaped. They are produced under ambient temperatures and pressures at high yield, 85% theoretical maximum. UV-visible and Fourier transform infrared spectroscopy, dynamic light scattering, X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy measurements confirmed the formation, optical and surface properties, purity and crystallinity of the synthesized particles. Further characterization revealed that the particles consist of spheres with a mean diameter of 9 {+-} 3.5 nm, and are capped by a detachable protein/peptide surface coat. Toxicity assessments of these biogenic Ag{sub 2}S nanoparticles on Gram-negative (Escherichia coli and S. oneidensis) and Gram-positive (Bacillus subtilis) bacterial systems, as well as eukaryotic cell lines including mouse lung epithelial (C 10) and macrophage (RAW-264.7) cells, showed that the particles were non-inhibitory and non-cytotoxic to any of these systems. Our results provide a facile, eco-friendly and economical route for the fabrication of technologically important semiconducting Ag{sub 2}S nanoparticles. These particles are dispersible and biocompatible, thus providing excellent potential for use in optical imaging, electronic devices and solar cell applications.

  1. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system

    SciTech Connect

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-09-11

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein 'small tetraheme c' replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-{angstrom}-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

  2. Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System

    PubMed Central

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-01-01

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein). PMID:22382353

  3. Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms.

    PubMed

    Sivakumar, Krishnakumar; Mukherjee, Manisha; Cheng, Hsin-I; Zhang, Yingdan; Ji, Lianghui; Cao, Bin

    2015-03-01

    Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations.

  4. System-level approach to studying oxygen stress and acclimation of Shewanella oneidensis to growth under aerobic conditions

    NASA Astrophysics Data System (ADS)

    Beliaev, A.

    2008-12-01

    Systems-level approaches have been proven extremely useful in elucidating the mechanisms involved in stress response and acclimation of microorganisms to different environments. Recent studies of Shewanella oneidensis, a dissimilatory metal reducer catalyzing biogeochemical cycling of Fe and Mn, demonstrate that this facultatively aerobic bacterium is inhibited by high concentrations of oxygen. Physiological and genomic studies demonstrated that growth under aerobic conditions triggers autoaggregation of S. oneidensis leading to significant physiological and morphological changes which are consistent with biofilm mode of growth. Global transcriptome profiling of the aggregates revealed coordinated upregulation of various attachment and adhesion factors which is governed through coordinate regulation by the RpoS, SpoIIA, and Crp transcription factors. The aerobic aggregated cells also revealed increased expression of putative anaerobic electron transfer and homologs of metal reduction genes. The experimental evidence indicates that aggregate formation in S. oneidensis may serve as an alternative or an addition to biochemical detoxification to reduce the oxidative stress associated with production of reactive oxygen species during aerobic metabolism while facilitating the development of hypoxic conditions within the aggregate interior.

  5. Real-Time Gene Expression Profiling of Live Shewanella Oneidensis Cells

    SciTech Connect

    Xiaoliang Sunney Xie

    2009-03-30

    The overall objective of this proposal is to make real-time observations of gene expression in live Shewanella oneidensis cells with high sensitivity and high throughput. Gene expression, a central process to all life, is stochastic because most genes often exist in one or two copies per cell. Although the central dogma of molecular biology has been proven beyond doubt, due to insufficient sensitivity, stochastic protein production has not been visualized in real time in an individual cell at the single-molecule level. We report the first direct observation of single protein molecules as they are generated, one at a time in a single live E. coli cell, yielding quantitative information about gene expression [Science 2006; 311: 1600-1603]. We demonstrated a general strategy for live-cell single-molecule measurements: detection by localization. It is difficult to detect single fluorescence protein molecules inside cytoplasm - their fluorescence is spread by fast diffusion to the entire cell and overwhelmed by the strong autofluorescence. We achieved single-molecule sensitivity by immobilizing the fluorescence protein on the cell membrane, where the diffusion is much slowed. We learned that under the repressed condition protein molecules are produced in bursts, with each burst originating from a stochastically-transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. We also simultaneously published a paper reporting a different method using β-glactosidase as a reporter [Nature 440, 358 (2006)]. Many important proteins are expressed at low levels, inaccessible by previous proteomic techniques. Both papers allowed quantification of protein expression with unprecedented sensitivity and received overwhelming acclaim from the scientific community. The Nature paper has been identified as one of the most-cited papers in the past year [http://esi-topics.com/]. We have also an analytical framework describing the

  6. Water Dynamics in Shewanella oneidensis at Ambient and High Pressure using Quasi-Elastic Neutron Scattering

    PubMed Central

    Foglia, Fabrizia; Hazael, Rachael; Simeoni, Giovanna G.; Appavou, Marie-Sousai; Moulin, Martine; Haertlein, Michael; Trevor Forsyth, V.; Seydel, Tilo; Daniel, Isabelle; Meersman, Filip; McMillan, Paul F.

    2016-01-01

    Quasielastic neutron scattering (QENS) is an ideal technique for studying water transport and relaxation dynamics at pico- to nanosecond timescales and at length scales relevant to cellular dimensions. Studies of high pressure dynamic effects in live organisms are needed to understand Earth’s deep biosphere and biotechnology applications. Here we applied QENS to study water transport in Shewanella oneidensis at ambient (0.1 MPa) and high (200 MPa) pressure using H/D isotopic contrast experiments for normal and perdeuterated bacteria and buffer solutions to distinguish intracellular and transmembrane processes. The results indicate that intracellular water dynamics are comparable with bulk diffusion rates in aqueous fluids at ambient conditions but a significant reduction occurs in high pressure mobility. We interpret this as due to enhanced interactions with macromolecules in the nanoconfined environment. Overall diffusion rates across the cell envelope also occur at similar rates but unexpected narrowing of the QENS signal appears between momentum transfer values Q = 0.7–1.1 Å−1 corresponding to real space dimensions of 6–9 Å. The relaxation time increase can be explained by correlated dynamics of molecules passing through Aquaporin water transport complexes located within the inner or outer membrane structures. PMID:26738409

  7. Water Dynamics in Shewanella oneidensis at Ambient and High Pressure using Quasi-Elastic Neutron Scattering.

    PubMed

    Foglia, Fabrizia; Hazael, Rachael; Simeoni, Giovanna G; Appavou, Marie-Sousai; Moulin, Martine; Haertlein, Michael; Trevor Forsyth, V; Seydel, Tilo; Daniel, Isabelle; Meersman, Filip; McMillan, Paul F

    2016-01-07

    Quasielastic neutron scattering (QENS) is an ideal technique for studying water transport and relaxation dynamics at pico- to nanosecond timescales and at length scales relevant to cellular dimensions. Studies of high pressure dynamic effects in live organisms are needed to understand Earth's deep biosphere and biotechnology applications. Here we applied QENS to study water transport in Shewanella oneidensis at ambient (0.1 MPa) and high (200 MPa) pressure using H/D isotopic contrast experiments for normal and perdeuterated bacteria and buffer solutions to distinguish intracellular and transmembrane processes. The results indicate that intracellular water dynamics are comparable with bulk diffusion rates in aqueous fluids at ambient conditions but a significant reduction occurs in high pressure mobility. We interpret this as due to enhanced interactions with macromolecules in the nanoconfined environment. Overall diffusion rates across the cell envelope also occur at similar rates but unexpected narrowing of the QENS signal appears between momentum transfer values Q = 0.7-1.1 Å(-1) corresponding to real space dimensions of 6-9 Å. The relaxation time increase can be explained by correlated dynamics of molecules passing through Aquaporin water transport complexes located within the inner or outer membrane structures.

  8. Graphene oxide as nanogold carrier for ultrasensitive electrochemical immunoassay of Shewanella oneidensis with silver enhancement strategy.

    PubMed

    Wen, Junlin; Zhou, Shungui; Yuan, Yong

    2014-02-15

    The genus Shewanella is ubiquitous in environment and has been extensively studied for their applications in bioremediation. A novel immunoassay for ultrasensitive detection of Shewanella oneidensis was presented based on graphene oxide (GO) as nanogold carrier with silver enhancement strategy. The enhanced sensitivity was achieved by employing conjugate-featuring gold nanoparticles (AuNPs) and antibodies (Ab) assembled on bovine serum albumin (BSA)-modified GO (Ab/AuNPs/BSA/GO). After a sandwich-type antigen-antibody reaction, Ab/AuNPs/BSA/GO conjugate binding on the target analyte produced an enhanced immune-recognition response by the reduction of silver ion in the present of hydroquinone. The deposited silver metal was dissolved with nitric acid and subsequently quantified by anodic stripping voltammetry. The high AuNPs loading capacity of GO and the obvious signal amplification by gold-catalyzed silver deposition offer an excellent detection method with a wide range of linear relationship between 7.0 × 10(1) and 7.0 × 10(7)cfu/mL. Furthermore, the immunoassay developed in this work exhibited high sensitivity, acceptable stability and reproducibility. This simple and sensitive assay method has promising application in various fields for rapid detection of bacteria, protein and DNA.

  9. Electron tunneling properties of outer-membrane decaheme cytochromes from Shewanella oneidensis

    SciTech Connect

    Wigginton, Nicholas S; Rosso, Kevin M; Lower, Brian H; Shi, Liang; Hochella, Michael F

    2007-02-01

    In this report, we describe the characterization of two outer-membrane decaheme cytochromes OmcA and MtrC purified from the metal-reducing bacterium Shewanella oneidensis using scanning tunneling microscopy (STM) and tunneling spectroscopy (TS). OmcA and MtrC were solubilized with a common detergent and irreversibly bound to Au (111) substrates as self-assembled cytochrome films. X-ray photoelectron spectroscopy (XPS) verified that OmcA and MtrC were covalently bound to the Au surface via thiol bonds to cysteine residues. Initial STM images show that a layer of detergent covers and protects the cytochrome films. Temporary application of high bias voltage causes the detergent film to reorganize around the tip, opening a window for direct STM imaging of the cytochrome layer underneath. The STM apparent sizes of both OmcA and MtrC are 58 nanometers in diameter consistent with expectations from their molecular masses. Current-voltage TS over individual cytochromes showed that OmcA and MtrC have different abilities to mediate the tunneling current, reflecting differences in their electronic structures. The data suggest that the two cytochromes could have different roles in the electron transport chain during metal reduction.

  10. Cell growth and protein expression of Shewanella oneidensis in biofilms and hydrogel-entrapped cultures.

    PubMed

    Zhang, Yingdan; Ng, Chun Kiat; Cohen, Yehuda; Cao, Bin

    2014-05-01

    The performance of biofilm-based bioprocesses is difficult to predict and control because of the intrinsic heterogeneous and dynamic properties of microbial biofilms. Biofilm mimics, such as microbial cells entrapped in polymeric scaffolds that are permeable for nutrients, have been proposed to replace real biofilms to achieve long-term robust performance in engineering applications. However, the physiological differences between cells that are physically entrapped in a synthetic polymeric matrix and biofilm cells that are encased in a self-produced polymeric matrix remain unknown. In this study, using Shewanella oneidensis as a model organism and alginate hydrogel as a model synthetic matrix, we compared the cell growth and protein expression in entrapped cultures and biofilms. The hydrogel-entrapped cultures were found to exhibit a growth rate comparable with biofilms. There was no substantial difference in cell viability, surface charge, as well as hydrophobicity between the cells grown in alginate hydrogel and those grown in biofilms. However, the gel-entrapped cultures were found to be physiologically different from biofilms. The gel-entrapped cultures had a higher demand for metabolic energy. The siderophore-mediated iron uptake was repressed in the gel-entrapped cells. The presence of the hydrogel matrix decreased the expression of proteins involved in biofilm formation, while inducing the production of extracellular DNA (eDNA) in the gel-entrapped cultures. These results advance the fundamental understanding of the physiology of hydrogel-entrapped cells, which can lead to more efficient biofilm mimic-based applications.

  11. Investigating different mechanisms for biogenic selenite transformations: Geobacter sulfurreducens, Shewanella oneidensis and Veillonella atypica

    USGS Publications Warehouse

    Pearce, C.I.; Pattrick, R.A.D.; Law, N.; Charnock, J.M.; Coker, V.S.; Fellowes, J.W.; Oremland, R.S.; Lloyd, J.R.

    2009-01-01

    The metal-reducing bacteria Geobacter sulfurreducens, Shewanella oneidensis and Veillonella atypica, use different mechanisms to transform toxic, bioavailable sodium selenite to less toxic, non-mobile elemental selenium and then to selenide in anaerobic environments, offering the potential for in situ and ex situ bioremediation of contaminated soils, sediments, industrial effluents, and agricultural drainage waters. The products of these reductive transformations depend on both the organism involved and the reduction conditions employed, in terms of electron donor and exogenous extracellular redox mediator. The intermediary phase involves the precipitation of elemental selenium nanospheres and the potential role of proteins in the formation of these structures is discussed. The bionanomineral phases produced during these transformations, including both elemental selenium nanospheres and metal selenide nanoparticles, have catalytic, semiconducting and light-emitting properties, which may have unique applications in the realm of nanophotonics. This research offers the potential to combine remediation of contaminants with the development of environmentally friendly manufacturing pathways for novel bionanominerals. ?? 2009 Taylor & Francis.

  12. Effect of biofilm coatings at metal-oxide/water interfaces II: Competitive sorption between Pb(II) and Zn(II) at Shewanella oneidensis/metal-oxide/water interfaces

    SciTech Connect

    Wang, Yingge; Gelabert, Alexandre; Michel, F. Marc; Choi, Yongseong; Eng, Peter J.; Spormann, Alfred M.; Brown, Jr., Gordon E.

    2016-05-07

    Competitive sorption of Pb(II) and Zn(II) on Shewanella oneidensis MR-1 biofilm-coated single-crystal α-Al2O3 (1 –1 0 2) and α-Fe2O3 (0 0 0 1) surfaces was investigated using long-period X-ray standing wave-florescence yield (LP-XSW-FY) spectroscopy. In situ partitioning of aqueous Pb(II) and Zn(II) between the biofilms and underlying metal-oxide substrates was probed following exposure of these complex interfaces to equi-molar Pb and Zn solutions (0.01 M NaNO3 as background electrolyte, pH = 6.0, and 3-h equilibration time). At higher Pb and Zn concentrations (≥10–5 M), more than 99% of these ions partitioned into the biofilms at S. oneidensis/α-Al2O3 (1 –1 0 2)/water interfaces, which is consistent with the partitioning behavior of both Pb(II) or Zn(II) in single-metal-ion experiments. Furthermore, no apparent competitive effects were found in this system at these relatively high metal-ion concentrations. However, at lower equi-molar concentrations (≤10–6 M), Pb(II) and Zn(II) partitioning in the same system changed significantly compared to the single-metal-ion systems. The presence of Zn(II) decreased Pb(II) partitioning onto α-Al2O3 (1 –1 0 2) substantially (~52% to ~13% at 10–7 M, and ~23% to ~5% at 10–6 M), whereas the presence of Pb(II) caused more Zn(II) to partition onto α-Al2O3 (1 –1 0 2) surfaces (~15% to ~28% at 10–7 M, and ~1% to ~7% at 10–6 M) .The higher observed partitioning of Zn(II) (~28%) at the α-Al2O3 (1 –1 0 2) surfaces compared to Pb(II) (~13%) in the mixed-metal-ion systems at the lowest concentration (10–7 M) suggests that Zn(II) is slightly favored over Pb(II) for sorption sites on α-Al2O3 (1 –1 0 2) surfaces under our experimental conditions.

  13. Comparative characterization and expression analysis of the four Old Yellow Enzyme homologues from Shewanella oneidensis indicate differences in physiological function

    PubMed Central

    Brigé, Ann; Van Den Hemel, Debbie; Carpentier, Wesley; De Smet, Lina; Van Beeumen, Jozef J.

    2005-01-01

    Shewanella oneidensis contains four genes that encode proteins that have high sequence identity with yeast OYE (Old Yellow Enzyme, an NADPH oxidoreductase), the well-studied archetype of the OYE protein family. The present paper describes the first comparative study of OYEs that are present in a single bacterial species, performed to gain insight into their biochemical properties and physiological importance. The four proteins [named SYE1–SYE4 (Shewanella Yellow Enzyme 1–4)] were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The yield of SYE2, however, was too low for further characterization, even after expression attempts in S. oneidensis. The SYE1, SYE3 and SYE4 proteins were found to have characteristics similar to those of other OYE family members. They were identified as flavoproteins that catalyse the reduction of different α,β-unsaturated carbonyl compounds and form charge transfer complexes with a range of phenolic compounds. Whereas the properties of SYE1 and SYE3 were very similar, those of SYE4 were clearly different in terms of ligand binding, catalytic efficiency and substrate specificity. Also, the activity of SYE4 was found to be NADPH-dependent, whereas SYE1 and SYE3 had a preference for NADH. It has been suggested that yeast OYE protects the actin cytoskeleton from oxidative stress. There are indications that bacterial OYEs are also involved in the oxidative stress response, but their exact role is unclear. Induction studies in S. oneidensis revealed that yeast and bacterial OYEs may share a common physiological role, i.e. the protection of cellular components against oxidative damage. As only SYE4 was induced under oxidative stress conditions, however, a functional divergence between bacterial OYEs is likely to exist. PMID:16293111

  14. The Effect of the Presence and Density of Shewanella oneidensis on Nuclear Magnetic Relaxation Measurements

    NASA Astrophysics Data System (ADS)

    Keating, K.; Halsey, J.

    2011-12-01

    A recent interest in the use of non-invasive geophysical methods to detect the presence of and measure the growth of microbes in the subsurface has arisen due to the potential use of such methods to monitor the progress of bioremediation. Previous research to this end has focused on electrical measurements, such as complex resistivity, which are sensitive to the presence of microbes but can be difficult to interpret. Nuclear magnetic resonance (NMR), an emerging near-surface geophysical method, is sensitive to the presence and physiochemical environment of hydrogen. Typically, NMR measurements in geophysics are used to detect hydrogen in water or hydrocarbons and to determine its pore environment; however, NMR imaging measurements have shown that NMR can also detect hydrogen in microbes. Geophysical NMR measurements thus have the potential to directly detect microbes in geologic material or indirectly detect the way in which the presence of microbes alters the physical and chemical properties of a water-saturated geologic material. This laboratory-scale study was designed to explore the effect of the presence and density of microbes on NMR relaxation measurements. Measurements were collected on microbial slurries and microbes in porous media both during microbial growth and on samples with known microbial density. Shewanella oneidensis was used as a representative environmental microbe in this study. The research shows that low field NMR measurements are sensitive to the presence and density of microbes and provides fundamental information required to determine if low-field NMR measurements can be used to monitor microbial growth during bioremediation.

  15. The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation†

    PubMed Central

    Pulcu, Gökçe Su; Frato, Katherine E.; Gupta, Rupal; Hsu, Hao-Ru; Levine, George A.; Hendrich, Michael P.; Elliott, Sean J.

    2012-01-01

    We report the characterization of the diheme cytochrome c peroxidase (CcP) from Shewanella oneidensis (So) using UV/Visible absorbance, Electron Paramagnetic Resonance Spectroscopy, and Michaelis-Menten kinetics. While sequence alignment with other bacterial diheme cytochrome c peroxidases suggests that So CcP may be active in the as-isolated state, we find that So CcP requires reductive activation for full activity, similar to the canonical Pseudomonas-type of bacterial CcP enzyme. Peroxide turnover initiated with oxidized So CcP shows a distinct lag-phase, which we interpret as reductive activation in situ. A simple kinetic model is sufficient to recapitulate the lag-phase behavior of the progress curves and separate the contributions of reductive activation and peroxide turnover. The rates of catalysis and activation differ between MBP-fusion and tag-free So CcP, and also depend on the identity of the electron donor. Combined with Michaelis-Menten analysis these data suggest that So CcP can accommodate electron donor binding in several possible orientations, and that the presence of the MBP tag affects the availability of certain binding sites. To further investigate the structural basis of reductive activation in So CcP we introduced mutations into two different regions of the protein that have been suggested to be important for reductive activation in homologous bacterial CcPs. Mutations in a flexible loop region neighboring the low-potential heme significantly increased the activation rate, confirming the importance of flexible loop regions of the protein in converting the inactive, as-isolated enzyme into the activated form. PMID:22239664

  16. Pressure effects on the chimeric 3-isopropylmalate dehydrogenases of the deep-sea piezophilic Shewanella benthica and the atmospheric pressure-adapted Shewanella oneidensis.

    PubMed

    Hamajima, Yuki; Nagae, Takayuki; Watanabe, Nobuhisa; Kato-Yamada, Yasuyuki; Imai, Takeo; Kato, Chiaki

    2014-01-01

    The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme.

  17. Conduction-band edge dependence of carbon-coated hematite stimulated extracellular electron transfer of Shewanella oneidensis in bioelectrochemical systems.

    PubMed

    Zhou, Shungui; Tang, Jiahuan; Yuan, Yong

    2015-04-01

    Bacteria-based bioelectrochemical systems (BESs) are promising technologies used for alternative energy generation, wastewater treatment, and environmental monitoring. However, their practical application is limited by the bioelectrode performance, mainly resulting from low extracellular electron transfer (EET) efficiency. In this study, a carbon-coated hematite (C/Hematite) electrode was successfully obtained by a green and solvent-free route, that is, heat treatment in an oxygen-rich environment using solid ferrocene as the precursor. The as-prepared C/Hematite electrode was evaluated as a high-performance electrode material in a Shewanella oneidensis-inoculated BES. The maximum biocurrent density of the Shewanella-attached C/Hematite electrode reached 0.22 ± 0.01 mA cm(-2), which is nearly 6-times higher than that of a bare carbon cloth (CC) electrode (0.036 ± 0.005 mA cm(-2)). Electrochemical measurements revealed that the enhanced conductivity and better energy matching between the outer membrane c-type cytochromes of S. oneidensis and the electrode contributed to the improved EET efficiency. The results of this study demonstrated that the semiconductive properties of iron oxides play important roles for the involved bacterial extracellular respiration activities.

  18. Polyphasic Taxonomy of the GenusShewanellaand Description ofShewanellaoneidensis sp. nov.

    SciTech Connect

    Venkateswaran, K.

    1999-01-01

    The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA--DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.

  19. Shewanella oneidensis FabB: A β-ketoacyl-ACP Synthase That Works with C16:1-ACP.

    PubMed

    Luo, Qixia; Li, Meng; Fu, Huihui; Meng, Qiu; Gao, Haichun

    2016-01-01

    It is established that Escherichia coli β-ketoacyl-ACP synthase (KAS) I (encoded by EcfabB) is the primary, if not exclusive, factor for elongation of the cis-3-decenoyl-ACP (C10:1-ACP) but not effective with C16:1- or longer-chain-ACPs. To test the extent to which these features apply to KAS I proteins in other species, in this study, we examined the physiological role of FabB in Shewanella oneidensis, an excellent model for researching type II fatty acid synthetic (FAS) system and its regulation. We showed that the loss of either FabA (the enzyme that introduces double bond) or FabB, in the absence of DesA which desaturizes C16 and C18 to generate respective C16:1 and C18:1, leads to a UFA auxotroph. However, fatty acid profiles of membrane phospholipid of the fabA and fabB mutants are significantly different, suggesting that FabB participates in steps beyond elongation of C10:1-ACP. Further analyses demonstrated that S. oneidensis FabB differs from EcFabB in that (i) it is not the only enzyme capable of catalyzing elongation of the cis-3-decenoyl-ACP produced by FabA, (ii) it plays a critical role in elongation of C16:1- and longer-chain-ACPs, and (iii) its overproduction is detrimental.

  20. Mutation Analysis of MR-1, SLC2A1, and CLCN1 in 28 PRRT2-negative Paroxysmal Kinesigenic Dyskinesia Patients

    PubMed Central

    Wang, Hong-Xia; Li, Hong-Fu; Liu, Gong-Lu; Wen, Xiao-Dan; Wu, Zhi-Ying

    2016-01-01

    Background: Paroxysmal kinesigenic dyskinesia (PKD) is the most common subtype of paroxysmal dyskinesias and is caused by mutations in PRRT2 gene. The majority of familial PKD was identified to harbor PRRT2 mutations. However, over two-third of sporadic PKD patients did not carry any PRRT2 mutation, suggesting an existence of additional genetic mutations or possible misdiagnosis due to clinical overlap. Methods: A cohort of 28 Chinese patients clinically diagnosed with sporadic PKD and excluded PRRT2 mutations were recruited. Clinical features were evaluated, and all subjects were screened for MR-1, SLC2A1, and CLCN1 genes, which are the causative genes of paroxysmal nonkinesigenic dyskinesia (PNKD), paroxysmal exertion-induced dyskinesia, and myotonia congenita (MC), respectively. In addition, 200 genetically matched healthy individuals were recruited as controls. Results: A total of 16 genetic variants including 4 in MR-1 gene, 8 in SLC2A1 gene, and 4 in CLCN1 gene were detected. Among them, SLC2A1 c.363G>A mutation was detected in one case, and CLCN1 c.1205C>T mutation was detected in other two cases. Neither of them was found in 200 controls as well as 1000 Genomes database and ExAC database. Both mutations were predicted to be pathogenic by SIFT and PolyPhen2. The SLC2A1 c.363G>A mutation was novel. Conclusions: The phenotypic overlap may lead to the difficulty in distinguishing PKD from PNKD and MC. For those PRRT2- negative PKD cases, screening of SLC2A1 and CLCN1 genes are useful in confirming the diagnosis. PMID:27098784

  1. The role of multihaem cytochromes in the respiration of nitrite in Escherichia coli and Fe(III) in Shewanella oneidensis.

    PubMed

    Clarke, Thomas A; Holley, Tracey; Hartshorne, Robert S; Fredrickson, Jim K; Zachara, John M; Shi, Liang; Richardson, David J

    2008-10-01

    The periplasmic nitrite reductase system from Escherichia coli and the extracellular Fe(III) reductase system from Shewanella oneidensis contain multihaem c-type cytochromes as electron carriers and terminal reductases. The position and orientation of the haem cofactors in multihaem cytochromes from different bacteria often show significant conservation despite different arrangements of the polypeptide chain. We propose that the decahaem cytochromes of the iron reductase system MtrA, MtrC and OmcA comprise pentahaem 'modules' similar to the electron donor protein, NrfB, from E. coli. To demonstrate this, we have isolated and characterized the N-terminal pentahaem module of MtrA by preparing a truncated form containing five covalently attached haems. UV-visible spectroscopy indicated that all five haems were low-spin, consistent with the presence of bis-His ligand co-ordination as found in full-length MtrA.

  2. The role of multihaem cytochromes in the respiration of nitrite in Escherichia coli and Fe(III) in Shewanella oneidensis

    SciTech Connect

    Clarke, Thomas A.; Holley, Tracey; Hartshorne, Robert S.; Fredrickson, Jim K.; Zachara, John M.; Shi, Liang; Richardson, David

    2008-10-01

    The periplasmic nitrite reductase system from Escherichia coli and the extracellular Fe(III) reductase system from Shewanella oneidensis contain multihaem c-type cytochromes as electron carriers and terminal reductases. The position and orientation of the haem cofactors in multihaem cytochromes from different bacteria often show significant conservation despite different arrangements of the polypeptide chain. We propose that the decahaem cytochromes of the iron reductase system MtrA, MtrC and OmcA comprise pentahaem ‘modules’ similar to the electron donor protein, NrfB, from E. coli. To demonstrate this, we have isolated and characterized the N-terminal pentahaem module of MtrA by preparing a truncated form containing five covalently attached haems. UV–visible spectroscopy indicated that all five haems were low-spin, consistent with the presence of bis-His ligand co-ordination as found in full-length MtrA.

  3. Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors.

    SciTech Connect

    Beliaev, A. S.; Thompson, D. K.; Khare, T.; Lim, H.; Brandt, C. C.; Li, G.; Murray, A. E.; Heidelberg, J. F.; Giometti, C. S.; Yates, J., III; Nealson, K. H.; Tiedje, J. M.; Zhou, J.; Biosciences Division; ORNL; Scripps Research Inst.; Michigan State Univ.; The Inst. for Genomic Research; Jet Propulsion Laboratory; California Inst. of Tech.

    2002-01-01

    Changes in mRNA and protein expression profiles of Shewanella oneidenesis MR-1 during switch from aerobic to fumarate-, Fe(III)-, or nitrate-reducing conditions were examined using DNA microarrays and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In response to changes in growth conditions, 121 of the 691 arrayed genes displayed at least a two-fold difference in transcript abundance as determined by microarray analysis. Genes involved in aerobic respiration encoding cytochrome c and d oxidases and TCA cycle enzymes were repressed under anaerobic conditions. Genes induced during anaerobic respiration included those involved in cofactor biosynthesis and assembly (moaACE, ccmHF, nosD, cysG), substrate transport (cysUP, cysTWA, dcuB), and anaerobic energy metabolism (dmsAB, psrC, pshA, hyaABC, hydA). Transcription of genes encoding a periplasmic nitrate reductase (napBHGA), cytochrome c{sub 552}, and prismane was elevated 8- to 56-fold in response to the presence of nitrate, while cymA, ifcA, and frdA were specifically induced three- to eightfold under fumarate-reducing conditions. The mRNA levels for two oxidoreductase-like genes of unknown function and several cell envelope genes involved in multidrug resistance increased two- to fivefold specifically under Fe(III)-reducing conditions. Analysis of protein expression profiles under aerobic and anaerobic conditions revealed 14 protein spots that showed significant differences in abundance on 2-D gels. Protein identification by mass spectrometry indicated that the expression of prismane, dihydrolipoamide succinyltransferase, and alcaligin siderophore biosynthesis protein correlated with the microarray data.

  4. Monodispersed biocompatible Ag2S nanoparticles: Facile extracellular bio-fabrication using the gamma-proteobacterium, S. oneidensis

    SciTech Connect

    Suresh, Anil K; Doktycz, Mitchel John; Wang, Wei; Moon, Ji Won; Gu, Baohua; Meyer III, Harry M; Hensley, Dale K; Retterer, Scott T; Allison, David P; Phelps, Tommy Joe; Pelletier, Dale A

    2011-01-01

    Interest in engineered metal and semiconductor nanocrystallites continues to grow due to their unique size and or shape dependent optoelectronic, physicochemical and biological properties. Therefore identifying novel non-hazardous nanoparticle synthesis routes that address hydrophilicity, size and shape control and production costs have become a priority. In the present illustration we report for the first time the efficient generation of extracellular Ag2S nanoparticles by the metal reducing bacterium, Shewanella oneidensis. The particles are nearly monodispersed with homogeneous shape distributions and are produced under ambient temperatures and pressures at high yield, 85 % theoretical maximum. UV-vis and Fourier transform infrared spectroscopy, dynamic light scattering, X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy measurements confirmed the formation, optical properties, purity, and crystallinity of the as-synthesized particles. Further characterization revealed that the particles consist of spheres in the size range of 1-22 nm, with an average size of 9 3 nm and are capped by a detachable protein/peptide surface coat. Toxicity assessments of these silver sulfide nanoparticles on Gram-negative Escherichia coli and Shewanella oneidensis and Gram-positive Bacillus subtilis bacterial systems as well as eukaryotic; mouse lung epithelial (C 10) and macrophage (RAW-264.7) cells showed that the particles were non-inhibitory or non-cytotoxic to both these systems. Our results provide a facile, eco-friendly and economical route for the fabrication of technologically important semiconducting Ag2S nanoparticles which are dispersible and biocompatible; thus providing excellent potential for their uses in optical imaging and electronic devices, and solar cell applications.

  5. Metabolite-enabled mutualistic interaction between Shewanella oneidensis and Escherichia coli in a co-culture using an electrode as electron acceptor

    PubMed Central

    Wang, Victor Bochuan; Sivakumar, Krishnakumar; Yang, Liang; Zhang, Qichun; Kjelleberg, Staffan; Loo, Say Chye Joachim; Cao, Bin

    2015-01-01

    Mutualistic interactions in planktonic microbial communities have been extensively studied. However, our understanding on mutualistic communities consisting of co-existing planktonic cells and biofilms is limited. Here, we report a planktonic cells-biofilm mutualistic system established by the fermentative bacterium Escherichia coli and the dissimilatory metal-reducing bacterium Shewanella oneidensis in a bioelectrochemical device, where planktonic cells in the anode media interact with the biofilms on the electrode. Our results show that the transfer of formate is the key mechanism in this mutualistic system. More importantly, we demonstrate that the relative distribution of E. coli and S. oneidensis in the liquid media and biofilm is likely driven by their metabolic functions towards an optimum communal metabolism in the bioelectrochemical device. RNA sequencing-based transcriptomic analyses of the interacting organisms in the mutualistic system potentially reveal differential expression of genes involved in extracellular electron transfer pathways in both species in the planktonic cultures and biofilms. PMID:26061569

  6. Physiological roles of ArcA, Crp, and EtrA and their interactive control on aerobic and anaerobic respiration in Shewanella oneidensis.

    PubMed

    Gao, Haichun; Wang, Xiaohu; Yang, Zamin K; Chen, Jingrong; Liang, Yili; Chen, Haijiang; Palzkill, Timothy; Zhou, Jizhong

    2010-12-28

    In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA have been identified. All these proteins deviate from their counterparts in E. coli significantly in terms of functionality and regulon. It is worth investigating the involvement and relationship of these global regulators in aerobic and anaerobic respiration in S. oneidensis. In this study, the impact of the transcriptional factors ArcA, Crp, and EtrA on aerobic and anaerobic respiration in S. oneidensis were assessed. While all these proteins appeared to be functional in vivo, the importance of individual proteins in these two major biological processes differed. The ArcA transcriptional factor was critical in aerobic respiration while the Crp protein was indispensible in anaerobic respiration. Using a newly developed reporter system, it was found that expression of arcA and etrA was not influenced by growth conditions but transcription of crp was induced by removal of oxygen. An analysis of the impact of each protein on transcription of the others revealed that Crp expression was independent of the other factors whereas ArcA repressed both etrA and its own transcription while EtrA also repressed arcA transcription. Transcriptional levels of arcA in the wild type, crp, and etrA strains under either aerobic or anaerobic conditions were further validated by quantitative immunoblotting with a polyclonal antibody against ArcA. This extensive survey demonstrated that all these three global regulators are functional in S. oneidensis. In addition, the reporter system constructed in this study will facilitate in vivo transcriptional analysis of targeted promoters.

  7. Physiological roles of ArcA, Crp, and EtrA and their interactive control on aerobic and anaerobic respiration in Shewanella oneidensis

    SciTech Connect

    Gao, Haichun; Wang, Xiaohu; Chen, Jingrong; Liang, Yili; Chen, Haijiang; Palzkill, Timothy; Zhou, Jizhong

    2010-01-01

    In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA have been identified. All these proteins deviate from their counterparts in E. coli significantly in terms of functionality and regulon. It is worth investigating the involvement and relationship of these global regulators in aerobic and anaerobic respiration in S. oneidensis. In this study, the impact of the transcriptional factors ArcA, Crp, and EtrA on aerobic and anaerobic respiration in S. oneidensis were assessed. While all these proteins appeared to be functional in vivo, the importance of individual proteins in these two major biological processes differed. The ArcA transcriptional factor was critical in aerobic respiration while the Crp protein was indispensible in anaerobic respiration. Using a newly developed reporter system, it was found that expression of arcA and etrA was not influenced by growth conditions but transcription of crp was induced by removal of oxygen. An analysis of the impact of each protein on transcription of the others revealed that Crp expression was independent of the other factors whereas ArcA repressed both etrA and its own transcription while EtrA also repressed arcA transcription. Transcriptional levels of arcA in the wild type, crp, and etrA strains under either aerobic or anaerobic conditions were further validated by quantitative immunoblotting with a polyclonal antibody against ArcA. This extensive survey demonstrated that all these three global regulators are functional in S. oneidensis. In addition, the reporter system constructed in this study will facilitate in vivo transcriptional analysis of targeted promoters.

  8. Effect of oxygen on the per‐cell extracellular electron transfer rate of Shewanella oneidensis MR‐1 explored in bioelectrochemical systems

    PubMed Central

    Lu, Mengqian; Chan, Shirley; Babanova, Sofia

    2016-01-01

    ABSTRACT Extracellular electron transfer (EET) is a mechanism that enables microbes to respire solid‐phase electron acceptors. These EET reactions most often occur in the absence of oxygen, since oxygen can act as a competitive electron acceptor for many facultative microbes. However, for Shewanella oneidensis MR‐1, oxygen may increase biomass development, which could result in an overall increase in EET activity. Here, we studied the effect of oxygen on S. oneidensis MR‐1 EET rates using bioelectrochemical systems (BESs). We utilized optically accessible BESs to monitor real‐time biomass growth, and studied the per‐cell EET rate as a function of oxygen and riboflavin concentrations in BESs of different design and operational conditions. Our results show that oxygen exposure promotes biomass development on the electrode, but significantly impairs per‐cell EET rates even though current production does not always decrease with oxygen exposure. Additionally, our results indicated that oxygen can affect the role of riboflavin in EET. Under anaerobic conditions, both current density and per‐cell EET rate increase with the riboflavin concentration. However, as the dissolved oxygen (DO) value increased to 0.42 mg/L, riboflavin showed very limited enhancement on per‐cell EET rate and current generation. Since it is known that oxygen can promote flavins secretion in S. oneidensis, the role of riboflavin may change under anaerobic and aerobic conditions. Biotechnol. Bioeng. 2017;114: 96–105. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:27399911

  9. Transcription factors FabR and FadR regulate both aerobic and anaerobic pathways for unsaturated fatty acid biosynthesis in Shewanella oneidensis.

    PubMed

    Luo, Qixia; Shi, Miaomiao; Ren, Yedan; Gao, Haichun

    2014-01-01

    As genes for type II fatty acid synthesis are essential to the growth of Escherichia coli, its sole (anaerobic) pathway has significant potential as a target for novel antibacterial drug, and has been extensively studied. Despite this, we still know surprisingly little about fatty acid synthesis in bacteria because this anaerobic pathway in fact is not widely distributed. In this study, we show a novel model of unsaturated fatty acid (UFA) synthesis in Shewanella, emerging human pathogens in addition to well-known metal reducers. We identify both anaerobic and aerobic UFA biosynthesis pathways in the representative species, S. oneidensis. Uniquely, the bacterium also contains two regulators FabR and FadR, whose counterparts in other bacteria control the anaerobic pathway. However, we show that in S. oneidensis these two regulators are involved in regulation of both pathways, in either direct or indirect manner. Overall, our results indicate that the UFA biosynthesis and its regulation are far more complex than previously expected, and S. oneidensis serves as a good research model for further work.

  10. Two Residues Predominantly Dictate Functional Difference in Motility between Shewanella oneidensis Flagellins FlaA and FlaB*

    PubMed Central

    Sun, Linlin; Dong, Yangyang; Shi, Miaomiao; Jin, Miao; Zhou, Qing; Luo, Zhao-Qing; Gao, Haichun

    2014-01-01

    Nearly half of flagellated microorganisms possess a multiple-flagellin system. Although a functional filament can be formed from one of multiple flagellins alone in many bacteria, it is more common that one flagellin is the major constituent and others contribute. Underlying mechanisms proposed for such scenarios cover flagellin regulation of various levels, including transcription, translation, post-translational modification, secretion, and filament assembly. In Shewanella oneidensis, the flagellar filament is composed of FlaA and FlaB flagellins; the latter is the major one in terms of motility. In this study, we showed that regulation of all levels except for filament assembly is indistinguishable between these two flagellins. Further analyses revealed that two amino acid residues predominantly dictated functional difference with respect to motility. Given that Shewanella prefer a solid surface-associated life style, of which filaments consisting of either FlaA or FlaB are equally supportive, we envision that roles of flagella in surface adhesion and formation of bacterial communities are particularly important for their survival and proliferation in these specific niches. PMID:24733391

  11. Positive regulation of the Shewanella oneidensis OmpS38, a major porin facilitating anaerobic respiration, by Crp and Fur

    PubMed Central

    Gao, Tong; Ju, Lili; Yin, Jianhua; Gao, Haichun

    2015-01-01

    Major porins are among the most abundant proteins embedded in the outer membrane (OM) of Gram-negative bacteria, playing crucial roles in maintenance of membrane structural integrity and OM permeability. Although many OM proteins (especially c-type cytochromes) in Shewanella oneidensis, a research model for respiratory versatility, have been extensively studied, physiological significance of major porins remains largely unexplored. In this study, we show that OmpS38 and OmpA are two major porins, neither of which is responsive to changes in osmolarity or contributes to the intrinsic resistance to β-lactam antibiotics. However, OmpS38 but not OmpA is largely involved in respiration of non-oxygen electron acceptors. We then provide evidence that expression of ompS38 is transcribed from two promoters, the major of which is favored under anaerobic conditions while the other appears constitutive. The major promoter is under the direct control of Crp, the master regulator dictating respiration. As a result, the increase in the level of OmpS38 correlates with an elevated activity in Crp under anaerobic conditions. In addition, we show that the activity of the major promoter is also affected by Fur, presumably indirectly, the transcription factor for iron-dependent gene expression. PMID:26381456

  12. Impaired cell envelope resulting from arcA mutation largely accounts for enhanced sensitivity to hydrogen peroxide in Shewanella oneidensis

    PubMed Central

    Wan, Fen; Mao, Yinting; Dong, Yangyang; Ju, Lili; Wu, Genfu; Gao, Haichun

    2015-01-01

    Oxidative stress is one of the major challenges that Shewanella encounter routinely because they thrive in redox-stratified environments prone to reactive oxygen species (ROS) formation, letting alone that ROS can be generated endogenously. As respiration is the predominant process for endogenous ROS, regulators mediating respiration have been demonstrated and/or implicated to play a role in oxidative stress response. In our efforts to unveil the involvement of global regulators for respiration in the oxidative stress response, we found that loss of the Arc system increases S. oneidensis sensitivity to H2O2 whereas neither Fnr nor Crp has a significant role. A comparison of transcriptomic profiles of the wild-type and its isogenic arcA mutant revealed that the OxyR regulon is independent of the Arc system. We then provided evidence that the enhanced H2O2 sensitivity of the arcA mutant is due to an increased H2O2 uptake rate, a result of a cell envelope defect. Although one of three proteases of the ArcA regulon when in excess is partially accountable for the envelope defect, the major contributors remain elusive. Overall, our data indicate that the Arc system influences the bacterial cell envelope biosynthesis, a physiological aspect that has not been associated with the regulator before. PMID:25975178

  13. Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye‐Sensitized TiO2

    PubMed Central

    Ainsworth, Emma V.; Lockwood, Colin W. J.; White, Gaye F.; Hwang, Ee Taek; Sakai, Tsubasa; Gross, Manuela A.; Richardson, David J.; Clarke, Thomas A.

    2016-01-01

    Abstract The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar‐assisted chemical synthesis. In Shewanella oneidensis MR‐1 (MR‐1), trans‐outer‐membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer‐membrane‐spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR‐1. We show photoreduction of MtrC and OmcA adsorbed on RuII‐dye‐sensitized TiO2 nanoparticles and that these protein‐coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer‐membrane‐associated cytochromes of MR‐1 for solar‐driven microbial synthesis in natural and engineered bacteria. PMID:27685371

  14. NapB in excess inhibits growth of Shewanella oneidensis by dissipating electrons of the quinol pool

    PubMed Central

    Jin, Miao; Zhang, Qianyun; Sun, Yijuan; Gao, Haichun

    2016-01-01

    Shewanella, a group of ubiquitous bacteria renowned for respiratory versatility, thrive in environments where various electron acceptors (EAs) of different chemical and physiological characteristics coexist. Despite being extensively studied, we still know surprisingly little about strategies by which multiple EAs and their interaction define ecophysiology of these bacteria. Previously, we showed that nitrite inhibits growth of the genus representative Shewanella oneidensis on fumarate and presumably some other CymA (quinol dehydrogenase)-dependent EAs by reducing cAMP production, which in turn leads to lowered expression of nitrite and fumarate reductases. In this study, we demonstrated that inhibition of fumarate growth by nitrite is also attributable to overproduction of NapB, the cytochrome c subunit of nitrate reductase. Further investigations revealed that excessive NapB per se inhibits growth on all EAs tested, including oxygen. When overproduced, NapB acts as an electron shuttle to dissipate electrons of the quinol pool, likely to extracellullar EAs, because the Mtr system, the major electron transport pathway for extracellular electron transport, is implicated. The study not only sheds light on mechanisms by which certain EAs, especially toxic ones, impact the bacterial ecophysiology, but also provides new insights into how electron shuttle c-type cytochromes regulate multi-branched respiratory networks. PMID:27857202

  15. Positive regulation of the Shewanella oneidensis OmpS38, a major porin facilitating anaerobic respiration, by Crp and Fur.

    PubMed

    Gao, Tong; Ju, Lili; Yin, Jianhua; Gao, Haichun

    2015-09-18

    Major porins are among the most abundant proteins embedded in the outer membrane (OM) of Gram-negative bacteria, playing crucial roles in maintenance of membrane structural integrity and OM permeability. Although many OM proteins (especially c-type cytochromes) in Shewanella oneidensis, a research model for respiratory versatility, have been extensively studied, physiological significance of major porins remains largely unexplored. In this study, we show that OmpS38 and OmpA are two major porins, neither of which is responsive to changes in osmolarity or contributes to the intrinsic resistance to β-lactam antibiotics. However, OmpS38 but not OmpA is largely involved in respiration of non-oxygen electron acceptors. We then provide evidence that expression of ompS38 is transcribed from two promoters, the major of which is favored under anaerobic conditions while the other appears constitutive. The major promoter is under the direct control of Crp, the master regulator dictating respiration. As a result, the increase in the level of OmpS38 correlates with an elevated activity in Crp under anaerobic conditions. In addition, we show that the activity of the major promoter is also affected by Fur, presumably indirectly, the transcription factor for iron-dependent gene expression.

  16. Impact of ArcA loss in Shewanella oneidensis revealed by comparative proteomics under aerobic and anaerobic conditions

    SciTech Connect

    Yuan, Jie; Wei, Buyun; Lipton, Mary S.; Gao, Haichun

    2012-06-01

    Shewanella inhabit a wide variety of niches in nature and can utilize a broad spectrum of electron acceptors under anaerobic conditions. How they modulate their gene expression to adapt is poorly understood. ArcA, homologue of a global regulator controlling hundreds of genes involved in aerobic and anaerobic respiration in E. coli, was shown to be important in aerobiosis/anaerobiosis of S. oneidensis as well. Loss of ArcA, in addition to altering transcription of many genes, resulted in impaired growth under aerobic condition, which was not observed in E. coli. To further characterize the impact of ArcA loss on gene expression on the level of proteome under aerobic and anaerobic conditions, liquid-chromatography-mass-spectrometry (LC-MS) based proteomic approach was employed. Results show that ArcA loss led to globally altered gene expression, generally consistent with that observed with transcripts. Comparison of transcriptomic and proteomic data permitted identification of 17 high-confidence ArcA targets. Moreover, our data indicate that ArcA is required for regulation of cytochrome c proteins, and the menaquinone level may play a role in regulating ArcA as in E. coli. Proteomic-data-guided growth assay revealed that the aerobic growth defect of ArcA mutant is presumably due to impaired peptide utilization.

  17. Reduced expression of cytochrome oxidases largely explains cAMP inhibition of aerobic growth in Shewanella oneidensis

    PubMed Central

    Yin, Jianhua; Meng, Qiu; Fu, Huihui; Gao, Haichun

    2016-01-01

    Inhibition of bacterial growth under aerobic conditions by elevated levels of cyclic adenosine 3′,5′-monophosphate (cAMP), first revealed more than 50 years ago, was attributed to accumulation of toxic methylglyoxal (MG). Here, we report a Crp-dependent mechanism rather than MG accumulation that accounts for the phenotype in Shewanella oneidensis, an emerging research model for the bacterial physiology. We show that a similar phenotype can be obtained by removing CpdA, a cAMP phosphodiesterase that appears more effective than its Escherichia coli counterpart. Although production of heme c and cytochromes c is correlated well with cAMP levels, neither is sufficient for the retarded growth. Quantities of overall cytochromes c increased substantially in the presence of elevated cAMP, a phenomenon resembling cells respiring on non-oxygen electron acceptors. In contrast, transcription of Crp-dependent genes encoding both cytochromes bd and cbb3 oxidases is substantially repressed under the same condition. Overall, our results suggest that cAMP of elevated levels drives cells into a low-energetic status, under which aerobic respiration is inhibited. PMID:27076065

  18. Cloning and sequence of cymA, a gene encoding a tetraheme cytochrome c required for reduction of iron(III), fumarate, and nitrate by Shewanella putrefaciens MR-1.

    PubMed Central

    Myers, C R; Myers, J M

    1997-01-01

    The cymA gene, which encodes a tetraheme cytochrome c, was cloned from Shewanella putrefaciens MR-1. This gene complemented a mutant which had a TnphoA insertion in cymA and which was deficient in the respiratory reduction of iron(III), nitrate, fumarate, and manganese(IV). The 561-bp nucleotide sequence of cymA encodes a protein of 187 amino acids with a predicted molecular mass of 20.8 kDa. No N-terminal signal sequence was readily apparent; consistent with this, a cytochrome with a size of 21 kDa was detected in the wild type but was absent in the insertional mutant. The cymA gene is transcribed into an mRNA; the major transcript was approximately 790 bases, suggesting that it is not part of a multicistronic operon. This RNA transcript was not detected in the cymA mutant. The CymA protein was found in the cytoplasmic membrane and soluble fraction of MR-1, and it shares partial amino acid sequence homology with multiheme c-type cytochromes from other bacteria. These cytochromes are ostensibly involved in the transfer of electrons from the cytoplasmic membrane to acceptors in the periplasm. The localization of the fumarate and iron(III) reductases to the periplasm and outer membrane of MR-1, respectively, suggests the possibility of a similar electron transfer role for CymA. PMID:9023196

  19. The X-ray crystal structure of Shewanella oneidensis OmcA reveals new insight at the microbe-mineral interface

    SciTech Connect

    Edwards, Marcus; Baiden, Nanakow; Johs, Alexander; Tomanicek, Stephen J.; Liang, Liyuan; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Gates, Andrew J.; Butt, Julea N.; Richardson, David; Clarke, Thomas A.

    2014-05-21

    The x-ray crystal structure of Shewanella oneidensis OmcA, an extracellular decaheme cytochrome involved in mineral reduction, was solved to a resolution of 2.7 Å. The four OmcA molecules in the asymmetric unit were arranged so the distance between heme-5 on adjacent OmcA monomers was less than 1 nm, indicative of a transient OmcA dimer capable of intermolecular electron transfer. A previously identified hematite binding motif was identified near heme 10, forming a hydroxylated surface that would bring a heme-10 electron egress site to ~ 1 nm of mineral surface.

  20. Reduction of Nitrate in Shewanella oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA

    SciTech Connect

    Gao, Haichun; Yang, Zamin; Barua, Soumitra; Reed, Samantha B.; Romine, Margaret F.; Nealson, Kenneth H.; Fredrickson, Jim K.; Tiedje, James M.; Zhou, Jizhong

    2009-04-23

    In the genome of Shewanella oneidensis, a napDAGHB gene cluster encoding periplasmic nitrate reductase (NapA) and accessory proteins and an nrfA gene encoding periplasmic nitrite reductase (NrfA) have been identified. These two systems seem to be atypical because the genome lacks genes encoding cytoplasmic membrane electron transport proteins, NapC for NAP and NrfBCD/NrfH for NRF, respectively. Here, we present evidence that reduction of nitrate to ammonium in S. oneidensis is carried out by these atypical systems in a two-step manner. Transcriptional and mutational analyses suggest that CymA, a cytoplasmic membrane electron transport protein, is likely to be the functional replacement of both NapC and NrfH in S. oneidensis. Surprisingly, a strain devoid of napB encoding the small subunit of nitrate reductase exhibited the maximum cell density sooner than the wild type. Further characterization of this strain showed that nitrite was not detected as a free intermediate in its culture and NapB provides a fitness gain for S. oneidensis to compete for nitrate in the environments. On the basis results from mutational analyses of napA, napB, nrfA and napBnrfA in-frame deletion mutants, we propose that NapB is able to favor nitrate reduction by routing electrons to NapA exclusively.

  1. Reduction of nitrate in Shewanella oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA.

    PubMed

    Gao, Haichun; Yang, Zamin K; Barua, Soumitra; Reed, Samantha B; Romine, Margaret F; Nealson, Kenneth H; Fredrickson, James K; Tiedje, James M; Zhou, Jizhong

    2009-08-01

    In the genome of Shewanella oneidensis, a napDAGHB gene cluster encoding periplasmic nitrate reductase (NapA) and accessory proteins and an nrfA gene encoding periplasmic nitrite reductase (NrfA) have been identified. These two systems seem to be atypical because the genome lacks genes encoding cytoplasmic membrane electron transport proteins, NapC for NAP and NrfBCD/NrfH for NRF, respectively. Here, we present evidence that reduction of nitrate to ammonium in S. oneidensis is carried out by these atypical systems in a two-step manner. Transcriptional and mutational analyses suggest that CymA, a cytoplasmic membrane electron transport protein, is likely to be the functional replacement of both NapC and NrfH in S. oneidensis. Surprisingly, a strain devoid of napB encoding the small subunit of nitrate reductase exhibited the maximum cell density sooner than the wild type. Further characterization of this strain showed that nitrite was not detected as a free intermediate in its culture and NapB provides a fitness gain for S. oneidensis to compete for nitrate in the environments. On the basis results from mutational analyses of napA, napB, nrfA and napBnrfA in-frame deletion mutants, we propose that NapB is able to favor nitrate reduction by routing electrons to NapA exclusively.

  2. Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis.

    PubMed

    Li, Meng; Meng, Qiu; Fu, Huihui; Luo, Qixia; Gao, Haichun

    2016-11-15

    As type II fatty acid synthesis is essential for the growth of Escherichia coli, its many components are regarded as potential targets for novel antibacterial drugs. Among them, β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) FabB is the exclusive factor for elongation of the cis-3-decenoyl-ACP (cis-3-C10-ACP). In our previous study, we presented evidence to suggest that this may not be the case in Shewanella oneidensis, an emerging model gammaproteobacterium renowned for its respiratory versatility. Here, we identified FabF1, another KAS, as a functional replacement for FabB in S. oneidensis In fabB(+) or desA(+) (encoding a desaturase) cells, which are capable of making unsaturated fatty acids (UFA), FabF1 is barely produced. However, UFA auxotroph mutants devoid of both fabB and desA genes can be spontaneously converted to suppressor strains, which no longer require exogenous UFAs for growth. Suppression is caused by a TGTTTT deletion in the region upstream of the fabF1 gene, resulting in enhanced FabF1 production. We further demonstrated that the deletion leads to transcription read-through of the terminator for acpP, an acyl carrier protein gene immediately upstream of fabF1 There are multiple tandem repeats in the region covering the terminator, and the TGTTTT deletion, as well as others, compromises the terminator efficacy. In addition, FabF2 also shows an ability to complement the FabB loss, albeit substantially less effectively than FabF1.

  3. High-and low-affinity binding sites for Cd on the bacterial cell walls of Bacillus subtilis and Shewanella oneidensis.

    SciTech Connect

    Mishra, B.; Boyanov, M.; Bunker, B. A.; Kelly, S. D.; Kemner, K. M.; Fein, J. B.; Biosciences Division; Univ. of Notre Dame

    2010-08-01

    Bulk Cd adsorption isotherm experiments, thermodynamic equilibrium modeling, and Cd K edge EXAFS were used to constrain the mechanisms of proton and Cd adsorption to bacterial cells of the commonly occurring Gram-positive and Gram-negative bacteria, Bacillus subtilis and Shewanella oneidensis, respectively. Potentiometric titrations were used to characterize the functional group reactivity of the S. oneidensis cells, and we model the titration data using the same type of non-electrostatic surface complexation approach as was applied to titrations of B. subtilis suspensions by Fein et al. (2005). Similar to the results for B. subtilis, the S. oneidensis cells exhibit buffering behavior from approximately pH 3-9 that requires the presence of four distinct sites, with pK{sub a} values of 3.3 {+-} 0.2, 4.8 {+-} 0.2, 6.7 {+-} 0.4, and 9.4 {+-} 0.5, and site concentrations of 8.9({+-}2.6) x 10{sup -5}, 1.3({+-}0.2) x 10{sup -4}, 5.9({+-}3.3) x 10{sup -5}, and 1.1({+-}0.6) x 10{sup -4} moles/g bacteria (wet mass), respectively. The bulk Cd isotherm adsorption data for both species, conducted at pH 5.9 as a function of Cd concentration at a fixed biomass concentration, were best modeled by reactions with a Cd:site stoichiometry of 1:1. EXAFS data were collected for both bacterial species as a function of Cd concentration at pH 5.9 and 10 g/L bacteria. The EXAFS results show that the same types of binding sites are responsible for Cd sorption to both bacterial species at all Cd loadings tested (1-200 ppm). Carboxyl sites are responsible for the binding at intermediate Cd loadings. Phosphoryl ligands are more important than carboxyl ligands for Cd binding at high Cd loadings. For the lowest Cd loadings studied here, a sulfhydryl site was found to dominate the bound Cd budgets for both species, in addition to the carboxyl and phosphoryl sites that dominate the higher loadings. The EXAFS results suggest that both Gram-positive and Gram-negative bacterial cell walls have a low

  4. The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate: insights into its biological function.

    PubMed

    Garvey, Graeme S; Rocco, Christopher J; Escalante-Semerena, Jorge C; Rayment, Ivan

    2007-07-01

    In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 A resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle.

  5. Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

    PubMed

    Stein, Natalia; Love, Daniel; Judd, Evan T; Elliott, Sean J; Bennett, Brian; Pacheco, A Andrew

    2015-06-23

    The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

  6. Integrated genome based studies of Shewanella ecophysiology

    SciTech Connect

    Saffarini, Daad A

    2013-03-07

    Progress is reported in these areas: Regulation of anaerobic respiration by cAMP receptor protein and role of adenylate cyclases; Identification of an octaheme c cytochrome as the terminal sulfite reductase in S. oneidensis MR-1; Identification and analysis of components of the electron transport chains that lead to reduction of thiosulfate, tetrathionate, and elemental sulfur in MR-1; Involvement of pili and flagella in metal reduction by S. oneidensis MR-1; and work suggesting that HemN1 is the major enzyme that is involved in heme biosynthesis under anaerobic conditions.

  7. Functional Analysis of Shewanella, a cross genome comparison.

    SciTech Connect

    Serres, Margrethe H.

    2009-05-15

    The bacterial genus Shewanella includes a group of highly versatile organisms that have successfully adapted to life in many environments ranging from aquatic (fresh and marine) to sedimentary (lake and marine sediments, subsurface sediments, sea vent). A unique respiratory capability of the Shewanellas, initially observed for Shewanella oneidensis MR-1, is the ability to use metals and metalloids, including radioactive compounds, as electron acceptors. Members of the Shewanella genus have also been shown to degrade environmental pollutants i.e. halogenated compounds, making this group highly applicable for the DOE mission. S. oneidensis MR-1 has in addition been found to utilize a diverse set of nutrients and to have a large set of genes dedicated to regulation and to sensing of the environment. The sequencing of the S. oneidensis MR-1 genome facilitated experimental and bioinformatics analyses by a group of collaborating researchers, the Shewanella Federation. Through the joint effort and with support from Department of Energy S. oneidensis MR-1 has become a model organism of study. Our work has been a functional analysis of S. oneidensis MR-1, both by itself and as part of a comparative study. We have improved the annotation of gene products, assigned metabolic functions, and analyzed protein families present in S. oneidensis MR-1. The data has been applied to analysis of experimental data (i.e. gene expression, proteome) generated for S. oneidensis MR-1. Further, this work has formed the basis for a comparative study of over 20 members of the Shewanella genus. The species and strains selected for genome sequencing represented an evolutionary gradient of DNA relatedness, ranging from close to intermediate, and to distant. The organisms selected have also adapted to a variety of ecological niches. Through our work we have been able to detect and interpret genome similarities and differences between members of the genus. We have in this way contributed to the

  8. Mechanism(s) of Electricity Production by Shewanella and Other Microbes: Understanding and Optimization

    DTIC Science & Technology

    2012-01-01

    Shewanella and other microbes: Understanding and Optimization FA9550-06-1-0292 61102 PI: Kenneth H. Nealson, Dept. of Earth Science, USC CoIs: Moh... Shewanella oneidensis (MR-1); 4) construction of conceptual models of extracellular electron transport (EET); 5) characterization and quantification of the...by MR-1 and other Shewanella strains and species; 9) characterization of the selective advantages of Shewanella cells in late stationary phase, and

  9. Structure of 2C-Methyl-D-erythritol-2,4-cyclodiphosphate Synthase from Shewanella oneidensis at 1.6 angstrom: Identification of Farnesyl pyrophosphate Trapped in a Hydrophobic Cavity

    SciTech Connect

    Ni, Shuisong; Robinson, Howard; Marsing, Gregory C.; Bussiere, Dirksen E.; Kennedy, Michael A.

    2004-11-01

    definitive analysis of the identity and mode of binding of the bound molecule. Kishida et al. (2003) reported that no cavity existed in a 1.6Å structure of the SO3437 homolog from Thermus thermophilus, presumably due to tighter packing of the protein from the thermophilic organism. Steinbacher et al. (2002) make no description of a hydrophobic cavity in a lower resolution (2.5-3.2Å) of the Escherichia coli protein. Here, we report a high-resolution (1.6Å) structure of MECDP synthase from Shewanella oneidensis in the absence of substrate in the active site. We provide unambiguous data that confirms the presence of Zn2+ in one of the metal binding sites and observe what appears to be farnesyl diphosphate (FPP) bound in the hydrophobic cavity along the non-crystallographic three-fold symmetry axis of the homotrimer. The high-resolution structure clarifies the mode of binding of the pyrophosphate of FPP in the arginine cluster that caps the hydrophobic cavity.

  10. Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

    SciTech Connect

    Shi, Liang; Squier, Thomas C.; Zachara, John M.; Fredrickson, Jim K.

    2007-07-01

    Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobactersulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1-and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope.

  11. Bioremediation of nanomaterials

    DOEpatents

    Chen, Frank Fanqing; Keasling, Jay D; Tang, Yinjie J

    2013-05-14

    The present invention provides a method comprising the use of microorganisms for nanotoxicity study and bioremediation. In some embodiment, the microorganisms are bacterial organisms such as Gram negative bacteria, which are used as model organisms to study the nanotoxicity of the fullerene compounds: E. coli W3110, a human related enterobacterium and Shewanella oneidensis MR-1, an environmentally important bacterium with versatile metabolism.

  12. Improved microbial electrocatalysis with osmium polymer modified electrodes.

    PubMed

    Patil, Sunil A; Hasan, Kamrul; Leech, Dónal; Hägerhäll, Cecilia; Gorton, Lo

    2012-10-21

    Using the well-known exoelectrogen Shewanella oneidensis MR-1, an osmium redox polymer modified anode exhibited ca. 4-fold increase in current generation. Additionally, a significant decrease in the start-up time for electrocatalysis was observed. The findings suggest that the inherent extracellular electron transfer capabilities of electrogens coupled with such polymers could enhance electrocatalysis.

  13. Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes.

    PubMed

    Shi, Liang; Squier, Thomas C; Zachara, John M; Fredrickson, James K

    2007-07-01

    Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope.

  14. Branched Nanowire Architectures for Compact Power Sources

    DTIC Science & Technology

    2009-04-13

    oriented protein/enzymes and/or single bacterial cells, such as Shewanella oneidensis MR-1. To address such fundamental issues a major emphasis of...electrode region exposed to solution is passivated with a dielectric layer to isolate the electrodes from solution. In our initial experiments

  15. Over-expression of Multi-heme C-type Cytochromes

    SciTech Connect

    Shi, Liang; Lin, Chiann Tso; Markillie, Lye Meng; Squier, Thomas C.; Hooker, Brian S.

    2005-02-01

    ABSTRACT-Because they contain covalently attached hemes, c-type cytochromes, especially those with multi-heme, are difficult to over-express. The gram negative bacterium Shewanella oneidensis MR-1 has been successfully used for over-expression of multi-heme c-type cytochromes...

  16. Molecular AND logic gate based on bacterial anaerobic respiration.

    PubMed

    Arugula, Mary Anitha; Shroff, Namita; Katz, Evgeny; He, Zhen

    2012-10-21

    Enzyme coding genes that integrate information for anaerobic respiration in Shewanella oneidensis MR-1 were used as input for constructing an AND logic gate. The absence of one or both genes inhibited electrochemically-controlled anaerobic respiration, while wild type bacteria were capable of accepting electrons from an electrode for DMSO reduction.

  17. Photoheterotrophic Fluxome in Synechocystis sp. Strain PCC 6803 and Its Implications for Cyanobacterial Bioenergetics

    PubMed Central

    You, Le; He, Lian

    2014-01-01

    This study investigated metabolic responses in Synechocystis sp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O2 evolution and ATP/NADPH generation by linear electron flow. Based on 13C-metabolic flux analysis (13C-MFA) and RNA sequencing, we have found that Synechocystis sp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO2 and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria. PMID:25535269

  18. Genomic and fluxomic analysis of carbohydrate metabolism in Bifidobacterium spp: human symbiotic bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bifidobacteria are gram-positive microorganisms widely applied in fermented dairy products due to their health-promoting effects. Bifidobacterium ssp. may also represent up to 91% of microbial gut population in the infant colon, but considerably less in adults. Fructose-6 phosphate phosphoketolase...

  19. Measurement uncertainty of isotopologue fractions in fluxomics determined via mass spectrometry.

    PubMed

    Guerrasio, R; Haberhauer-Troyer, C; Steiger, M; Sauer, M; Mattanovich, D; Koellensperger, G; Hann, S

    2013-06-01

    Metabolic flux analysis implies mass isotopomer distribution analysis and determination of mass isotopologue fractions (IFs) of proteinogenic amino acids of cell cultures. In this work, for the first time, this type of analysis is comprehensively investigated in terms of measurement uncertainty by calculating and comparing budgets for different mass spectrometric techniques. The calculations addressed amino acids of Pichia pastoris grown on 10% uniformly (13)C labeled glucose. Typically, such experiments revealed an enrichment of (13)C by at least one order of magnitude in all proteinogenic amino acids. Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The samples were diluted to fit the linear dynamic range of the mass spectrometers used (10 μM amino acid concentration). The total combined uncertainties of IFs as well as the major uncertainty contributions affecting the IFs were determined for phenylalanine, which was selected as exemplary model compound. A bottom-up uncertainty propagation was performed according to Quantifying Uncertainty in Analytical Measurement and using the Monte Carlo method by considering all factors leading to an IF, i.e., the process of measurement and the addition of (13)C-glucose. Excellent relative expanded uncertainties (k = 1) of 0.32, 0.75, and 0.96% were obtained for an IF value of 0.7 by LC-MS/MS, GC-MS, and LC-TOFMS, respectively. The major source of uncertainty, with a relative contribution of 20-80% of the total uncertainty, was attributed to the signal intensity (absolute counts) uncertainty calculated according to Poisson counting statistics, regardless which of the mass spectrometry platforms was used. Uncertainty due to measurement repeatability was of importance in LC-MS/MS, showing a relative contribution up to 47% of the total uncertainty, whereas for GC-MS and LC-TOFMS the average contribution was lower (30 and 15%, respectively). Moreover, the IF actually present also depends on the isotopic purity of the carbon sources. Therefore, in the uncertainty calculation a carbon source purity factor was introduced and a minor contribution to the total uncertainty was observed. The results obtained by uncertainty calculation performed according to the Monte Carlo method were in agreement with the uncertainty value of the Kragten approach and showed a Gaussian distribution.

  20. Integrated Investigation on the Production and Fate of Organo-Cr(III) Complexes from Microbial Reduction of Chromate

    SciTech Connect

    Xun, Luying

    2004-06-01

    The screening of different genera of bacteria for production of soluble Cr(III) complexes has been completed. A total of eight organisms were screened for production of soluble Cr(III); three were Gram positive and five were Gram negative. The Gram positive bacteria were Cellulomonas sp. ES 6, Rhodococcus sp., and Leafsonia sp., while Shewanella. oneidensis MR 1, Desulfovibrio desulfuricans G20, D. vulgaris Hildenborough, Pseudomonas putida MK 1 and Ps. aeruginosa PAO 1 were Gram negative. S. oneidensis MR 1 and Cellulomonas sp ES 6 were grown in minimal media, GWM (Ground Water Medium with lactate/fumarate) and SGM (Simulated Groundwater Medium with sucrose), respectively. Other bacteria were screened under non-growth conditions with sucrose, lactate, or glycerol as electron donor. All experiments were carried out for a period of 15-30 days, with different organisms reaching a maximum soluble Cr(III) concentrations at different times: S. oneidensis, 2d; Cellulomonas sp., 8d; Leafsonia, 6d; Rhodococcus, 9d; Ps. putida MK 1, 6d, Ps. aeruginosa PAO 1, 3d; D. vulgaris Hildenborough, 3d; and D. desulfuricans G20, 21d. Initial characterization indicates that the soluble Cr(III) fraction produced by both S. oneidensis MR 1 and Cellulomonas sp. ES 6 passes through a 1-Kd cut off filter.

  1. [Bion-M1. Biological activities of microorganisms under the conditions of a 30-day space flight].

    PubMed

    Voeĭkova, T A; Tiaglov, B V; Novikova, L M; Krest'ianova, I N; Emel'ianova, L K; Korshunov, D V; Morozova, Iu A; Il'in, V K

    2014-01-01

    It was stated that spaceflight factors (SFF) affect the chromosomal DNA interchange during Streptomyces crossing. Cross polarity and primary input of a parent chromosome fragment in recombinant generation imply a more lasting cells contact in microgravity and a broader horizontal transport of genetic material. SFF had no effect on recombination frequency and mutation in a model of parental auxotrophic markers reversion to prototrophism. It was demonstrated that SFF boosted the fC31 phage exit from S. lividans 66 (fC31) and did not influence phage induction in S. coelicolor A3(2) (fC31). SFF inhibited synthesis of antiobiotic actinorhodin in lisogenic S. coelicolor A3(2), and tylosin and desmicosin in S. fradiae. Survivability of electrogenic bacteria Shewanella oneidensis MR-1 in space flight was higher compared with the synchronous control experiment. The reduction activity of S. oneidensis MR-1 as an indicator of electron generation effectiveness was identical in flight and laboratory samples.

  2. [Microbial fuel cells as an alternative power supply].

    PubMed

    Il'in, V K; Smirnov, I A; Soldatov, P É; Korshunov, D V; Tiurin-Kuz'min, A Iu; Starkova, L V; Chumakov, P E; Emel'ianova, L K; Novikova, L M; Debabov, V G; Voeĭkova, T A

    2012-01-01

    Purpose of the work was designing and prototyping of microbial fuel cells (MFC) and comparative evaluation of the electrogenic activity of wastewater autochthonous microorganisms as well as bacterial monocultures. Objects were model electrogenic strain Shewanella oneidensis MR-1, and an Ochrobactrum sp. strain isolated from the active anode biofilm of MFC composed as an electricity generating system. The study employed the methods typically used for aerobic and anaerobic strains, current measurement, identification of new electrogenic strains in microbial association of wastewater sludge and species definition by rRNA 16-S. As a result, two MFCs prototypes were tried out. Besides, it was shown that electrogenic activity of S. oneidensis MR-1 and Ochrobactrum sp. monocultures is similar but differs from that of the microbial association of the anode biofilm.

  3. Probing Single- to Multi-Cell Level Charge Transport in Geobacter sulfurreducens DL-1

    DTIC Science & Technology

    2013-11-08

    nanoelectrodes in which an array of nanoholes (200 400 nm2) precludes or single window (6 10 mm2) allows for direct microbe/electrode contacts (Supplementary...previous measurement with S. oneidensis MR-1 cells, which gave almost identical current output between window and nanohole electrodes30. At longer times, it...is interesting to note that the nanohole electrode was also able to yield a current, albeit at a much smaller magnitude, which could be attributed to

  4. K-shell Analysis Reveals Distinct Functional Parts in an Electron Transfer Network and Its Implications for Extracellular Electron Transfer

    PubMed Central

    Ding, Dewu; Li, Ling; Shu, Chuanjun; Sun, Xiao

    2016-01-01

    Shewanella oneidensis MR-1 is capable of extracellular electron transfer (EET) and hence has attracted considerable attention. The EET pathways mainly consist of c-type cytochromes, along with some other proteins involved in electron transfer processes. By whole genome study and protein interactions inquisition, we constructed a large-scale electron transfer network containing 2276 interactions among 454 electron transfer related proteins in S. oneidensis MR-1. Using the k-shell decomposition method, we identified and analyzed distinct parts of the electron transfer network. We found that there was a negative correlation between the ks (k-shell values) and the average DR_100 (disordered regions per 100 amino acids) in every shell, which suggested that disordered regions of proteins played an important role during the formation and extension of the electron transfer network. Furthermore, proteins in the top three shells of the network are mainly located in the cytoplasm and inner membrane; these proteins can be responsible for transfer of electrons into the quinone pool in a wide variety of environmental conditions. In most of the other shells, proteins are broadly located throughout the five cellular compartments (cytoplasm, inner membrane, periplasm, outer membrane, and extracellular), which ensures the important EET ability of S. oneidensis MR-1. Specifically, the fourth shell was responsible for EET and the c-type cytochromes in the remaining shells of the electron transfer network were involved in aiding EET. Taken together, these results show that there are distinct functional parts in the electron transfer network of S. oneidensis MR-1, and the EET processes could achieve high efficiency through cooperation through such an electron transfer network. PMID:27148219

  5. Effects of a Strong Static Magnetic Field on Bacterium Shewanellaoneidensis: An Assessment by Using Whole Genome Microarray.

    SciTech Connect

    Gao, W.; Liu, Y.; Zhou, J.-Z.; Hongjun, P.

    2007-04-02

    The effect of a strong static 14.1 T magnetic field on logphase cells of bacterial strain Shewanella oneidensis MR-1 was evaluatedby using whole genome microarray of this bacterium. Although differenceswere not observed between the treatment and control by measuring theoptical density (OD), colony forming unit (CFU), as well as post-exposuregrowth of cells, transcriptional expression levels of 65 genes werealtered according to our microarray data. Among these genes, 21 wereupregulated while other 44were downregulated, compared withcontrol.

  6. Lactate Oxidation Coupled to Iron or Electrode Reduction by Geobacter sulfurreducens PCA▿

    PubMed Central

    Call, Douglas F.; Logan, Bruce E.

    2011-01-01

    Geobacter sulfurreducens PCA completely oxidized lactate and reduced iron or an electrode, producing pyruvate and acetate intermediates. Compared to the current produced by Shewanella oneidensis MR-1, G. sulfurreducens PCA produced 10-times-higher current levels in lactate-fed microbial electrolysis cells. The kinetic and comparative analyses reported here suggest a prominent role of G. sulfurreducens strains in metal- and electrode-reducing communities supplied with lactate. PMID:22003020

  7. Metabolic Profiling Directly from the Petri Dish Using Nanospray Desorption Electrospray Ionization Imaging Mass Spectrometry

    SciTech Connect

    Watrous, Jeramie D.; Roach, Patrick J.; Heath, Brandi S.; Alexandrov, Theodore; Laskin, Julia; Dorrestein, Pieter C.

    2013-11-05

    Understanding molecular interaction pathways in complex biological systems constitutes a treasure trove of knowledge that might facilitate the specific, chemical manipulation of the countless microbiological systems that occur throughout our world. However, there is a lack of methodologies that allow the direct investigation of chemical gradients and interactions in living biological systems, in real time. Here, we report the use of nanospray desorption electrospray ionization (nanoDESI) imaging mass spectrometry for in vivo metabolic profiling of living bacterial colonies directly from the Petri dish with absolutely no sample preparation needed. Using this technique, we investigated single colonies of Shewanella oneidensis MR-1, Bacillus subtilis 3610, and Streptomyces coelicolor A3(2) as well as a mixed biofilm of S. oneidensis MR-1 and B. subtilis 3610. Data from B. subtilis 3610 and S. coelicolor A3(2) provided a means of validation for the method while data from S. oneidensis MR-1 and the mixed biofilm showed a wide range of compounds that this bacterium uses for the dissimilatory reduction of extracellular metal oxides, including riboflavin, iron-bound heme and heme biosynthetic intermediates, and the siderophore putrebactin.

  8. Pathway confirmation and flux analysis of central metabolicpathways in Desulfovibrio vulgaris Hildenborough using gaschromatography-mass spectrometry and fourier transform-ion cyclotronresonance mass spectrometry

    SciTech Connect

    Tang, Yinjie; Pingitore, Francesco; Mukhopadhyay, Aindrila; Phan,Richard; Hazen, Terry C.; Keasling, Jay D.

    2006-07-11

    It has been proposed that during growth under anaerobic oroxygen-limited conditions Shewanella oneidensis MR-1 uses theserine-isocitrate lyase pathway common to many methylotrophic anaerobes,in which formaldehyde produced from pyruvate is condensed with glycine toform serine. The serine is then transformed through hydroxypyruvate andglycerate to enter central metabolism at phosphoglycerate. To examine itsuse of the serine-isocitrate lyase pathway under anaerobic conditions, wegrew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source witheither trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor.Analysis of cellular metabolites indicates that a large percentage(>75 percent) of lactate was partially oxidized to either acetate orpyruvate. The 13C isotope distributions in amino acids and other keymetabolites indicate that, under anaerobic conditions, a complete serinepathway is not present, and lactate is oxidized via a highly reversibleserine degradation pathway. The labeling data also suggest significantactivity in the anaplerotic (malic enzyme and phosphoenolpyruvatecarboxylase) and glyoxylate shunt (isocitrate lyase and malate synthase)reactions. Although the tricarboxylic acid (TCA) cycle is often observedto be incomplete in many other anaerobes (absence of 2-oxoglutaratedehydrogenase activity), isotopic labeling supports the existence of acomplete TCA cycle in S. oneidensis MR-1 under TMAO reductioncondition.

  9. Diurnal Regulation of Cellular Processes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Insights from Transcriptomic, Fluxomic, and Physiological Analyses

    PubMed Central

    Saha, Rajib; Liu, Deng; Hoynes-O’Connor, Allison; Liberton, Michelle; Yu, Jingjie; Bhattacharyya-Pakrasi, Maitrayee; Balassy, Andrea; Zhang, Fuzhong; Maranas, Costas D.

    2016-01-01

    ABSTRACT Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper) were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H), and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP+ showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium. PMID:27143387

  10. Combined fluxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H.

    PubMed

    Hanke, Tanja; Nöh, Katharina; Noack, Stephan; Polen, Tino; Bringer, Stephanie; Sahm, Hermann; Wiechert, Wolfgang; Bott, Michael

    2013-04-01

    In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by (13)C-based metabolic flux analysis ((13)C-MFA) in combination with transcriptomics and enzyme assays. For (13)C-MFA, cells were cultivated with specifically (13)C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. Since G. oxydans lacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP). (13)C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.

  11. Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

    PubMed Central

    Hanke, Tanja; Noack, Stephan; Polen, Tino; Bringer, Stephanie; Sahm, Hermann; Wiechert, Wolfgang

    2013-01-01

    In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by 13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For 13C-MFA, cells were cultivated with specifically 13C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. Since G. oxydans lacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP). 13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II. PMID:23377928

  12. Enhanced Shewanella biofilm promotes bioelectricity generation.

    PubMed

    Liu, Ting; Yu, Yang-Yang; Deng, Xiao-Peng; Ng, Chun Kiat; Cao, Bin; Wang, Jing-Yuan; Rice, Scott A; Kjelleberg, Staffan; Song, Hao

    2015-10-01

    Electroactive biofilms play essential roles in determining the power output of microbial fuel cells (MFCs). To engineer the electroactive biofilm formation of Shewanella oneidensis MR-1, a model exoelectrogen, we herein heterologously overexpressed a c-di-GMP biosynthesis gene ydeH in S. oneidensis MR-1, constructing a mutant strain in which the expression of ydeH is under the control of IPTG-inducible promoter, and a strain in which ydeH is under the control of a constitutive promoter. Such engineered Shewanella strains had significantly enhanced biofilm formation and bioelectricity generation. The MFCs inoculated with these engineered strains accomplished a maximum power density of 167.6 ± 3.6 mW/m(2) , which was ∼ 2.8 times of that achieved by the wild-type MR-1 (61.0 ± 1.9 mW/m(2) ). In addition, the engineered strains in the bioelectrochemical system at poised potential of 0.2 V vs. saturated calomel electrode (SCE) generated a stable current density of 1100 mA/m(2) , ∼ 3.4 times of that by wild-type MR-1 (320 mA/m(2) ).

  13. A synthetic microbial consortium of Shewanella and Bacillus for enhanced generation of bioelectricity.

    PubMed

    Liu, Ting; Yu, Yang-Yang; Chen, Tao; Chen, Wei Ning

    2017-03-01

    In this study, a synthetic microbial consortium containing exoelectrogen Shewanella oneidensis MR-1 and riboflavin-producing strain, Bacillus subtilis RH33, was rationally designed and successfully constructed, enabling a stable, multiple cycles of microbial fuel cells (MFCs) operation for more than 500 h. The maximum power density of MFCs with this synthetic microbial consortium was 277.4 mW/m(2) , which was 4.9 times of that with MR-1 (56.9 mW/m(2) ) and 40.2 times of RH33 (6.9 mW/m(2) ), separately. At the same time, the Coulombic efficiency of the synthetic microbial consortium (5.6%) was higher than MR-1 (4.1%) and RH33 (2.3%). Regardless the high concentration of riboflavin produced by RH33, the power density of RH33 was rather low. The low bioelectricity generation can be ascribed to the low efficiency of RH33 in utilizing riboflavin for extracellular electron transfer (EET). In the synthetic microbial consortium of MR-1 and RH33, it was found that both mediated and direct electron transfer efficiencies were enhanced. By exchanging the anolyte of MR-1 and RH33, it was confirmed that the improved MFC performance with the synthetic microbial consortium was because MR-1 could efficiently utilize the high concentration of riboflavin produced by RH33. Biotechnol. Bioeng. 2017;114: 526-532. © 2016 Wiley Periodicals, Inc.

  14. Sulfur-mediated electron shuttling during bacterial iron reduction.

    PubMed

    Flynn, Theodore M; O'Loughlin, Edward J; Mishra, Bhoopesh; DiChristina, Thomas J; Kemner, Kenneth M

    2014-05-30

    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.

  15. Structural and functional studies of multiheme cytochromes C involved in extracellular electron transport in bacterial dissimilatory metal reduction.

    PubMed

    Tikhonova, T V; Popov, V O

    2014-12-01

    Bacteria utilizing insoluble mineral forms of metal oxides as electron acceptors in respiratory processes are widespread in the nature. The electron transfer from a pool of reduced quinones in the cytoplasmic membrane across the periplasm to the bacterial outer membrane and then to an extracellular acceptor is a key step in bacterial dissimilatory metal reduction. Multiheme cytochromes c play a crucial role in the extracellular electron transfer. The bacterium Shewanella oneidensis MR-1 was used as a model organism to study the mechanism of extracellular electron transport. In this review, we discuss recent data on the composition, structures, and functions of multiheme cytochromes c and their functional complexes responsible for extracellular electron transport in Shewanella oneidensis.

  16. Utilization of DNA as a Sole Source of Phosphorus, Carbon, and Energy by Shewanella spp.: Ecological and Physiological Implications for Dissimilatory Metal Reduction

    SciTech Connect

    Pinchuk, Grigoriy E.; Ammons, Christine G.; Culley, David E.; Li, Shu-Mei; McLean, Jeffrey S.; Romine, Margaret F.; Nealson, Kenneth H.; Fredrickson, Jim K.; Beliaev, Alex S.

    2008-02-15

    As a constituent of dissolved organic matter, DNA may be consumed by microorganisms inhabiting various freshwater and marine environments. In this study, we demonstrate that dissolved extracellular DNA can serve as a sole source of carbon, energy, nitrogen, and phosphorus for microorganisms residing in the upper layer of Columbia River (WA, USA) water column as well as a sole source of phosphorus for the dissimilatory metal-reducing bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens and for Bacillus subtilis ATCC 49760. Our results suggest that DNA assimilation by S. oneidensis is linked to the activity of Ca2+-dependent nuclease(s) and extracellular phosphatase(s). The ability to use DNA as the sole source of phosphorus may be of particular ecological advantage for microorganisms living under Fe(III)-reducing conditions where bioavailability of inorganic phosphate may be limited by the formation of vivianite [Fe3(PO4)2•8H20].

  17. Bacterial Nanowires Facilitate Extracellular Electron Transfer

    NASA Astrophysics Data System (ADS)

    Gorby, Y.

    2005-12-01

    Dissimilatory metal reducing bacteria, including Shewanella oneidensis and Geobacter sulfurreducens, produce electrically conductive nanowires that facilitate electron transfer to solid phase iron oxides. Nanowires produced by S. oneidensis strain MR-1 are functionalized by decaheme cytochromes MtrC and OmcA that are distributed along the length of the nanowires, as confirmed by immunolocalization experiments using peptide specific antibodies. Mutants lacking MtrC and OmcA produce nanowires that were poorly conductive, are unable to reduce solid phase iron oxides, and do not produce electric current in microbial fuel cells. Although less completely characterized, nanowires are also produced by organisms throughout a broad metabolic spectrum, from sulfate reducing bacteria to oxygenic, phototrophic cyanobacteria. Our research suggests that electrically conductive nanowires may be common throughout the microbial world and may serve as structures for efficient electron transfer and energy dissemination in complex communities such as microbial mats and biofilms.

  18. In Situ Environmental Cell-Transmission Electron Microscopy Study of Microbial Reduction of Chromium(VI) Using Electron Energy Loss Spectroscopy.

    PubMed

    Daulton, Tyrone L.; Little, Brenda J.; Lowe, Kristine; Jones-Meehan, Joanne

    2001-11-01

    Reduction of Cr(VI) by the bacterium, Shewanella oneidensis (previously classified Shewanella putrefaciens strain MR-1), was studied by absorption spectrophotometry and in situ, environmental cell-transmission electron microscopy (EC-TEM) coupled with electron energy loss spectroscopy (EELS). Bacteria from rinsed cultures were placed directly in the environmental cell of the transmission electron microscope and examined under 100 Torr pressure. Bright field EC-TEM images show two distinct populations of S. oneidensis in incubated cultures containing Cr(VI)O2- 4: those that exhibit low image contrast and heavily precipitate-encrusted cells exhibiting high image contrast. Several EELS techniques were applied to determine the oxidation state of Cr associated with encrusted cells. The encrusted cells are shown to contain a reduced form of Cr in oxidation state +3 or lower. These results demonstrate the capability to determine the chemistry and valence state of reduction products associated with unfixed, hydrated bacteria in an environmental cell transmission electron microscope.

  19. Single-Cell Imaging and Spectroscopic Analyses of Cr(VI) Reduction on the Surface of Bacterial Cells

    SciTech Connect

    Wang, Yuanmin; Sevinc, Papatya C.; Belchik, Sara M.; Fredrickson, Jim K.; Shi, Liang; Lu, H. Peter

    2013-01-22

    We investigate single-cell reduction of toxic Cr(VI) by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), an important bioremediation process, using Raman spectroscopy and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Our experiments indicate that the toxic and highly soluble Cr(VI) can be efficiently reduced to the less toxic and non-soluble Cr2O3 nanoparticles by MR-1. Cr2O3 is observed to emerge as nanoparticles adsorbed on the cell surface and its chemical nature is identified by EDX imaging and Raman spectroscopy. Co-localization of Cr2O3 and cytochromes by EDX imaging and Raman spectroscopy suggests a terminal reductase role for MR-1 surface-exposed cytochromes MtrC and OmcA. Our experiments revealed that the cooperation of surface proteins OmcA and MtrC makes the reduction reaction most efficient, and the sequence of the reducing reactivity of the MR-1 is: wild type > single mutant @mtrC or mutant @omcA > double mutant (@omcA-@mtrC). Moreover, our results also suggest that the direct microbial Cr(VI) reduction and Fe(II) (hematite)-mediated Cr(VI) reduction mechanisms may co-exist in the reduction processes.

  20. Cation-limited kinetic model for microbial extracellular electron transport via an outer membrane cytochrome C complex

    PubMed Central

    Okamoto, Akihiro; Tokunou, Yoshihide; Saito, Junki

    2016-01-01

    Outer-membrane c-type cytochrome (OM c-Cyt) complexes in several genera of iron-reducing bacteria, such as Shewanella and Geobacter, are capable of transporting electrons from the cell interior to extracellular solids as a terminal step of anaerobic respiration. The kinetics of this electron transport has implications for controlling the rate of microbial electron transport during bioenergy or biochemical production, iron corrosion, and natural mineral cycling. Herein, we review the findings from in-vivo and in-vitro studies examining electron transport kinetics through single OM c-Cyt complexes in Shewanella oneidensis MR-1. In-vitro electron flux via a purified OM c-Cyt complex, comprised of MtrA, B, and C proteins from S. oneidensis MR-1, embedded in a proteoliposome system is reported to be 10- to 100-fold faster compared with in-vivo estimates based on measurements of electron flux per cell and OM c-Cyts density. As the proteoliposome system is estimated to have 10-fold higher cation flux via potassium channels than electrons, we speculate that the slower rate of electron-coupled cation transport across the OM is responsible for the significantly lower electron transport rate that is observed in-vivo. As most studies to date have primarily focused on the energetics or kinetics of interheme electron hopping in OM c-Cyts in this microbial electron transport mechanism, the proposed model involving cation transport provides new insight into the rate detemining step of EET, as well as the role of self-secreted flavin molecules bound to OM c-Cyt and proton management for energy conservation and production in S. oneidensis MR-1. PMID:27924259

  1. Cr isotope fractionation in metal-mineral-microbe interactions

    NASA Astrophysics Data System (ADS)

    Zhang, Qiong; Porcelli, Don; Thompson, Ian; Amor, Ken; Galer, Stephen

    2016-04-01

    Microbes interact with metals and minerals in the environments, altering their physical and chemical state whilst in turn the metals and minerals affect microbial growth, activity and survival. The interactions between Cr, Fe minerals and bacteria were investigated in this study. Cr(VI) reduction experiments by two iron-reducing bacteria, Pseudomonas fluorescens LB 300 and Shewanella oneidensis MR 1, in the presence of two iron oxide minerals, goethite and hematite, were conducted. Both minerals were found to inhibit the Cr(VI) reduction rate by Pseudomonas fluorescens LB 300 but accelerated Shewanella oneidensis MR 1. The Cr isotopic fractionation factor generated by both bacteria was mostly independent of the presence of the minerals, except for hematite with Pseudomonas fluorescens LB 300, where the ɛ was much higher. Aqueous Fe(III) in the solution did not have any detectable impact on either bacterial Cr reduction rates or the isotopic fractionation factors, indicating that the reduction of Cr(VI) occurred prior to that of Fe(III). The presence of aqueous Fe(II) induced a very fast abiotic reduction of Cr, but had little impact on the bacterial Cr reduction rates or its isotope fractionations. The evidence suggests that the different impact that Fe minerals had on the bacteria were related to the way they attached to the minerals and the difference in the reduction mechanism. SEM images confirmed that the attachment of Pseudomonas fluorescens LB 300 on the mineral surfaces were much more tightly packed than that of Shewanella oneidensis MR 1, so reducing mineral-metal interactions.

  2. Modeling of Sustainable Base Production by Microbial Electrolysis Cell.

    PubMed

    Blatter, Maxime; Sugnaux, Marc; Comninellis, Christos; Nealson, Kenneth; Fischer, Fabian

    2016-07-07

    A predictive model for the microbial/electrochemical base formation from wastewater was established and compared to experimental conditions within a microbial electrolysis cell. A Na2 SO4 /K2 SO4 anolyte showed that model prediction matched experimental results. Using Shewanella oneidensis MR-1, a strong base (pH≈13) was generated using applied voltages between 0.3 and 1.1 V. Due to the use of bicarbonate, the pH value in the anolyte remained unchanged, which is required to maintain microbial activity.

  3. NMR methods for in-situ biofilm metabolism studies: spatial and temporal resolved measurements

    SciTech Connect

    Majors, Paul D.; Mclean, Jeffrey S.; Fredrickson, Jim K.; Wind, Robert A.

    2005-11-01

    We are developing nuclear magnetic resonance (NMR) microscopy, spectroscopy and combined NMR/optical techniques to the study of biofilms. Objectives include: time and depth-resolved metabolite concentrations with isotropic spatial resolution on the order of 10 microns, metabolic pathways and flux rates, mass transport and ultimately their correlation with gene expression by optical microscopy in biofilms. These methods are being developed with Shewanella oneidensis MR-1 as a model system, but are equally applicable to other biofilm systems of interest. Thus, spatially resolved NMR of biofilms is expected to contribute significantly to the understanding of adherent cell metabolism.

  4. Comparisons of Shewanella strains based on genome annotations, modeling and experiments

    SciTech Connect

    Ong, Wai Kit; Vu, Trang; Lovendahl, Klaus N.; Llull, Jenna; Serres, Margaret; Romine, Margaret F.; Reed, Jennifer L.

    2014-01-01

    Shewanella is a genus of facultatively anaerobic, Gram-negative bacteria that have highly adaptable metabolism which allows them to thrive in diverse environments. This quality makes them attractive target bacteria for research in bioremediation and microbial fuel cell applications. Constraint-based modeling is a useful tool for helping researchers gain insights into the metabolic capabilities of these bacteria. However, Shewanella oneidensis MR-1 is the only strain with a genome-scale metabolic model constructed out of the 22 sequenced Shewanella strains.

  5. Towards Environmental Systems Biology of Shewanella

    SciTech Connect

    Fredrickson, Jim K.; Romine, Margaret F.; Beliaev, Alex S.; Auchutung, Jennifer M.; Driscoll, Michael E.; Gardner, Timothy S.; Nealson, Kenneth H.; Osterman, Andrei L.; Pinchuk, Grigoriy E.; Reed, Jennifer L.; Rodionov, Dmitry; Rodrigues, Jorge L.M.; Saffarini, Daad; Serres, Margrethe H.; Spormann, Alfred M.; Zhulin, Igor B.; Tiedje, James M.

    2008-07-07

    Abstract: Shewanella are known for versatility in their electron accepting capacities, which allow them to couple decomposition of organic matter to reduction of the variety of terminal electron acceptors encountered in the stratified environments they inhabit. Due to their diverse metabolic capabilities, shewanellae play important roles in carbon cycling and have considerable potential for remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species, S. oneidensis MR-1, and other members of this genus have provided significant insights into the signal transduction proteins, regulators, and metabolic and respiratory subsystems governing the remarkable versatility of the shewanellae.

  6. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

  7. Identification and Characterization of UndA-HRCR-6, an Outer Membrane Endecaheme c-Type Cytochrome of Shewanella sp. Strain HRCR-6

    SciTech Connect

    Shi, Liang; Belchik, Sara M.; Wang, Zheming; Kennedy, David W.; Dohnalkova, Alice; Marshall, Matthew J.; Zachara, John M.; Fredrickson, Jim K.

    2011-08-01

    The outer membrane decaheme c-type cytochromes (c-Cyt) MtrC and OmcA of Shewanella oneidensis MR-1(MR-1) play critical roles in extracellular reduction of iron [Fe(III)] oxides and uranium [ U(VI)]. To identify and characterize the outer membrane c-Cyts found in the metal-reducing Shewanella strains isolated from the Hanford Reach of the Columbia River (HRCR), 7 HRCR isolates were tested for the presence of mtrC, omcA and undA1 (a gene encoding a putative 11-heme c-Cyt) homologues in their genomes. All 7 tested strains possessed an mtrC homologue, while 3 strains had an omcA homologue and the remaining 4 strains contained an undA1 homologue. The coding region of an undA1 homologue from HRCR isolate 6 was cloned and sequenced. Because it was 93% identical to the UndA of S. baltica OS223, the protein product encoded by this sequenced gene was named as UndA-HRCR6. In MR-1, UndA-HRCR6 (i) restored an MR-1 mutant’s ability to reduce solid phase ferrihydrite at 40% of that for MR-1 wild type, (ii) increased extracellular formation of UO2 associated with the outer membrane and extracellular polymeric substances in a U(VI) reduction assay and (iii) was secreted to the extracellular environment by bacterial type II secretion system. UndA-HRCR6 was purified from the membrane fraction following its overexpression in MR-1 cells. Purified UndA-HRCR6 possessed 11 heme-Fe and reduced ferric complexes. Collectively, these results show that UndA-HRCR6 is an outer membrane endecaheme c-Cyt and can serve an extracellular metal reductase with functions similar to that of MR-1 MtrC and OmcA.

  8. Aggregation Kinetics of Hematite Particles in the Presence of Outer Membrane Cytochrome OmcA of Shewanella oneidenesis MR-1.

    PubMed

    Sheng, Anxu; Liu, Feng; Shi, Liang; Liu, Juan

    2016-10-06

    The aggregation behavior of 9, 36, and 112 nm hematite particles was studied in the presence of OmcA, a bacterial extracellular protein, in aqueous dispersions at pH 5.7 through time-resolved dynamic light scattering, electrophoretic mobility, and circular dichroism spectra, respectively. At low salt concentration, the attachment efficiencies of hematite particles in all sizes first increased, then decreased, and finally remained stable with the increase of OmcA concentration, indicating the dominant interparticle interaction changed along with the increase in the protein-to-particle ratio. Nevertheless, at high salt concentration, the attachment efficiencies of all hematite samples gradually decreased with increasing OmcA concentration, which can be attributed to increasing steric force. Additionally, the aggregation behavior of OmcA-hematite conjugates was more correlated to total particle-surface area than primary particle size. It was further established that OmcA could stabilize hematite nanoparticles more efficiently than bovine serum albumin (BSA), a model plasma protein, due to the higher affinity of OmcA to hematite surface. This study highlighted the effects of particle properties, solution conditions, and protein properties on the complicated aggregation behavior of protein-nanoparticle conjugates in aqueous environments.

  9. CeCaFDB: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13C-fluxomics.

    PubMed

    Zhang, Zhengdong; Shen, Tie; Rui, Bin; Zhou, Wenwei; Zhou, Xiangfei; Shang, Chuanyu; Xin, Chenwei; Liu, Xiaoguang; Li, Gang; Jiang, Jiansi; Li, Chao; Li, Ruiyuan; Han, Mengshu; You, Shanping; Yu, Guojun; Yi, Yin; Wen, Han; Liu, Zhijie; Xie, Xiaoyao

    2015-01-01

    The Central Carbon Metabolic Flux Database (CeCaFDB, available at http://www.cecafdb.org) is a manually curated, multipurpose and open-access database for the documentation, visualization and comparative analysis of the quantitative flux results of central carbon metabolism among microbes and animal cells. It encompasses records for more than 500 flux distributions among 36 organisms and includes information regarding the genotype, culture medium, growth conditions and other specific information gathered from hundreds of journal articles. In addition to its comprehensive literature-derived data, the CeCaFDB supports a common text search function among the data and interactive visualization of the curated flux distributions with compartmentation information based on the Cytoscape Web API, which facilitates data interpretation. The CeCaFDB offers four modules to calculate a similarity score or to perform an alignment between the flux distributions. One of the modules was built using an inter programming algorithm for flux distribution alignment that was specifically designed for this study. Based on these modules, the CeCaFDB also supports an extensive flux distribution comparison function among the curated data. The CeCaFDB is strenuously designed to address the broad demands of biochemists, metabolic engineers, systems biologists and members of the -omics community.

  10. Defining How a Microbial Cell Senses and Responds to a Redox Active Environment

    SciTech Connect

    Kenneth H. Nealson

    2012-06-22

    This grant was for four years, and the work was designed to look at the mechanisms of extracellular electron transfer by the dissimilatory iron reducing bacteria Shewanella oneidensis MR-1, and other closely related Shewanella strains and species. During this work, we defined many of the basic physiological and biochemical properties of the Shewanella group, Much of which was summarized in review articles. We also finished and published the genome sequence of strain MR-1, the first of the shewanellae to have its genome sequenced. Control at the transcriptional and translational level was studied in collaboration with colleagues at PNNL and ANL. We utilized synchrotron X-ray radiation to image both the bacteria and the metal oxide particles via a technique called STXM, synchrotron X-ray absorption (ref. No.9), and X-ray microbeam analysis. We purified several of the cytochromes involved with metal reduction, and improved gene annotation of the MR-1 genome. The conductive appendages (nanowires) of MR-1 were described and characterized. Comparative genomics and biochemistry revealed that the pathway for the utilization of N-acetyl glucosamine in the various strains of Shewanella exhibited great variability, and had a number of previously unknown genes.

  11. Diffusion in biofilms respiring on electrodes

    SciTech Connect

    Renslow, Ryan S.; Babauta, Jerome T.; Majors, Paul D.; Beyenal, Haluk

    2012-11-15

    The goal of this study was to measure spatially and temporally resolved effective diffusion coefficients (De) in biofilms respiring on electrodes. Two model electrochemically active biofilms, Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1, were investigated. A novel nuclear magnetic resonance microimaging perfusion probe capable of simultaneous electrochemical and pulsed-field gradient nuclear magnetic resonance (PFG-NMR) techniques was used. PFG-NMR allowed for noninvasive, nondestructive, high spatial resolution in situ De measurements in living biofilms respiring on electrodes. The electrodes were polarized so that they would act as the sole terminal electron acceptor for microbial metabolism. We present our results as both two-dimensional De heat maps and surface-averaged relative effective diffusion coefficient (Drs) depth profiles. We found that (1) Drs decreases with depth in G. sulfurreducens biofilms, following a sigmoid shape; (2) Drs at a given location decreases with G. sulfurreducens biofilm age; (3) average De and Drs profiles in G. sulfurreducens biofilms are lower than those in S. oneidensis biofilms—the G. sulfurreducens biofilms studied here were on average 10 times denser than the S. oneidensis biofilms; and (4) halting the respiration of a G. sulfurreducens biofilm decreases the De values. Density, reflected by De, plays a major role in the extracellular electron transfer strategies of electrochemically active biofilms.

  12. DIFFUSION IN BIOFILMS RESPIRING ON ELECTRODES

    PubMed Central

    Renslow, RS; Babauta, JT; Majors, PD; Beyenal, H

    2013-01-01

    The goal of this study was to measure spatially and temporally resolved effective diffusion coefficients (De) in biofilms respiring on electrodes. Two model electrochemically active biofilms, Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1, were investigated. A novel nuclear magnetic resonance microimaging perfusion probe capable of simultaneous electrochemical and pulsed-field gradient nuclear magnetic resonance (PFG-NMR) techniques was used. PFG-NMR allowed noninvasive, nondestructive, high spatial resolution in situ De measurements in living biofilms respiring on electrodes. The electrodes were polarized so that they would act as the sole terminal electron acceptor for microbial metabolism. We present our results as both two-dimensional De heat maps and surface-averaged relative effective diffusion coefficient (Drs) depth profiles. We found that 1) Drs decreases with depth in G. sulfurreducens biofilms, following a sigmoid shape; 2) Drs at a given location decreases with G. sulfurreducens biofilm age; 3) average De and Drs profiles in G. sulfurreducens biofilms are lower than those in S. oneidensis biofilms—the G. sulfurreducens biofilms studied here were on average 10 times denser than the S. oneidensis biofilms; and 4) halting the respiration of a G. sulfurreducens biofilm decreases the De values. Density, reflected by De, plays a major role in the extracellular electron transfer strategies of electrochemically active biofilms. PMID:23420623

  13. Differential arsenic mobilization from As-bearing ferrihydrite by iron-respiring Shewanella strains with different arsenic-reducing activities.

    PubMed

    Jiang, Shenghua; Lee, Ji-Hoon; Kim, Donghun; Kanaly, Robert A; Kim, Min-Gyu; Hur, Hor-Gil

    2013-08-06

    Arsenic immobilization and release in the environment is significantly influenced by bacterial oxidation and reduction of arsenic and arsenic-bearing minerals. In this study, we tested three iron-reducing bacteria, Shewanella oneidensis MR-1, Shewanella sp. HN-41, and Shewanella putrefaciens 200, which have diverse arsenate-reducing activities with regard to reduction of an As-bearing ferrihydrite slurry. In the cultures of S. oneidensis MR-1 and Shewanella sp. HN-41, which are not capable of respiratory reduction of As(V) to As(III), arsenic was maintained predominantly in its pentavalent form, existing in particulate poorly crystalline As-bearing ferrihydrite and formed small quantities of a stable ferrous arsenate [Fe3(AsO4)2] precipitate. However, in the culture of the As(V) reducer, S. putrefaciens 200, As(V) was reduced to As(III) and a small fraction of As-bearing ferrihydrite was transformed into ribbon-shaped siderite that subsequently re-released arsenic into the liquid phase. Our results indicated that release of arsenic and formation of diverse secondary nanoscale Fe-As minerals are specifically closely related to the arsenic-reducing abilities of different bacteria. Therefore, bacterial arsenic reduction appears to significantly influence As mobilization in soils, minerals, and other Fe-rich environments.

  14. Extracellular Polymeric Substances from Shewanella sp. HRCR-1 Biofilms: Characterization by Infrared Spectroscopy and Proteomics

    SciTech Connect

    Cao, Bin; Shi, Liang; Brown, Roslyn N.; Xiong, Yijia; Fredrickson, Jim K.; Romine, Margaret F.; Marshall, Matthew J.; Lipton, Mary S.; Beyenal, Haluk

    2011-04-01

    This study characterizes the composition of extracellular polymeric substances (EPS) from Shewanella sp. HRCR-1 biofilms to provide insight into potential interactions of EPS with redox-active metals and radionuclides. Both bound and loosely associated EPS were extracted from Shewanella sp. HRCR-1 biofilms prepared using a hollow-fiber membrane biofilm reactor (HfMBR). FTIR spectra revealed the presence of proteins, polysaccharides, nucleic acids, membrane lipids, and fatty acids in both bound and loosely associated EPS. Using a global proteomic approach, a total of 58 extracellular and outer membrane proteins were identified in the EPS. These included homologues of multiple S. oneidensis MR-1 proteins that potentially contribute to key physiological biofilm processes, such as biofilm-promoting protein BpfA, surface-associated serine protease, nucleotidases (CpdB and UshA), an extracellular lipase, and oligopeptidases (PtrB and a M13 family oligopeptidase lipoprotein). In addition, 20 redox proteins were found in extracted EPS. Among the detected redox proteins were the homologues of two S. oneidensis MR-1 c-type cytochromes, MtrC and OmcA, which have been implicated in extracellular electron transfer. Given their detection in the EPS of Shewanella sp. HRCR 1 biofilms, c-type cytochromes may contribute to the possible redox activity of the biofilm matrix and play important roles in extracellular electron transfer reactions.

  15. In-situ growth of graphene/polyaniline for synergistic improvement of extracellular electron transfer in bioelectrochemical systems.

    PubMed

    Sun, De-Zhen; Yu, Yang-Yang; Xie, Rong-Rong; Zhang, Chun-Lian; Yang, Yuan; Zhai, Dan-Dan; Yang, Guodong; Liu, Lei; Yong, Yang-Chun

    2017-01-15

    Graphene composite has been widely used in various bioelectrochemical systems (BES). However, it is suffered from tedious fabrication procedure and ambiguous mechanism for its effect on BES. Here, a one-step and in-situ strategy for simultaneously graphene exfoliation and aniline polymerization was developed for fabrication of graphene/PANI composite electrode (GO/PANIOS). This GO/PANIOS outperformed graphite paper (GP), GP with PANI (GP/PANI) and GP with electrochemical exfoliated graphene (GOH2SO4) in Shewanella oneidensis MR-1 inoculated BES (improved the power density output, i.e., 24, 3.4 and 5.7 times of GP , GP/PANI and GOH2SO4, respectively). Further analysis revealed a synergistic improvement on both direct and mediated extracellular electron transfer of S. oneidensis MR-1 by GO/PANIOS contributed to its performance enhancement in BES. This work not only provided a simple strategy for graphene composite fabrication, but also unveiled the underlying mechanism for its stimulation on BES, which promises new opportunity of graphene composite application in various biosystems.

  16. In search of experimental evidence for the biogeobattery

    NASA Astrophysics Data System (ADS)

    Hubbard, Christopher G.; West, L. Jared; Morris, Katherine; Kulessa, Bernd; Brookshaw, Diana; Lloyd, Jonathan R.; Shaw, Samuel

    2011-12-01

    Recent work has suggested that the electrical self-potential (SP) geophysical technique may be used to noninvasively map redox conditions associated with contaminant plumes or bioremediation schemes. The proposed mechanism linking SP response and redox involves the generation of a current source and sink in the subsurface whereby electrons are transferred between anoxic and oxic environments via a conductive biofilm and/or biominerals, creating a biogeobattery. To investigate the conditions required for biogeobattery formation, we successfully created contrasting redox zones in a flow-through column setup. In this setup, an oxic section, containing clean sand, transitioned into an Fe(III)-reducing section. Fe(III) reduction was mediated by either a natural microbial community or a pure culture of the model organism Shewanella oneidensis MR-1 in two different column experiments. Visual observations and electron microscopy showed that ferrihydrite was sequentially transformed to goethite and magnetite; despite this change, no SP signal was generated in either column. Electron microscopy suggested that in the pure culture column, S. oneidensis MR-1 cells did not form a continuous, interconnected biofilm but rather interacted with the iron (oxyhydr)oxide surfaces as individual cells. In our experiments we therefore did not form the conductor of the biogeobattery. We thus conclude that generation of a biogeobattery is nontrivial and requires specific geochemical and microbiological conditions that will not occur at every contaminated site undergoing microbially mediated redox processes. This conclusion suggests that SP cannot be used in isolation to monitor subsurface biogeochemical conditions.

  17. Physiological and transcriptional approaches reveal connection between nitrogen and manganese cycles in Shewanella algae C6G3

    PubMed Central

    Aigle, Axel; Bonin, Patricia; Iobbi-Nivol, Chantal; Méjean, Vincent; Michotey, Valérie

    2017-01-01

    To explain anaerobic nitrite/nitrate production at the expense of ammonium mediated by manganese oxide (Mn(IV)) in sediment, nitrate and manganese respirations were investigated in a strain (Shewanella algae C6G3) presenting these features. In contrast to S. oneidensis MR-1, a biotic transitory nitrite accumulation at the expense of ammonium was observed in S. algae during anaerobic growth with Mn(IV) under condition of limiting electron acceptor, concomitantly, with a higher electron donor stoichiometry than expected. This low and reproducible transitory accumulation is the result of production and consumption since the strain is able to dissimilative reduce nitrate into ammonium. Nitrite production in Mn(IV) condition is strengthened by comparative expression of the nitrate/nitrite reductase genes (napA, nrfA, nrfA-2), and rates of the nitrate/nitrite reductase activities under Mn(IV), nitrate or fumarate conditions. Compared with S. oneidensis MR-1, S. algae contains additional genes that encode nitrate and nitrite reductases (napA-α and nrfA-2) and an Outer Membrane Cytochrome (OMC)(mtrH). Different patterns of expression of the OMC genes (omcA, mtrF, mtrH and mtrC) were observed depending on the electron acceptor and growth phase. Only gene mtrF-2 (SO1659 homolog) was specifically expressed under the Mn(IV) condition. Nitrate and Mn(IV) respirations seem connected at the physiological and transcriptional levels. PMID:28317859

  18. Comparative Genomics Analysis and Phenotypic Characterization of Shewanella putrefaciens W3-18-1: Anaerobic Respiration, Bacterial Microcompartments, and Lateral Flagella

    SciTech Connect

    Qiu, D.; Tu, Q.; He, Zhili; Zhou, Jizhong

    2010-05-17

    Respiratory versatility and psychrophily are the hallmarks of Shewanella. The ability to utilize a wide range of electron acceptors for respiration is due to the large number of c-type cytochrome genes present in the genome of Shewanella strains. More recently the dissimilatory metal reduction of Shewanella species has been extensively and intensively studied for potential applications in the bioremediation of radioactive wastes of groundwater and subsurface environments. Multiple Shewanella genome sequences are now available in the public databases (Fredrickson et al., 2008). Most of the sequenced Shewanella strains were isolated from marine environments and this genus was believed to be of marine origin (Hau and Gralnick, 2007). However, the well-characterized model strain, S. oneidensis MR-1, was isolated from the freshwater lake sediment of Lake Oneida, New York (Myers and Nealson, 1988) and similar bacteria have also been isolated from other freshwater environments (Venkateswaran et al., 1999). Here we comparatively analyzed the genome sequence and physiological characteristics of S. putrefaciens W3-18-1 and S. oneidensis MR-1, isolated from the marine and freshwater lake sediments, respectively. The anaerobic respirations, carbon source utilization, and cell motility have been experimentally investigated. Large scale horizontal gene transfers have been revealed and the genetic divergence between these two strains was considered to be critical to the bacterial adaptation to specific habitats, freshwater or marine sediments.

  19. Microbial mediated iron redox cycling in Fe (hydr)oxides for nitrite removal.

    PubMed

    Lu, Yongsheng; Xu, Lu; Shu, Weikang; Zhou, Jizhi; Chen, Xueping; Xu, Yunfeng; Qian, Guangren

    2017-01-01

    Nitrite, at an environmentally relevant concentration, was significantly reduced with iron (hydr)oxides mediated by Shewanella oneidensis MR-1. The average nitrite removal rates of 1.28±0.08 and 0.65±0.02(mgL(-1))h(-1) were achieved with ferrihydrite and magnetite, respectively. The results showed that nitrite removal was able to undergo multiple redox cycles with iron (hydr)oxides mediated by Shewanella oneidensis MR-1. During the bioreduction of the following cycles, biogenic Fe(II) was subsequently chemically oxidized to Fe(III), which is associated with nitrite reduction. There was 11.18±1.26mgL(-1) of NH4(+)-N generated in the process of redox cycling of ferrihydrite. Additionally, results obtained by using X-ray diffraction showed that ferrihydrite and magnetite remained mainly stable in the system. This study indicated that redox cycling of Fe in iron (hydr)oxides was a potential process associated with NO2(-)-N removal from solution, and reduced most nitrite abiotically to gaseous nitrogen species.

  20. Physiological and transcriptional approaches reveal connection between nitrogen and manganese cycles in Shewanella algae C6G3

    NASA Astrophysics Data System (ADS)

    Aigle, Axel; Bonin, Patricia; Iobbi-Nivol, Chantal; Méjean, Vincent; Michotey, Valérie

    2017-03-01

    To explain anaerobic nitrite/nitrate production at the expense of ammonium mediated by manganese oxide (Mn(IV)) in sediment, nitrate and manganese respirations were investigated in a strain (Shewanella algae C6G3) presenting these features. In contrast to S. oneidensis MR-1, a biotic transitory nitrite accumulation at the expense of ammonium was observed in S. algae during anaerobic growth with Mn(IV) under condition of limiting electron acceptor, concomitantly, with a higher electron donor stoichiometry than expected. This low and reproducible transitory accumulation is the result of production and consumption since the strain is able to dissimilative reduce nitrate into ammonium. Nitrite production in Mn(IV) condition is strengthened by comparative expression of the nitrate/nitrite reductase genes (napA, nrfA, nrfA-2), and rates of the nitrate/nitrite reductase activities under Mn(IV), nitrate or fumarate conditions. Compared with S. oneidensis MR-1, S. algae contains additional genes that encode nitrate and nitrite reductases (napA-α and nrfA-2) and an Outer Membrane Cytochrome (OMC)(mtrH). Different patterns of expression of the OMC genes (omcA, mtrF, mtrH and mtrC) were observed depending on the electron acceptor and growth phase. Only gene mtrF-2 (SO1659 homolog) was specifically expressed under the Mn(IV) condition. Nitrate and Mn(IV) respirations seem connected at the physiological and transcriptional levels.

  1. A Thermodynamic Study of Heavy Metal Adsorption at the Biofilm/Mineral Interface: Comparison Between Thermodynamic Model Results and In-situ Measurements

    NASA Astrophysics Data System (ADS)

    Gelabert, A.; Ha, J.; Wang, Y.; Ona-Nguema, G.; Cordova-Ardy, C.; Gescher, J.; Bargar, J. R.; Rogers, J.; Eng, P. J.; Ghose, S. K.; Farges, F.; Spormann, A. M.; Brown, G. E.

    2007-12-01

    Fe- and Al-(oxyhydr)oxides are among the most reactive mineral surfaces in water-rock systems. Bacteria are effective adsorbents of metal ions and can form colonies on the surfaces of minerals. The resulting biofilm coatings may create local microenvironments that could change significantly trace metal ion sorption compared to biofilm-free mineral surfaces. In this study, ATR-FTIR spectroscopy were used to identify the different types of metal binding sites in Shewanella oneidensis strain MR-1 (wild type) as well as the nature of the functional groups involved in metal complexation. Adsorption affinities of these sites for Pb(II) and Zn(II) as well as site densities were determined by fitting metal adsorption data as a function of pH and ionic strength using a constant capacitance model in the FITEQL computer code. Electrokinetic techniques were used to determine the biofilm influence on the overall mineral surface charge. This influence was quantified by determining the in-situ partitioning of Pb(II) between S. oneidensis MR-1 biofilms and highly polished and oriented single crystal surfaces of α-alumina (1-102) and hematite (0001) using the long-period X-ray standing wave-fluorescent yield (XSW-FY) method. ATR-FTIR spectra of S. oneidensis strain MR-1 revealed the presence of carboxyl, amide, and phosphate groups, as well as carbohydrate moieties. Electrophoretic mobility measurements of S. oneidensis MR-1 cell surfaces indicate that the bacterial surfaces become negatively charged at pH ~ 3.5, suggesting a high Pb(II) and Zn(II) adsorption capacity of the biofilm. Changes in ionic strengths from 1M to 0.01M NaNO3 had no effect on the interactions of the metal ions with the bacteria, suggesting that the physical structure of the cell wall does not change over this range of electrolyte concentrations. The thermodynamic stability of metal complexes on S. oneidensis and the binding sites concentrations for these metal complexes have been determined by FITEQL

  2. The Shewanella Federation: Functional Genomic Investigations of Dissimilatory Metal-Reducing Shewanella

    SciTech Connect

    Zhou, Jizhong; He, Zhili

    2009-01-30

    Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display. An ORF clone set for S. oneidensis was created using the lambda recombinase system. ORFs within entry vectors in this system can be readily transferred into multiple destination vectors, making the clone set a useful resource for research groups studying this microorganism. To establish that the S. oneidensis clone set could be used for protein expression and functional studies, three sets of ORFs were examined for expression of His-tag proteins, expression of His/GST-tag proteins, or for effective display on phage. A total of 21 out of 30 (70%) predicted two-component transcriptional regulators from S. oneidensis were successfully expressed in the His-tag format. The use of the S. oneidensis clone set for functional studies was tested using a phage display system. The method involves the fusion of peptides or proteins to a coat protein of a bacteriophage. This results in display of the fused protein on the exterior of the phage, while the DNA encoding the fusion resides within the virion. The physical linkage between the displayed protein and the DNA encoding it allows screening of vast numbers of proteins for ligand-binding properties. With this technology, a phage clone encoding thioredoxin TrxA was isolated from a sub-library consisting of 80 clones. It is evident that the S. oneidensis clone set can be used for expression of functional S. oneidensis proteins in E. coli using the appropriate destination vectors. Characterization of ArcA. In Escherichia coli, metabolic transitions between aerobic and anaerobic growth states occur when cells enter an oxygen-limited condition. Many of these metabolic transitions are controlled at the transcriptional level by the activities of the global regulatory proteins ArcA (aerobic respiration control) and Fnr (fumarate nitrate regulator). A homolog of ArcA (81% amino acid sequence identity) was identified in S

  3. Photoenhanced electrochemical interaction between Shewanella and a hematite nanowire photoanode.

    PubMed

    Qian, Fang; Wang, Hanyu; Ling, Yichuan; Wang, Gongming; Thelen, Michael P; Li, Yat

    2014-06-11

    Here we report the investigation of interplay between light, a hematite nanowire-arrayed photoelectrode, and Shewanella oneidensis MR-1 in a solar-assisted microbial photoelectrochemical system (solar MPS). Whole cell electrochemistry and microbial fuel cell (MFC) characterization of Shewanella oneidensis strain MR-1 showed that these cells cultured under (semi)anaerobic conditions expressed substantial c-type cytochrome outer membrane proteins, exhibited well-defined redox peaks, and generated bioelectricity in a MFC device. Cyclic voltammogram studies of hematite nanowire electrodes revealed active electron transfer at the hematite/cell interface. Notably, under a positive bias and light illumination, the hematite electrode immersed in a live cell culture was able to produce 150% more photocurrent than that in the abiotic control of medium or dead culture, suggesting a photoenhanced electrochemical interaction between hematite and Shewanella. The enhanced photocurrent was attributed to the additional redox species associated with MR-1 cells that are more thermodynamically favorable to be oxidized than water. Long-term operation of the hematite solar MPS with light on/off cycles showed stable current generation up to 2 weeks. Fluorescent optical microscope and scanning electron microscope imaging revealed that the top of the hematite nanowire arrays were covered by a biofilm, and iron determination colorimetric assay revealed 11% iron loss after a 10-day operation. To our knowledge, this is the first report on interfacing a photoanode directly with electricigens in a MFC system. Such a system could open up new possibilities in solar-microbial device that can harvest solar energy and recycle biomass simultaneously to treat wastewater, produce electricity, and chemical fuels in a self-sustained manner.

  4. Electrochemically Active Soluble Mediators from Shewanella oneidensis: Relevance to Microbial Fuel Cells and Extracellular Electron Transfer

    DTIC Science & Technology

    2008-05-01

    A second approach is the use of soluble mediators such as, quinones, phenazines , and riboflavin, which are able to shuttle electrons from the cell...done using the equivalent graphite felt or graphite felt coated with platinum nanoparticles . Fuel cell chambers were separated using a gas-permeable

  5. Standardized Microbial Fuel Cell Anodes of Silica-Immobilized Shewanella oneidensis

    DTIC Science & Technology

    2010-03-01

    The association between bacteria and electrodes, however, is inconsistent due to inherent variations in bacterial growth due to changes in...Fredrickson, J. Microbiol. Methods, 2008, 74, 47–56; H. Yi, K. P. Nevin, B.-C. Kim, A. E. Franks, A. Klimes , L. M. Tender and D. R. Lovley, Biosens...Biosens. Bioelectron., 2008, 23, 820–826. 12 Y. A. Gorby, S. Yanina, J. S. McLean, K. M. Rosso, D. Moyles, A. Dohnalkova, T. J. Beveridge, I. S. Chang

  6. Arsenic biotransformation in solid waste residue: comparison of contributions from bacteria with arsenate and iron reducing pathways.

    PubMed

    Tian, Haixia; Shi, Qiantao; Jing, Chuanyong

    2015-02-17

    Arsenic- and iron-reducing bacteria play an important role in regulating As redox transformation and mobility. The motivation of this study was to compare the contributions of different As- and Fe-reducing bacteria to As biotransformation. In this work, three bacteria strains with different functional genes were employed including Pantoea sp. IMH with the arsC gene, Alkaliphilus oremlandii OhILAs possessing the arrA gene, and Shewanella oneidensis MR-1, an iron reducer. The incubation results showed that Pantoea sp. IMH aerobically reduced 100% of As(V) released from waste residues, though total As release was not enhanced. Similarly, strain OhILAs anaerobically reduced dissolved As(V) but could not enhance As release. In contrast, strain MR-1 substantially enhanced As mobilization because of iron reduction, but without changing the As speciation. The formation of the secondary iron mineral pyrite in the MR-1 incubation experiments, as evidenced by the X-ray absorption near-edge spectroscopy (XANES) analysis, contributed little to the uptake of the freed As. Our results suggest that the arsC gene carriers mainly control the As speciation in the aqueous phase in aerobic environments, whereas in anaerobic conditions, the As speciation should be regulated by arrA gene carriers, and As mobility is greatly enhanced by iron reduction.

  7. Bion M1. Peculiarities of life activities of microbes in 30-day spaceflight

    NASA Astrophysics Data System (ADS)

    Viacheslav, Ilyin; Korshunov, Denis; Morozova, Julia; Voeikova, Tatiana; Tyaglov, Boris; Novikova, Liudmila; Krestyanova, Irina; Emelyanova, Lydia

    The aim of this work was to analyze the influence of space flight factors ( SFF) to microorganism strains , exposed inside unmanned spacecraft Bion M-1 during the 30- day space flight. Objectives of the work - the study of the influence of the SFF exchange chromosomal DNA in crosses microorganisms of the genus Streptomyces; the level of spontaneous phage induction of lysogenic strains fS31 from Streptomyces lividans 66 and Streptomyces coelicolor A3 ( 2 ) on the biosynthesis of the antibiotic tylosin strain of Streptomyces fradiae; survival electrogenic bacteria Shewanella oneidensis MR- 1 is used in the microbial fuel cell As a result of this work it was found that the SFF affect the exchange of chromosomal DNA by crossing strains of Streptomyces. Was detected polarity crossing , expressed in an advantageous contribution chromosome fragment of one of the parent strains in recombinant offspring. This fact may indicate a more prolonged exposure of cells in microgravity and , as a consequence, the transfer of longer fragments of chromosomal DNA This feature is the transfer of genetic material in microgravity could lead to wider dissemination and horizontal transfer of chromosomal and plasmid DNA of symbiotic microflora astronauts and other strains present in the spacecraft. It was shown no effect on the frequency of recombination PCF and the level of mutation model reversion of auxotrophic markers to prototrophy It was demonstrated that PCF increase the level of induction of cell actinophage fS31 lysogenic strain of S. lividans 66, but did not affect the level of induction of this phage cells S. coelicolor A3 ( 2). It is shown that the lower the level of synthesis PCF antibiotic aktinorodina (actinorhodin) in lysogenic strain S. coelicolor A3 ( 2). 66 Strains of S. lividans and S. coelicolor A3 ( 2 ) can be used as a biosensor for studying the effect on microorganisms PCF It is shown that the effect of the PCF reduces synthesis of tylosin and desmicosyn S. fradiae at

  8. Integrated genome-based studies of Shewanella ecophysiology

    SciTech Connect

    Segre Daniel; Beg Qasim

    2012-02-14

    This project was a component of the Shewanella Federation and, as such, contributed to the overall goal of applying the genomic tools to better understand eco-physiology and speciation of respiratory-versatile members of Shewanella genus. Our role at Boston University was to perform bioreactor and high throughput gene expression microarrays, and combine dynamic flux balance modeling with experimentally obtained transcriptional and gene expression datasets from different growth conditions. In the first part of project, we designed the S. oneidensis microarray probes for Affymetrix Inc. (based in California), then we identified the pathways of carbon utilization in the metal-reducing marine bacterium Shewanella oneidensis MR-1, using our newly designed high-density oligonucleotide Affymetrix microarray on Shewanella cells grown with various carbon sources. Next, using a combination of experimental and computational approaches, we built algorithm and methods to integrate the transcriptional and metabolic regulatory networks of S. oneidensis. Specifically, we combined mRNA microarray and metabolite measurements with statistical inference and dynamic flux balance analysis (dFBA) to study the transcriptional response of S. oneidensis MR-1 as it passes through exponential, stationary, and transition phases. By measuring time-dependent mRNA expression levels during batch growth of S. oneidensis MR-1 under two radically different nutrient compositions (minimal lactate and nutritionally rich LB medium), we obtain detailed snapshots of the regulatory strategies used by this bacterium to cope with gradually changing nutrient availability. In addition to traditional clustering, which provides a first indication of major regulatory trends and transcription factors activities, we developed and implemented a new computational approach for Dynamic Detection of Transcriptional Triggers (D2T2). This new method allows us to infer a putative topology of transcriptional dependencies

  9. Aggregation Kinetics of Hematite Particles in the Presence of Outer Membrane Cytochrome OmcA of Shewanella oneidenesis MR-1

    SciTech Connect

    Sheng, Anxu; Liu, Feng; Shi, Liang; Liu, Juan

    2016-09-20

    The aggregation behavior of 9, 36, and 112 nm hematite particles was studied in the presence of OmcA, a bacterial extracellular protein, in aqueous dispersions at pH 5.7 through time-resolved dynamic light scattering, electrophoretic mobility, and circular dichroism spectra, respectively. At low salt concentration, the attachment efficiencies of hematite particles in all sizes first increased, then decreased, and finally remained stable with the increase of OmcA concentration, indicating the dominant interparticle interaction changed along with the increase in the protein-to-particle ratio. Nevertheless, at high salt concentration, the attachment efficiencies of all hematite samples gradually decreased with increasing OmcA concentration, which can be attributed to increasing steric force. Additionally, the aggregation behavior of OmcA-hematite conjugates was more correlated to total particle-surface area than primary particle size. It was further established that OmcA could stabilize hematite nanoparticles more efficiently than bovine serum albumin (BSA), a model plasma protein, due to the higher affinity of OmcA to hematite surface. This study highlighted the effects of particle properties, solution conditions, and protein properties on the complicated aggregation behavior of protein-nanoparticle conjugates in aqueous environments.

  10. 78 FR 33406 - Clean River Power MR-1, LLC, Clean River Power MR-2, LLC, Clean River Power MR-3, LLC, Clean...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-04

    ... Clean River Power MR-7, LLC (Clean River Power), subsidiaries of Free Flow Power Corporation. e. Names.... Applicant Contacts: Ramya Swaminathan, Chief Operating Officer, Free Flow Power Corporation, 239 Causeway Street, Suite 300, Boston, MA 02114; or at (978) 283-2822. Daniel Lissner, General Counsel, Free...

  11. 77 FR 68757 - Clean River Power MR-1, LLC; Clean River Power MR-2, LLC; Clean River Power MR-3, LLC; Clean...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-16

    ... analysis at this time. n. The proposed Beverly Lock and Dam Water Power Project would be located on the... 17,853 megawatt-hours (MWh). The proposed Devola Lock and Dam Water Power Project would be located at... Dam Water Power Project would be located at RM 13.6, and consist of the following new facilities:...

  12. Identification and analysis of a highly conserved chemotaxis gene cluster in Shewanella species.

    SciTech Connect

    Li, J.; Romine, Margaret F.; Ward, M.

    2007-08-01

    A conserved cluster of chemotaxis genes was identified from the genome sequences of fifteen Shewanella species. An in-frame deletion of the cheA-3 gene, which is located in this cluster, was created in S. oneidensis MR-1 and the gene shown to be essential for chemotactic responses to anaerobic electron acceptors. The CheA-3 protein showed strong similarity to Vibrio cholerae CheA-2 and P. aeruginosa CheA-1, two proteins that are also essential for chemotaxis. The genes encoding these proteins were shown to be located in chemotaxis gene clusters closely related to the cheA-3-containing cluster in Shewanella species. The results of this study suggest that a combination of gene neighborhood and homology analyses may be used to predict which cheA genes are essential for chemotaxis in groups of closely related microorganisms.

  13. High Performance Reduction of H2O2 with an Electron Transport Decaheme Cytochrome on a Porous ITO Electrode.

    PubMed

    Reuillard, Bertrand; Ly, Khoa H; Hildebrandt, Peter; Jeuken, Lars J C; Butt, Julea N; Reisner, Erwin

    2017-03-08

    The decaheme cytochrome MtrC from Shewanella oneidensis MR-1 immobilized on an ITO electrode displays unprecedented H2O2 reduction activity. Although MtrC showed lower peroxidase activity in solution compared to horseradish peroxidase, the ten heme cofactors enable excellent electronic communication and a superior activity on the electrode surface. A hierarchical ITO electrode enabled optimal immobilization of MtrC and a high current density of 1 mA cm(-2) at 0.4 V vs SHE could be obtained at pH 6.5 (Eonset = 0.72 V). UV-visible and Resonance Raman spectroelectrochemical studies suggest the formation of a high valent iron-oxo species as the catalytic intermediate. Our findings demonstrate the potential of multiheme cytochromes to catalyze technologically relevant reactions and establish MtrC as a new benchmark in biotechnological H2O2 reduction with scope for applications in fuel cells and biosensors.

  14. Physical constraints on charge transport through bacterial nanowires

    PubMed Central

    Polizzi, Nicholas F.; Skourtis, Spiros S.

    2012-01-01

    Extracellular appendages of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 were recently shown to sustain currents of 1010 electrons per second over distances of 0.5 microns [El-Naggar et al., Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 18127]. However, the identity of the charge localizing sites and their organization along the “nanowire” remain unknown. We use theory to predict redox cofactor separation distances that would permit charge flow at rates of 1010 electrons per second over 0.5 microns for voltage biases of ≤1V, using a steady-state analysis governed by a non-adiabatic electron transport mechanism. We find the observed currents necessitate a multi-step hopping transport mechanism, with charge localizing sites separated by less than 1 nm and reorganization energies that rival the lowest known in biology. PMID:22470966

  15. Mechanism of Fluorescence Enhancement of Biosynthesized XFe2O4-BiFeO3 (X = Cr, Mn, Co, or Ni) Membranes

    NASA Astrophysics Data System (ADS)

    Bian, Liang; Li, Hai-long; Dong, Hai-liang; Dong, Fa-qin; Song, Mian-xin; Wang, Li-sheng; Hou, Wen-ping; Gao, Lei; Zhang, Xiao-yan; Zhou, Tian-liang; Sun, Guang-ai; Li, Xin-xi; Xie, Lei

    2016-12-01

    Ferrites-bismuth ferrite is an intriguing option for medical diagnostic imaging device due to its magnetoelectric and enhanced near-infrared fluorescent properties. However, the embedded XFO nanoparticles are randomly located on the BFO membranes, making implementation in devices difficult. To overcome this, we present a facile bio-approach to produce XFe2O4-BiFeO3 (XFO-BFO) (X = Cr, Mn, Co, or Ni) membranes using Shewanella oneidensis MR-1. The perovskite BFO enhances the fluorescence intensity (at 660 and 832 nm) and surface potential difference (-469 385 meV and -80 525 meV) of the embedded spinel XFO. This mechanism is attributed to the interfacial coupling of the X-Fe (e- or h+) and O-O (h+) interfaces. Such a system could open up new ideas in the design of environmentally friendly fluorescent membranes.

  16. Fluorescent enhancement of bio-synthesized X-Zn-ferrite-bismuth ferrite (X = Mg, Mn or Ni) membranes: Experiment and theory

    NASA Astrophysics Data System (ADS)

    Bian, Liang; Li, Hai-long; Dong, Hai-liang; Dong, Fa-qin; Song, Mian-xin; Wang, Li-sheng; Zhou, Tian-liang; Li, Wei-min; Hou, Wen-ping; Zhang, Xiao-yan; Lu, Xi-rui; Li, Xin-Xi; Xie, Lei

    2017-02-01

    Large areas of high-photostability fluorescent X-Zn-ferrite (XZn)Fe2O4 (X = Mg, Mn or Ni) nanoparticles embedded in a bismuth ferrite (BiFeO3) membrane were successfully fabricated via a facile bio-approach using Shewanella oneidensis MR-1. The results revealed that the perovskite BiFeO3 enhances the fluorescent intensity (at 635 and 795 nm) and surface potential difference (14 meV and -40 meV) of the embedded spinel (XZn)Fe2O4. This mechanism is attributed to the interfacial coupling of the Fesbnd Osbnd Osbnd Bi and Fesbnd Osbnd Fe interfaces. Such a system could open up new ideas in the design of environmentally friendly fluorescent sensors for water environments.

  17. Nontronite (NAu-1) Structure Associated with Microbial Fe(III) Reduction in Various Redox Conditions

    NASA Astrophysics Data System (ADS)

    Koo, T.; Kim, S.; Kim, J.

    2011-12-01

    Shewanella oneidensis MR-1 respires the structural Fe(III) of smectite and promotes illite formation in O2-free environment (Kostka et al., 1996, Kim et al., 2004). Since S. oneidensis is a facultative iron reducing bacterium, it is crucial to understand the structural changes induced by bio-reduction of structural Fe(III) in various redox conditions. Furthermore, the changes in cation exchange capacity (CEC) of bio-reduced nontronite upon the modification of mineral structure has not been extensively studied in terms of Fe-cycling. In this present study, we reported the evolution of nontronite structure at various time points in various redox conditions and corresponding CEC upon reduction and re-oxidation. S. oneidensis MR-1 was incubated in M1 medium with Na-lactate as the electron donor and Fe in nontronite (NAu-1) as the sole electron acceptor at pH 7 in anaerobic chamber for 3 hrs, 12 hrs, 1 day, 2 days, 4 days, 7 days, 14 days, and 21 days. O2 gas bubbling was then applied to the sample at each time point for 24 hours for re-oxidation. The triplet samples at each time point for both reduction and re-oxidation experiments were prepared. The extent of Fe(III) reduction measured by 1,10-phenanthroline method (Stucki and Anderson, 1981) indicated that the structural Fe(III) was reduced up to 8.8% of total Fe(III) within 21 days. XRD data with various treatments such as air dried, glycolated and lithium-saturated showed that K-nontronite may be formed because no discrete 10-Å illite peak was observed in Li-saturated sample upon glycolation. The CEC increased from 747 meg/kg to 1145 meg/kg during Fe(III) reduction and decreased to 954 meg/kg upon re-oxidation, supporting the possible formation of K-nontronite. The direct observation by electron microscopy verified the structural changes in nontonite in various redox conditions. The long-term experiment for 6 months, is in progress in anaerobic chamber, and results will be discussed. Kim, J. W., Dong, H., Seabaugh

  18. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    SciTech Connect

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

    2014-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

  19. Molecular Mechanism of Bacterial Attachment to Fe(III)-Oxide Surfaces

    SciTech Connect

    Neal, Andrew

    2006-06-01

    To date, our studies have concentrated upon two aspects of the chemistry and architecture of the Shewanella outer membrane surface: the influence of terminal electron acceptor during anaerobic respiration, and the role that protein secretion systems play in determining the presence and chemistry of exopolymers. Using Shewanella oneidensis strain MR-1 and S. putrefaciens strain 200R as model organisms we have developed a microelectrophoresis approach to estimating cell mobility in solution over a range of ionic strengths. From this data, and applying Ohshima's soft particle theory, we have successfully estimated the net electrostatic charge and relative thickness of any capsular material of the two model strains under various terminal electron acceptor availabilities. Additionally, by employing state-of-the-art cryo-electron microscopy techniques we have been able to not only confirm presence or absence of capsular material but also visualize the outer cell surface architecture in a completely hydrated state.

  20. Electrode assemblies composed of redox cascades from microbial respiratory electron transfer chains

    SciTech Connect

    Gates, Andrew J.; Marritt, Sophie; Bradley, Justin; Shi, Liang; McMillan, Duncan G.; Jeuken, Lars J.; Richardson, David; Butt, Julea N.

    2013-10-01

    Respiratory and photosynthetic electron transfer chains are dependent on vectorial electron transfer through a series of redox proteins. Examples include electron transfer from NapC to NapAB nitrate reductase in Paracoccus denitrificans and from CymA to Fcc3 (flavocytochrome c3) fumarate reductase in Shewanella oneidensis MR-1. In the present article, we demonstrate that graphite electrodes can serve as surfaces for the stepwise adsorption of NapC and NapAB, and the stepwise adsorption of CymA and Fcc3. Aspects of the catalytic properties of these assemblies are different from those of NapAB and Fcc3 adsorbed in isolation. We propose that this is due to the formation of NapC-NapAB and of CymA-Fcc3 complexes that are capable of supporting vectorial electron transfer.

  1. Physical constraints on charge transport through bacterial nanowires

    SciTech Connect

    Polizzi, Nicholas F.; Skourtis, Spiros S.; Beratan, David N.

    2012-01-01

    Extracellular appendages of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 were recently shown to sustain currents of 10{sup 10} electrons per second over distances of 0.5 microns [El-Naggar et al., Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 18127]. However, the identity of the charge localizing sites and their organization along the “nanowire” remain unknown. We use theory to predict redox cofactor separation distances that would permit charge flow at rates of 10{sup 10} electrons per second over 0.5 microns for voltage biases of ≤1V, using a steady-state analysis governed by a non-adiabatic electron transport mechanism. We find the observed currents necessitate a multi-step hopping transport mechanism, with charge localizing sites separated by less than 1 nm and reorganization energies that rival the lowest known in biology.

  2. Physical constraints on charge transport through bacterial nanowires

    SciTech Connect

    Polizzi, Nicholas F.; Skourtis, Spiros S.; Beratan, David N.

    2011-10-17

    Extracellular appendages of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 were recently shown to sustain currents of 10¹⁰ electrons per second over distances of 0.5 microns [El-Naggar et al., Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 18127]. However, the identity of the charge localizing sites and their organization along the “nanowire” remain unknown. We use theory to predict redox cofactor separation distances that would permit charge flow at rates of 10¹⁰ electrons per second over 0.5 microns for voltage biases of ≤1V, using a steady-state analysis governed by a non-adiabatic electron transport mechanism. We find the observed currents necessitate a multi-step hopping transport mechanism, with charge localizing sites separated by less than 1 nm and reorganization energies that rival the lowest known in biology.

  3. Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus

    PubMed Central

    2011-01-01

    Background Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in bacteria is one of the critical tasks of modern genomics. The Shewanella genus is comprised of metabolically versatile gamma-proteobacteria, whose lifestyles and natural environments are substantially different from Escherichia coli and other model bacterial species. The comparative genomics approaches and computational identification of regulatory sites are useful for the in silico reconstruction of transcriptional regulatory networks in bacteria. Results To explore conservation and variations in the Shewanella transcriptional networks we analyzed the repertoire of transcription factors and performed genomics-based reconstruction and comparative analysis of regulons in 16 Shewanella genomes. The inferred regulatory network includes 82 transcription factors and their DNA binding sites, 8 riboswitches and 6 translational attenuators. Forty five regulons were newly inferred from the genome context analysis, whereas others were propagated from previously characterized regulons in the Enterobacteria and Pseudomonas spp.. Multiple variations in regulatory strategies between the Shewanella spp. and E. coli include regulon contraction and expansion (as in the case of PdhR, HexR, FadR), numerous cases of recruiting non-orthologous regulators to control equivalent pathways (e.g. PsrA for fatty acid degradation) and, conversely, orthologous regulators to control distinct pathways (e.g. TyrR, ArgR, Crp). Conclusions We tentatively defined the first reference collection of ~100 transcriptional regulons in 16 Shewanella genomes. The resulting regulatory network contains ~600 regulated genes per genome that are mostly involved in metabolism of carbohydrates, amino acids, fatty acids, vitamins, metals, and stress responses. Several reconstructed regulons including NagR for N-acetylglucosamine catabolism were experimentally validated in S. oneidensis MR-1. Analysis of

  4. Conservation of Transcription Start Sites within Genes across a Bacterial Genus

    SciTech Connect

    Shao, Wenjun; Price, Morgan N.; Deutschbauer, Adam M.; Romine, Margaret F.; Arkin, Adam P.

    2014-07-01

    Transcription start sites (TSSs) lying inside annotated genes, on the same or opposite strand, have been observed in diverse bacteria, but the function of these unexpected transcripts is unclear. Here, we use the metal-reducing bacterium Shewanella oneidensis MR-1 and its relatives to study the evolutionary conservation of unexpected TSSs. Using high-resolution tiling microarrays and 5'-end RNA sequencing, we identified 2,531 TSSs in S. oneidensis MR-1, of which 18% were located inside coding sequences (CDSs). Comparative transcriptome analysis with seven additional Shewanella species revealed that the majority (76%) of the TSSs within the upstream regions of annotated genes (gTSSs) were conserved. Thirty percent of the TSSs that were inside genes and on the sense strand (iTSSs) were also conserved. Sequence analysis around these iTSSs showed conserved promoter motifs, suggesting that many iTSS are under purifying selection. Furthermore, conserved iTSSs are enriched for regulatory motifs, suggesting that they are regulated, and they tend to eliminate polar effects, which confirms that they are functional. In contrast, the transcription of antisense TSSs located inside CDSs (aTSSs) was significantly less likely to be conserved (22%). However, aTSSs whose transcription was conserved often have conserved promoter motifs and drive the expression of nearby genes. Overall, our findings demonstrate that some internal TSSs are conserved and drive protein expression despite their unusual locations, but the majority are not conserved and may reflect noisy initiation of transcription rather than a biological function.

  5. Solid State Electron Transfer via Bacterial Nanowires: Contributions Toward a Mechanistic Understanding of Geophysical Response of Biostimulated Subsurface

    SciTech Connect

    Estella Atekwana

    2012-05-08

    The degradation of organic matter by microorganisms provides a source of electrical potential or so-called 'self potential' (SP) that can be measured by using a voltmeter. During this process electrons are being produced as a waste-product and bacterial cells have to dispose of these to allow for the complete biodegradation of organic matter. Especially in anaerobic microbial communities, exo-cellular electron transfer is the most important driving force behind this process and organisms have developed different, but also similar, ways to transfer electrons to other microorganisms. Recently, it has been postulated that direct electron transfer from cell-to-cell is actually done by 'hard-wired' microorganisms. This shuttling of electrons is most likely done by certain c-type cytochromes that form the functional part of electrically conductive nanowires. In this study we investigated if nanowires can explain the geoelectrical (self potential and spectral induced polarization) signals observed at some biostimulated environments such as DOE sites. The objectives of our project are to: (1) investigate any temporal changes in the geophysical signatures (Self Potential (SP) and Induced Polarization (IP)) associated with nanowires of the bacterium Shewanella oneidensis MR-1, wild type and mtrc/omcA deletion mutant, (2) demonstrate that mutant strains of bacteria that produce nonconductive nanowires do not contribute to geoelectrical responses. We accomplished the following: (1) Provided training to students and a postdoctoral fellow that worked on the project, (2) Conducted several SP & IP measurements correlating the distribution of nanowires and SIP/SP signals in partial fulfillment of object No. 1 and 2. On the following we will report and discuss the results of our last experiment with some emphasis on the source mechanisms of both SP and IP associated with Shewanella oneidensis MR-1, wild type in sand columns.

  6. Informatics Strategies for Large-Scale Novel Cross-Linking Analysis

    PubMed Central

    Anderson, Gordon A.; Tolic, Nikola; Tang, Xiaoting; Zheng, Chunxiang; Bruce, James E.

    2008-01-01

    The detection of protein interactions in biological systems represents a significant challenge for today’s technology. Chemical cross-linking provides the potential to impart new chemical bonds in a complex system that result in mass changes in a set of tryptic peptides detected by mass spectrometry. However, system complexity and cross-linking product heterogeneity have precluded widespread chemical cross-linking use for large-scale identification of protein–protein interactions. The development of mass spectrometry identifiable cross-linkers called protein interaction reporters (PIRs) has enabled on-cell chemical cross-linking experiments with product type differentiation. However, the complex datasets resultant from PIR experiments demand new informatics capabilities to allow interpretation. This manuscript details our efforts to develop such capabilities and describes the program X-links, which allows PIR product type differentiation. Furthermore, we also present the results from Monte Carlo simulation of PIR-type experiments to provide false discovery rate estimates for the PIR product type identification through observed precursor and released peptide masses. Our simulations also provide peptide identification calculations based on accurate masses and database complexity that can provide an estimation of false discovery rates for peptide identification. Overall, the calculations show a low rate of false discovery of PIR product types due to random mass matching of approximately 12% with 10 ppm mass measurement accuracy and spectral complexity resulting from 100 peptides. In addition, consideration of a reduced database resulting from stage 1 analysis of Shewanella oneidensis MR-1 containing 367 proteins resulted in a significant reduction of expected identification false discovery rate estimation compared to that from the entire Shewanella oneidensis MR-1 proteome. PMID:17676784

  7. Biofilm formation on a TiO2 nanotube with controlled pore diameter and surface wettability

    NASA Astrophysics Data System (ADS)

    Anitha, V. C.; Lee, Jin-Hyung; Lee, Jintae; Narayan Banerjee, Arghya; Joo, Sang Woo; Min, Bong Ki

    2015-02-01

    Titania (TiO2) nanotube arrays (TNAs) with different pore diameters (140 - 20 nm) are fabricated via anodization using hydrofluoric acid (HF) containing ethylene glycol (EG) by changing the HF-to-EG volume ratio and the anodization voltage. To evaluate the effects of different pore diameters of TiO2 nanotubes on bacterial biofilm formation, Shewanella oneidensis (S. oneidensis) MR-1 cells and a crystal-violet biofilm assay are used. The surface roughness and wettability of the TNA surfaces as a function of pore diameter, measured via the contact angle and AFM techniques, are correlated with the controlled biofilm formation. Biofilm formation increases with the decreasing nanotube pore diameter, and a 20 nm TiO2 nanotube shows the maximum biofilm formation. The measurements revealed that 20 nm surfaces have the least hydrophilicity with the highest surface roughness of ˜17 nm and that they show almost a 90% increase in the effective surface area relative to the 140 nm TNAs, which stimulate the cells more effectively to produce the pili to attach to the surface for more biofilm formation. The results demonstrate that bacterial cell adhesion (and hence, biofilm formation) can effectively be controlled by tuning the roughness and wettability of TNAs via controlling the pore diameters of TNA surfaces. This biofilm formation as a function of the surface properties of TNAs can be a potential candidate for both medical applications and as electrodes in microbial fuel cells.

  8. Enhancement of survival and electricity production in an engineered bacterium by light-driven proton pumping.

    PubMed

    Johnson, Ethan T; Baron, Daniel B; Naranjo, Belén; Bond, Daniel R; Schmidt-Dannert, Claudia; Gralnick, Jeffrey A

    2010-07-01

    Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane potential and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependent proton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probe microbial physiology and to engineer microbes with new light-driven properties. Here, we describe functional expression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanella oneidensis strain MR-1. We report that there were significant increases in electrical current generation during illumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidence that an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistent with the hypothesis that proteorhodopsin activity enhances lactate uptake by increasing the proton motive force. Our results demonstrate that there is coupling of a light-driven process to electricity generation in a nonphotosynthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium under nutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain the widespread distribution of proteorhodopsin in marine environments.

  9. Hydrogen production using single-chamber membrane-free microbial electrolysis cells.

    PubMed

    Hu, Hongqiang; Fan, Yanzhen; Liu, Hong

    2008-09-01

    Microbial electrohydrogenesis provides a new approach for hydrogen generation from renewable biomass. Membranes were used in all the reported microbial electrolysis cells (MECs) to separate the anode and cathode chambers. To reduce the potential losses associated with membrane and increase the energy recovery of this process, single-chamber membrane-free MECs were designed and used to investigate hydrogen production by one mixed culture and one pure culture: Shewanella oneidensis MR-1. At an applied voltage of 0.6 V, this system with a mixed culture achieved a hydrogen production rate of 0.53 m(3)/day/m(3) (0.11 m(3)/day/m(2)) with a current density of 9.3A/m(2) at pH 7 and 0.69 m(3)/day/m(3) (0.15m(3)/day/m(2)) with a current density of 14 A/m(2) at pH 5.8. Stable hydrogen production from lactic acid by S. oneidensis was also observed. Methane was detected during the hydrogen production process with the mixed culture and negatively affected hydrogen production rate. However, by employing suitable approaches, such as exposure of cathodes to air, the hydrogenotrophic methanogens can be suppressed. The current density and volumetric hydrogen production rate of this system have potential to increase significantly by further reducing the electrode spacing and increasing the ratio of electrode surface area/cell volume.

  10. A miniature microbial fuel cell with conducting nanofibers-based 3D porous biofilm

    NASA Astrophysics Data System (ADS)

    Jiang, Huawei; Halverson, Larry J.; Dong, Liang

    2015-12-01

    Miniature microbial fuel cell (MFC) technology has received growing interest due to its potential applications in high-throughput screening of bacteria and mutants to elucidate mechanisms of electricity generation. This paper reports a novel miniature MFC with an improved output power density and short startup time, utilizing electrospun conducting poly(3,4-ethylenedioxythiophene) (PEDOT) nanofibers as a 3D porous anode within a 12 μl anolyte chamber. This device results in 423 μW cm-3 power density based on the volume of the anolyte chamber, using Shewanella oneidensis MR-1 as a model biocatalyst without any optimization of bacterial culture. The device also excels in a startup time of only 1hr. The high conductivity of the electrospun nanofibers makes them suitable for efficient electron transfer. The mean pore size of the conducting nanofibers is several micrometers, which is favorable for bacterial penetration and colonization of surfaces of the nanofibers. We demonstrate that S. oneidensis can fully colonize the interior region of this nanofibers-based porous anode. This work represents a new attempt to explore the use of electrospun PEDOT nanofibers as a 3D anode material for MFCs. The presented miniature MFC potentially will provide a high-sensitivity, high-throughput tool to screen suitable bacterial species and mutant strains for use in large-size MFCs.

  11. In Situ Analysis of a Silver Nanoparticle-Precipitating Shewanella Biofilm by Surface Enhanced Confocal Raman Microscopy

    PubMed Central

    Schkolnik, Gal; Schmidt, Matthias; Mazza, Marco G.; Harnisch, Falk; Musat, Niculina

    2015-01-01

    Shewanella oneidensis MR-1 is an electroactive bacterium, capable of reducing extracellular insoluble electron acceptors, making it important for both nutrient cycling in nature and microbial electrochemical technologies, such as microbial fuel cells and microbial electrosynthesis. When allowed to anaerobically colonize an Ag/AgCl solid interface, S. oneidensis has precipitated silver nanoparticles (AgNp), thus providing the means for a surface enhanced confocal Raman microscopy (SECRaM) investigation of its biofilm. The result is the in-situ chemical mapping of the biofilm as it developed over time, where the distribution of cytochromes, reduced and oxidized flavins, polysaccharides and phosphate in the undisturbed biofilm is monitored. Utilizing AgNp bio-produced by the bacteria colonizing the Ag/AgCl interface, we could perform SECRaM while avoiding the use of a patterned or roughened support or the introduction of noble metal salts and reducing agents. This new method will allow a spatially and temporally resolved chemical investigation not only of Shewanella biofilms at an insoluble electron acceptor, but also of other noble metal nanoparticle-precipitating bacteria in laboratory cultures or in complex microbial communities in their natural habitats. PMID:26709923

  12. Electrokinesis is a microbial behavior that requires extracellular electron transport

    SciTech Connect

    Harris, Howard W.; El-Naggar, Mohamed Y.; Bretschger, Orianna; Ward, Melissa J.; Romine, Margaret F.; Obraztsova, Anna; Nealson, Kenneth H.

    2010-01-05

    Shewanella species are widespread in nature, enjoying a cosmopolitan distribution in marine,freshwater, sedimentary and soil environments (1), and have attracted considerable attention in recent years because of their ability to reduce an extensive number of different electron 3 acceptors, including the solid (oxy)hydroxides of iron and manganese, such as Fe(OH)3 and MnO2, using one or more proposed mechanisms of extracellular electron transport (EET) (2, 3). The EET ability of Shewanella species is consistent with their ability to generate electric current in microbial fuel cells in the absence of exogenous electron shuttles (4). Various strategies of extracellular electron transfer have been proposed in metal-reducing microbes, including naturally-occurring (2) or biogenic (5-7) soluble mediators that ‘shuttle’ electrons from cells to acceptors, as well as direct transfer using multiheme cytochromes located on the cell exterior (8) and transfer via conductive nanowires (9-11). S. oneidensis MR-1 features several proteins that are involved with the transport of electrons to the exterior of the cell where they play an important role with regard to the reduction of solid electron acceptors such as metal oxides. These include two outer-membrane decaheme c-type cytochromes (MtrC and OmcA), a membrane spanning protein (MtrB), and two periplasmic multi-heme c-type cytochromes (MtrA and CymA). Deletion of the genes encoding any of these proteins leads to phenotypes that are greatly inhibited with regard to metal-oxide reduction and current production in microbial fuel cells (MFCs) (12, 13). The mutation of genes that code for proteins involved in the movement of cytochromes to the outer membrane also results in loss of metal-reducing phenotypes (13). The shewanellae are highly motile, by virtue of a single polar flagellum, and individual S. oneidensis MR-1 cells have been tracked swimming at speeds of up to, and sometimes over, 100 μm/sec, although the average

  13. Combined Genomics and Experimental Analyses of Respiratory Characteristics of Shewanella putrefaciens W3-18-1

    PubMed Central

    Qiu, Dongru; Wei, Hehong; Tu, Qichao; Yang, Yunfeng; Xie, Ming; Chen, Jingrong; Pinkerton, Mark H.; Liang, Yili; He, Zhili

    2013-01-01

    It has previously been shown that the Shewanella putrefaciens W3-18-1 strain produces remarkably high current in microbial fuel cells (MFCs) and can form magnetite at 0°C. To explore the underlying mechanisms, we developed a genetic manipulation method by deleting the restriction-modification system genes of the SGI1 (Salmonella genome island 1)-like prophage and analyzed the key genes involved in bacterial respiration. W3-18-1 has less respiratory flexibility than the well-characterized S. oneidensis MR-1 strain, as it possesses fewer cytochrome c genes and lacks the ability to oxidize sulfite or reduce dimethyl sulfoxide (DMSO) and timethylamine oxide (TMAO). W3-18-1 lacks the hydrogen-producing Fe-only hydrogenase, and the hydrogen-oxidizing Ni-Fe hydrogenase genes were split into two separate clusters. Two periplasmic nitrate reductases (NapDAGHB and NapDABC) were functionally redundant in anaerobic growth of W3-18-1 with nitrate as the electron acceptor, though napDABC was not regulated by Crp. Moreover, nitrate respiration started earlier in W3-18-1 than in MR-1 (with NapDAGHB only) under microoxic conditions. These results indicate that Shewanella putrefaciens W3-18-1 is well adapted to habitats with higher oxygen levels. Taken together, the results of this study provide valuable insights into bacterial genome evolution. PMID:23811511

  14. Heterogeneity of the electron exchange capacity of kitchen waste compost-derived humic acids based on fluorescence components.

    PubMed

    Yuan, Ying; Tan, Wen-Bing; He, Xiao-Song; Xi, Bei-Dou; Gao, Ru-Tai; Zhang, Hui; Dang, Qiu-Ling; Li, Dan

    2016-11-01

    Composting is widely used for recycling of kitchen waste to improve soil properties, which is mainly attributed to the nutrient and structural functions of compost-derived humic acids (HAs). However, the redox properties of compost-derived HAs are not fully explored. Here, a unique framework is employed to investigate the electron exchange capacity (EEC) of HAs during kitchen waste composting. Most components of compost-derived HAs hold EEC, but nearly two-thirds of them are found to be easily destroyed by Shewanella oneidensis MR-1 and thus result in an EEC lower than the electron - donating capacity in compost-derived HAs. Fortunately, a refractory component also existed within compost-derived HAs and could serve as a stable and effective electron shuttle to promote the MR-1 involved in Fe(III) reduction, and its EEC was significantly correlated with the aromaticity and the amount of quinones. Nevertheless, with the increase of composting time, the EEC of the refractory component did not show an increasing trend. These results implied that there was an optimal composting time to maximize the production of HAs with more refractory and redox molecules. Recognition of the heterogeneity of EEC of the compost-derived HAs enables an efficient utilization of the composts for a variety of environmental applications. Graphical abstract Microbial reduction of compost-derived HAs.

  15. Metabolic Activity of Bacteria at High Pressure

    NASA Astrophysics Data System (ADS)

    Picard, A.; Daniel, I.; Oger, P.

    2008-12-01

    Over the last 20 years, there has been increasing evidence for the presence of a large number of microbes in the oceanic subsurface. Such a habitat has a very low energy input because it is deprived of light. A few meters below the sediment surface, conditions are already anoxic in most cases, sulfate reduction and/or methanogenesis becoming thus the primary respiratory reactions of organic matter. Neither the fate of methanogenesis, nor the fate of Dissimilatory Metal-Reduction (DMR) has been investigated so far as a function of pressure. For this reason, we measured experimentally the pressure limits of microbial anaerobic energetic metabolism. In practice, we measured in situ the kinetics of selenite respiration by the bacterial model Shewanella oneidensis MR-1 under high hydrostatic pressure (HHP) between 0 and 150 MPa at 30°C. MR-1 stationary-phase cells were used in Luria-Bertani (LB) medium amended with lactate as an additional electron donor and sodium selenite as an electron acceptor. In situ measurements were performed by X- ray Absorption Near-Edge Structure (XANES) spectroscopy in both a diamond-anvil cell and an autoclave. A red precipitate of amorphous Se(0) was virtually observed at any pressure to 150 MPa. A progressive reduction of selenite Se(IV) into selenium Se(0) was also observed in the evolution of XANES spectra with time. All kinetics between 0.1 and 150 MPa can be adjusted to a first order kinetic law. MR-1 respires all available selenite up to 60 MPa. Above 60 MPa, the respiration yield decreases linearly as a function of pressure and reaches 0 at 155 ±5 MPa. This indicates that selenite respiration by Shewanella oneidensis MR-1 stops at about 155 MPa, whereas its growth is arrested at 50 MPa. Hence, the present results show that the respiration of selenium by the strain MR-1 occurs efficiently up to 60 MPa and 30°C, i.e. from the surface of a continental sediment to an equivalent depth of about 2 km, or beneath a 5-km water column and

  16. Integrated Investigation on the Production and Fate of Organo-Cr(III) Complexes from Microbial Reduction of Chromate

    SciTech Connect

    Xun, Luying

    2005-06-01

    Our objective is to investigate the complexity of chromium biogeocycling. Our results clearly support more complexity. In short, the chromium cycle is not as simple as the conversion between Cr(III) and Cr(VI) in inorganic forms. We have obtained more evidence to prove the formation of soluble organo-Cr(III) complexes from microbial reduction of Cr(VI). The complexes are relatively stable due to the slow ligand exchange of Cr(III). However, some microorganisms can consume the organic ligands and release Cr(III), which then precipitates. Efforts are being made to characterize the organo-Cr(III) complexes and investigate their behavior in soil. Progress and efforts are summarized for each task. Task 1. Production of soluble organo-Cr(III) complexes by selected microorganisms A total of eight organisms were screened for production of soluble organo-Cr(III) complexes by culturing in both growth and non growth media containing 4 mg/L of Cr(VI); three were Gram positive and five were Gram negative. The Gram-positive bacteria were Cellulomonas sp. ES 6, Rhodococcus sp., and Leafsonia sp., while Shewanella oneidensis MR 1, Desulfovibrio desulfuricans G20, D. vulgaris Hildenborough, Pseudomonas putida MK 1 and Ps. aeruginosa PAO 1 were Gram negative. Purifications of the soluble organo-Cr(III) complexes produced by Cellulomonas sp. ES 6, Shewanella. oneidensis MR 1, Rhodococcus sp., and D. vulgaris Hildenborough were carried out. The culture supernatants were lyophilized and extracted first with methanol followed by water. The extracts were then analyzed for soluble Cr. The majority of the Cr(III) was present in the water-soluble fraction for all of the bacteria tested (data not shown), revealing a general phenomenon of soluble Cr(III) production. Cellulomonas sp. ES6 produced the highest amount of soluble Cr(III) (364 ppm) and D. vulgaris Hildenborough produced the least (143 ppm). Seventy eight percent of the soluble Cr(III) produced by Shewanella. oneidensis MR 1 was

  17. Biofilm Shows Spatially Stratified Metabolic Responses to Contaminant Exposure

    SciTech Connect

    Cao, Bin; Majors, Paul D.; Ahmed, B.; Renslow, Ryan S.; Sylvia, Crystal P.; Shi, Liang; Kjelleberg, Staffan; Fredrickson, Jim K.; Beyenal, Haluk

    2012-11-01

    The objective of this study was to elucidate the spatiotemporal responses of live S. oneidensis MR-1 biofilms to U(VI) (uranyl, UO22+) and Cr(VI) (chromate, CrO42-), important environmental contaminants at DOE contaminated sites. Toward this goal, we applied noninvasive nuclear magnetic resonance (NMR) imaging, diffusion, relaxation and spectroscopy techniques to monitor in situ spatiotemporal responses of S. oneidensis biofilms to U(VI) and Cr(VI) exposure in terms of changes in biofilm structures, diffusion properties, and cellular metabolism. Exposure to U(VI) or Cr(VI) did not appear to change the overall biomass distribution but caused changes in the physicochemical microenvironments inside the biofilm as indicated by diffusion measurements. Changes in the diffusion properties of the biofilms in response to U(VI) and Cr(VI) exposure imply a novel function of the extracellular polymeric substances (EPS) affecting the biotransformation and transport of contaminants in the environment. In the presence of U(VI) or Cr(VI), the anaerobic metabolism of lactate was inhibited significantly, although the biofilms were still capable of reducing U(VI) and Cr(VI). Local concentrations of Cr(III)aq in the biofilm suggested relatively high Cr(VI) reduction activities at the top of the biofilm, near the medium-biofilm interface. The depth-resolved metabolic activities of the biofilm suggested higher diversion effects of gluconeogenesis and C1 metabolism pathways at the bottom of the biofilm and in the presence of U(VI). This study provides a noninvasive means to investigate spatiotemporal responses of biofilms, including surface-associated microbial communities in engineering, natural and medical settings, to various environmental perturbations including exposure to environmental contaminants and antimicrobials.

  18. The effects of non-metabolizing bacterial cells on the precipitation of U, Pb and Ca phosphates

    NASA Astrophysics Data System (ADS)

    Dunham-Cheatham, Sarrah; Rui, Xue; Bunker, Bruce; Menguy, Nicolas; Hellmann, Roland; Fein, Jeremy

    2011-05-01

    In this study, we test the potential for passive cell wall biomineralization by determining the effects of non-metabolizing bacteria on the precipitation of uranyl, lead, and calcium phosphates from a range of over-saturated conditions. Experiments were performed using Gram-positive Bacillus subtilis and Gram-negative Shewanella oneidensis MR-1. After equilibration, the aqueous phases were sampled and the remaining metal and P concentrations were analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES); the solid phases were collected and analyzed using X-ray diffractometry (XRD), transmission electron microscopy (TEM), and X-ray absorption spectroscopy (XAS). At the lower degrees of over-saturation studied, bacterial cells exerted no discernable effect on the mode of precipitation of the metal phosphates, with homogeneous precipitation occurring exclusively. However, at higher saturation states in the U system, we observed heterogeneous mineralization and extensive nucleation of hydrogen uranyl phosphate (HUP) mineralization throughout the fabric of the bacterial cell walls. This mineral nucleation effect was observed in both B. subtilis and S. oneidensis cells. In both cases, the biogenic mineral precipitates formed under the higher saturation state conditions were significantly smaller than those that formed in the abiotic controls. The cell wall nucleation effects that occurred in some of the U systems were not observed under any of the saturation state conditions studied in the Pb or Ca systems. The presence of B. subtilis significantly decreased the extent of precipitation in the U system, but had little effect in the Pb and Ca systems. At least part of this effect is due to higher solubility of the nanoscale HUP precipitate relative to macroscopic HUP. This study documents several effects of non-metabolizing bacterial cells on the nature and extent of metal phosphate precipitation. Each of these effects likely contributes to higher

  19. Biological Recovery of Platinum Complexes from Diluted Aqueous Streams by Axenic Cultures

    PubMed Central

    Maes, Synthia; Props, Ruben; Fitts, Jeffrey P.; De Smet, Rebecca; Vanhaecke, Frank; Boon, Nico; Hennebel, Tom

    2017-01-01

    The widespread use of platinum in high-tech and catalytic applications has led to the production of diverse Pt loaded wastewaters. Effective recovery strategies are needed for the treatment of low concentrated waste streams to prevent pollution and to stimulate recovery of this precious resource. The biological recovery of five common environmental Pt-complexes was studied under acidic conditions; the chloro-complexes PtCl42- and PtCl62-, the amine-complex Pt(NH3)4Cl2 and the pharmaceutical complexes cisplatin and carboplatin. Five bacterial species were screened on their platinum recovery potential; the Gram-negative species Shewanella oneidensis MR-1, Cupriavidus metallidurans CH34, Geobacter metallireducens, and Pseudomonas stutzeri, and the Gram-positive species Bacillus toyonensis. Overall, PtCl42- and PtCl62- were completely recovered by all bacterial species while only S. oneidensis and C. metallidurans were able to recover cisplatin quantitatively (99%), all in the presence of H2 as electron donor at pH 2. Carboplatin was only partly recovered (max. 25% at pH 7), whereas no recovery was observed in the case of the Pt-tetraamine complex. Transmission electron microscopy (TEM) revealed the presence of both intra- and extracellular platinum particles. Flow cytometry based microbial viability assessment demonstrated the decrease in number of intact bacterial cells during platinum reduction and indicated C. metallidurans to be the most resistant species. This study showed the effective and complete biological recovery of three common Pt-complexes, and estimated the fate and transport of the Pt-complexes in wastewater treatment plants and the natural environment. PMID:28046131

  20. Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy

    PubMed Central

    McLean, Jeffrey S; Ona, Ositadinma N; Majors, Paul D

    2015-01-01

    Bacterial biofilms are complex, three-dimensional communities found nearly everywhere in nature and are also associated with many human diseases. Detailed metabolic information is critical to understand and exploit beneficial biofilms as well as combat antibiotic-resistant, disease-associated forms. However, most current techniques used to measure temporal and spatial metabolite profiles in these delicate structures are invasive or destructive. Here, we describe imaging, transport and metabolite measurement methods and their correlation for live, non-invasive monitoring of biofilm processes. This novel combination of measurements is enabled by the use of an integrated nuclear magnetic resonance (NMR) and confocal laser scanning microscope (CLSM). NMR methods provide macroscopic structure, metabolic pathway and rate data, spatially resolved metabolite concentrations and water diffusion profiles within the biofilm. In particular, current depth-resolved spectroscopy methods are applied to detect metabolites in 140–190 nl volumes within biofilms of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1 and the oral bacterium implicated in caries disease, Streptococcus mutans strain UA159. The perfused sample chamber also contains a transparent optical window allowing for the collection of complementary fluorescence information using a unique, in-magnet CLSM. In this example, the entire three-dimensional biofilm structure was imaged using magnetic resonance imaging. This was then correlated to a fluorescent CLSM image by employing a green fluorescent protein reporter construct of S. oneidensis. Non-invasive techniques such as described here, which enable measurements of dynamic metabolic processes, especially in a depth-resolved fashion, are expected to advance our understanding of processes occurring within biofilm communities. PMID:18253132

  1. Towards Electrosynthesis in Shewanella: Energetics of Reversing the Mtr Pathway for Reductive Metabolism

    PubMed Central

    Ross, Daniel E.; Flynn, Jeffrey M.; Baron, Daniel B.; Gralnick, Jeffrey A.; Bond, Daniel R.

    2011-01-01

    Bioelectrochemical systems rely on microorganisms to link complex oxidation/reduction reactions to electrodes. For example, in Shewanella oneidensis strain MR-1, an electron transfer conduit consisting of cytochromes and structural proteins, known as the Mtr respiratory pathway, catalyzes electron flow from cytoplasmic oxidative reactions to electrodes. Reversing this electron flow to drive microbial reductive metabolism offers a possible route for electrosynthesis of high value fuels and chemicals. We examined electron flow from electrodes into Shewanella to determine the feasibility of this process, the molecular components of reductive electron flow, and what driving forces were required. Addition of fumarate to a film of S. oneidensis adhering to a graphite electrode poised at −0.36 V versus standard hydrogen electrode (SHE) immediately led to electron uptake, while a mutant lacking the periplasmic fumarate reductase FccA was unable to utilize electrodes for fumarate reduction. Deletion of the gene encoding the outer membrane cytochrome-anchoring protein MtrB eliminated 88% of fumarate reduction. A mutant lacking the periplasmic cytochrome MtrA demonstrated more severe defects. Surprisingly, disruption of menC, which prevents menaquinone biosynthesis, eliminated 85% of electron flux. Deletion of the gene encoding the quinone-linked cytochrome CymA had a similar negative effect, which showed that electrons primarily flowed from outer membrane cytochromes into the quinone pool, and back to periplasmic FccA. Soluble redox mediators only partially restored electron transfer in mutants, suggesting that soluble shuttles could not replace periplasmic protein-protein interactions. This work demonstrates that the Mtr pathway can power reductive reactions, shows this conduit is functionally reversible, and provides new evidence for distinct CymA:MtrA and CymA:FccA respiratory units. PMID:21311751

  2. Beta-propeller phytases in the aquatic environment.

    PubMed

    Cheng, Chiwai; Lim, Boon L

    2006-03-01

    Phytate, which is one of the dominant organic phosphorus compounds in nature, is very stable in soils. Although a substantial amount of phytate is carried from terrestrial to aquatic systems, it is a minor component of organic phosphorus in coastal sediments. The ephemeral nature of phytate implies the rapid hydrolysis of phytate under aquatic conditions. Among the four classes of known phytases that have been identified in terrestrial organisms, only beta-propeller phytase-like sequences have been identified in the aquatic environment. A novel beta-propeller phytase gene (phyS), cloned from Shewanella oneidensis MR-1, was found to encode a protein with two beta-propeller phytase domains. The characterization of recombinant full-length PhyS and its domains demonstrated that Domain II was the catalytic domain responsible for phytate hydrolysis. The full-length PhyS displayed a K(m) of 83 microM with a kcat of 175.9 min(-1) and the Domain II displayed a K(m) of 474 microM with a kcat of 10.6 min(-1). These results confirm that the phyS gene encodes a functional beta-propeller phytase, which is expressed in S. oneidensis under phosphorus deficient condition. The presence of multiple sequences with a high similarity to phyS in aquatic environmental samples and the widespread occurrence of the Shewanella species in nature suggest that the beta-propeller phytase family is the major class of phytases in the aquatic environment, and that it may play an important role in the recycling of phosphorus.

  3. Genome-Facilitated Analyses of Geomicrobial Processes

    SciTech Connect

    Kenneth H. Nealson

    2012-05-02

    This project had the goal(s) of understanding the mechanism(s) of extracellular electron transport (EET) in the microbe Shewanella oneidensis MR-1, and a number of other strains and species in the genus Shewanella. The major accomplishments included sequencing, annotation, and analysis of more than 20 Shewanella genomes. The comparative genomics enabled the beginning of a systems biology approach to this genus. Another major contribution involved the study of gene regulation, primarily in the model organism, MR-1. As part of this work, we took advantage of special facilities at the DOE: e.g., the synchrotron radiation facility at ANL, where we successfully used this system for elemental characterization of single cells in different metabolic states (1). We began work with purified enzymes, and identification of partially purified enzymes, leading to initial characterization of several of the 42 c-type cytochromes from MR-1 (2). As the genome became annotated, we began experiments on transcriptome analysis under different conditions of growth, the first step towards systems biology (3,4). Conductive appendages of Shewanella, called bacterial nanowires were identified and characterized during this work (5, 11, 20,21). For the first time, it was possible to measure the electron transfer rate between single cells and a solid substrate (20), a rate that has been confirmed by several other laboratories. We also showed that MR-1 cells preferentially attach to cells at a given charge, and are not attracted, or even repelled by other charges. The interaction with the charged surfaces begins with a stimulation of motility (called electrokinesis), and eventually leads to attachment and growth. One of the things that genomics allows is the comparative analysis of the various Shewanella strains, which led to several important insights. First, while the genomes predicted that none of the strains looked like they should be able to degrade N-acetyl glucosamine (NAG), the monomer

  4. Monitoring microbial growth and activity using spectral induced polarization and low-field nuclear magnetic resonance

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Keating, Kristina; Revil, Andre

    2015-04-01

    Microbes and microbial activities in the Earth's subsurface play a significant role in shaping subsurface environments and are involved in environmental applications such as remediation of contaminants in groundwater and oil fields biodegradation. Stimulated microbial growth in such applications could cause wide variety of changes of physical/chemical properties in the subsurface. It is critical to monitor and determine the fate and transportation of microorganisms in the subsurface during such applications. Recent geophysical studies demonstrate the potential of two innovative techniques, spectral induced polarization (SIP) and low-field nuclear magnetic resonance (NMR), for monitoring microbial growth and activities in porous media. The SIP measures complex dielectric properties of porous media at low frequencies of exciting electric field, and NMR studies the porous structure of geologic media and characterizes fluids subsurface. In this laboratory study, we examined both SIP and NMR responses from bacterial growth suspension as well as suspension mixed with silica sands. We focus on the direct contribution of microbes to the SIP and NMR signals in the absence of biofilm formation or biomineralization. We used Zymomonas mobilis and Shewanella oneidensis (MR-1) for SIP and NMR measurements, respectively. The SIP measurements were collected over the frequency range of 0.1 - 1 kHz on Z. mobilis growth suspension and suspension saturated sands at different cell densities. SIP data show two distinct peaks in imaginary conductivity spectra, and both imaginary and real conductivities increased as microbial density increased. NMR data were collected using both CPMG pulse sequence and D-T2 mapping to determine the T2-distribution and diffusion properties on S. oneidensis suspension, pellets (live and dead), and suspension mixed with silica sands. NMR data show a decrease in the T2-distribution in S. oneidensis suspension saturated sands as microbial density increase. A

  5. Metabolic flexibility of the Fe(II)-oxidizing phototropic strain Rhodopseudomonas palustris TIE1 and its potential role in microbial iron cycling

    NASA Astrophysics Data System (ADS)

    Schmidt, C.; Oswald, K.; Melton, E. D.; Kappler, A.

    2012-04-01

    The biogeochemical conversion of iron(II) and iron(III) is widespread in many aquatic and terrestrial environments. In the anoxic regime of soils and sediments the conversion and alternation of the iron redox state is predominantly run by microorganisms that are thought to gain life-sustaining energy by the oxidation and/or reduction of ferrous/ferric components. The spatial arrangement of microbial iron(II) oxidation and iron(III) reduction is largely controlled by the availability of the required electron acceptor and electron donor, as well as the essential source of energy (i.e. light or chemical energy). The physico-chemical patterns of many microbial environments undergo dynamic variations (i.e. diurnal and seasonal changes) as a function of natural external forces (i.e. seasonality, storm events, algae blooms) which strongly affects the local budget of organic carbon and nutrients, as well as the day light penetration. Such fluctuations force microorganisms either to follow the flow of substrate or to switch their metabolism to alternative electron acceptors and/or donors. Different photoferrotrophic bacteria have been shown to be able to grow either on organic (heterotrophic) or inorganic (autotrophic) substrates while exploiting light as their energy source. Within the frame of this study the preference for organic substrates (lactate and acetate) and/or ferrous iron (in simultaneous presence) for photo(ferro)trophic growth of Rhodopseudomonas palustris TIE1 has been investigated in detail. Rates of iron oxidation, acetate/lactate consumption and growth have been followed over time as a function of different lactate to acetate to iron(II) ratios. Additional experiments have been designed to evaluate the potential of Rhodopseudomonas palustris TIE1 to contribute to the redox cycling of iron. TIE1 has been grown in a batch set-up in which the iron(III)-reducing strain Shewanella oneidensis MR1 has been incubated at different ferrihydrite concentrations in

  6. Identification and characterization of a highly variable region in mitochondrial genomes of fusarium species and analysis of power generation from microbial fuel cells

    NASA Astrophysics Data System (ADS)

    Hamzah, Haider Mousa

    In the microbial fuel cell (MFC) project, power generation from Shewanella oneidensis MR-1 was analyzed looking for a novel system for both energy generation and sustainability. The results suggest the possibility of generating electricity from different organic substances, which include agricultural and industrial by-products. Shewanella oneidensis MR-1 generates usable electrons at 30°C using both submerged and solid state cultures. In the MFC biocathode experiment, most of the CO2 generated at the anodic chamber was converted into bicarbonate due the activity of carbonic anhydrase (CA) of the Gluconobacter sp.33 strain. These findings demonstrate the possibility of generation of electricity while at the same time allowing the biomimetic sequestration of CO2 using bacterial CA. In the mitochondrial genomes project, the filamentous fungal species Fusarium oxysporum was used as a model. This species causes wilt of several important agricultural crops. A previous study revealed that a highly variable region (HVR) in the mitochondrial DNA (mtDNA) of three species of Fusarium contained a large, variable unidentified open reading frame (LV-uORF). Using specific primers for two regions of the LV-uORF, six strains were found to contain the ORF by PCR and database searches identified 18 other strains outside of the Fusarium oxysporum species complex. The LV-uORF was also identified in three isolates of the F. oxysporum species complex. Interestingly, several F. oxysporum isolates lack the LV-uORF and instead contain 13 ORFs in the HVR, nine of which are unidentified. The high GC content and codon usage of the LV-uORF indicate that it did not co-evolve with other mt genes and was horizontally acquired and was introduced to the Fusarium lineage prior to speciation. The nonsynonymous/synonymous (dN/dS) ratio of the LV-uORFs (0.43) suggests it is under purifying selection and the putative polypeptide is predicted to be located in the mitochondrial membrane. Growth assays

  7. The Role of Nanoscience in the Field of Environmental Mineralogy

    NASA Astrophysics Data System (ADS)

    Hochella, M. F.; Madden, A. S.; Bose, S.; Wigginton, N. S.

    2006-05-01

    The behavior of minerals with at least one dimension in the nanometer range deviates, sometimes dramatically, from larger particles of the same phase. Given that nanominerals are widespread and abundant in nature, it is important to understand these deviations in the context of essentially all ecosystems. As a result, nanomineralogy is an emerging field that is becoming a key component of environmental mineralogy in terrestrial, ocean, and atmospheric studies. To date, our group has observed the following examples of mineral behavior variations as a function of particle size: 1) the surface area normalized oxidation rate of aqueous Mn2+ on hematite nanoparticle surfaces can increase by up to two orders of magnitude as the hematite particles become smaller; 2) high concentrations of toxic heavy metals are consistently associated with iron and manganese oxyhydroxide nanoparticles within the Clark Fork Superfund site (Montana, USA), but not with macroscopic metal oxyhydroxides; and 3) the surface area normalized reduction rates of hematite nanoparticles by Shewanella oneidensis MR-1 decreases by approximately one order of magnitude as the nanoparticles become smaller. Although mineralogists have just begun to measure property changes as a function of size in the nanoregime, we anticipate that this property variability is essentially universal, and that large deviations from bulk properties should be expected in particle sizes less than a few tens of nanometers. Aggregation should not affect these property changes, as long as the particles remain discrete. There are a number of attributes of nanoparticles that may result in characteristic changes that deviate from the bulk, including electronic structure modification (distribution and population of electronic states), surface site variation, defect densities and distributions, and surface and edge nanotopography. Methods for studying nanoparticles continue to improve, and this field has even moved into applications to

  8. Empirical Establishment of Oligonucleotide Probe Design Criteria; Use of Microarrays with Different Probe Sizes for Monitoring Gene Expression; Temporal Transcriptomic Analysis as Desulfovibrio vulgaris Hildenborough Transitions into Stationary Phase during Electron Donor Depletion

    SciTech Connect

    He, Q.; He, Z.; Huang, K. H.; Alm, E. J.; Wan, X. F.; Hazen, T. C.; Arkin, A. P.; Wall, J. D.; Zhou, J. Z.; Fields, M. W.

    2005-07-15

    In order to experimentally establish the criteria for designing gene-specific and group-specific oligonucleotide probes, an oligonucleotide array was constructed that contained perfect match (PM) and mismatch (MM) probes (50mers and 70mers) based upon 4 genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were examined. Little hybridization was observed at a probe-target identity of <85% for both 50mer and 70mer probes........Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and these criteria should provide valuable information for the development of new software and algorithms for microarray-based studies.; Microarrays with oligonucleotides of different lengths were used to monitor gene expression at a wholegenome level. To determine what length of oligonucleotide is a better alternative to PCR-generated probes, the performance of oligonucleotide probes was systematically compared to that of their PCR-generated counterparts for 96 genes from Shewanella oneidensis MR-1 in terms of overall signal intensity, numbers of genes detected, specificity, sensitivity, and differential gene expression under experimental conditions. .......To evaluate differential gene expression under experimental conditions, S. oneidensis MR-1 cells were exposed to low- or high-pH conditions for 30 and 60 min, and the transcriptional profiles detected by oligonucleotide probes (50-mer, 60-mer, and 70-mer) were closely correlated with those detected by the PCR probes. The results demonstrated that 70-mer oligonucleotides can provide the performance most comparable to the performance obtained with PCR-generated probes.; Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the

  9. Microbial synthesis of Pd/Fe3O4, Au/Fe3O4 and PdAu/Fe3O4 nanocomposites for catalytic reduction of nitroaromatic compounds

    PubMed Central

    Tuo, Ya; Liu, Guangfei; Dong, Bin; Zhou, Jiti; Wang, Aijie; Wang, Jing; Jin, Ruofei; Lv, Hong; Dou, Zeou; Huang, Wenyu

    2015-01-01

    Magnetically recoverable noble metal nanoparticles are promising catalysts for chemical reactions. However, the chemical synthesis of these nanocatalysts generally causes environmental concern due to usage of toxic chemicals under extreme conditions. Here, Pd/Fe3O4, Au/Fe3O4 and PdAu/Fe3O4 nanocomposites are biosynthesized under ambient and physiological conditions by Shewanella oneidensis MR-1. Microbial cells firstly transform akaganeite into magnetite, which then serves as support for the further synthesis of Pd, Au and PdAu nanoparticles from respective precursor salts. Surface-bound cellular components and exopolysaccharides not only function as shape-directing agent to convert some Fe3O4 nanoparticles to nanorods, but also participate in the formation of PdAu alloy nanoparticles on magnetite. All these three kinds of magnetic nanocomposites can catalyze the reduction of 4-nitrophenol and some other nitroaromatic compounds by NaBH4. PdAu/Fe3O4 demonstrates higher catalytic activity than Pd/Fe3O4 and Au/Fe3O4. Moreover, the magnetic nanocomposites can be easily recovered through magnetic decantation after catalysis reaction. PdAu/Fe3O4 can be reused in at least eight successive cycles of 4-nitrophenol reduction. The biosynthesis approach presented here does not require harmful agents or rigorous conditions and thus provides facile and environmentally benign choice for the preparation of magnetic noble metal nanocatalysts. PMID:26310728

  10. The Detoxification and Degradation of Benzothiazole from the Wastewater in Microbial Electrolysis Cells.

    PubMed

    Liu, Xianshu; Ding, Jie; Ren, Nanqi; Tong, Qingyue; Zhang, Luyan

    2016-12-20

    In this study, the high-production-volume chemical benzothiazole (BTH) from synthetic water was fully degraded into less toxic intermediates of simple organic acids using an up-flow internal circulation microbial electrolysis reactor (UICMER) under the hydraulic retention time (HRT) of 24 h. The bioelectrochemical system was operated at 25 ± 2 °C and continuous-flow mode. The BTH loading rate varied during experiments from 20 g·m(-3)·day(-1) to 110 g·m(-3)·day(-1). BTH and soluble COD (Chemical Oxygen Demand) removal efficiency reached 80% to 90% under all BTH loading rates. Bioluminescence based Shewanella oneidensis strain MR-1 ecotoxicity testing demonstrated that toxicity was largely decreased compared to the BTH wastewater influent and effluent of two control experiments. The results indicated that MEC (Microbial Electrolysis Cell) was useful and reliable for improving BTH wastewater treatment efficiency, enabling the microbiological reactor to more easily respond to the requirements of higher loading rate, which is meaningful for economic and efficient operation in future scale-up.

  11. A general system for studying protein-protein interactions in gram-negative bacteria

    SciTech Connect

    Pelletier, Dale A.; Hurst, G. B.; Foote, Linda J.; Lankford, Patricia K.; McKeown, Cathy K.; Lu, Tse-Yuan S.; Schmoyer, Denise D.; Shah, Manesh B.; Hervey IV, W. J.; McDonald, W. Hayes; Hooker, Brian S.; Cannon, William R.; Daly, Don S.; Gilmore, Jason M.; Wiley, H. S.; Auberry, Deanna L.; Wang, Yisong; Larimer, Frank; Kennel, S. J.; Doktycz, M. J.; Morrell-Falvey, Jennifer; Owens, Elizabeth T.; Buchanan, M. V.

    2008-08-01

    One of the most promising of the emerging methods for large-scale studies of interactions among proteins is co-isolation of an affinity-tagged protein and its interaction partners, followed by mass spectrometric identification of the co-purifying proteins. We describe a methodology for systematically identifying the proteins that interact with affinity-tagged “bait” proteins expressed from a medium copy plasmid, which are based on a broad host range (pBBR1MCS5) vector backbone that has been modified to incorporate the Gateway DEST plasmid multiple cloning region. This construct was designed to facilitate expression of fusion proteins bearing an affinity tag, across a range of Gram negative bacterial hosts. We demonstrate the performance of this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results from the RNA polymerase complex from these two species compared favorably with those for both plasmid- and chromosomally-encoded affinity-tagged fusion proteins expressed in a model organism, E. coli.

  12. Biochar-Facilitated Microbial Reduction of Hematite.

    PubMed

    Xu, Shengnan; Adhikari, Dinesh; Huang, Rixiang; Zhang, Hua; Tang, Yuanzhi; Roden, Eric; Yang, Yu

    2016-03-01

    As an important component of soil organic matter (SOM), the transformation of pyrogenic carbon plays a critical role in the biogeochemical cycles of carbon and other redox-active elements such as iron (Fe). Herein, we studied the influences of wheat straw-derived biochars on the microbial reduction of 100 mM of hematite by the dissimilatory metal reducing bacteria Shewanella oneidensis MR-1 under anoxic conditions. The long-term microbial reduction extent and initial reduction rate of hematite were accelerated by more than 2-fold in the presence of 10 mg L(-1) biochar. Soluble leachate from 10 mg L(-1) biochar enhanced Fe(III) reduction to a similar degree. Microbially prereduced biochar leachate abiotically reduced hematite, consistent with the apparent electron shuttling capacity of biochar leachate. Electron paramagnetic resonance (EPR) analysis suggested that biochar leachate-associated semiquinone functional groups were likely involved in the redox reactions. In addition to electron shuttling effects, biochar particles sorbed 0.5-1.5 mM biogenic Fe(II) and thereby increased the long-term extent of hematite reduction by 1.4-1.7 fold. Our results suggest that Fe redox cycling may be strongly impacted by pyrogenic carbon in soils with relatively high content of indigenous pyrogenic carbon or substantial application of biochar.

  13. Characterization of the Decaheme c-type Cytochrome OmcA in Solution and on Hematite Surfaces by Small Angle X-ray Scattering and Neutron Reflectometry

    SciTech Connect

    Johs, Alexander; Shi, Liang; Droubay, Timothy; Ankner, John Francis; Liang, Liyuan

    2010-01-01

    The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate electron shuttling, generated by the bacteria s metabolism, to extracellular acceptors that include solid metal oxides such as hematite ( -Fe2O3). To investigate the mechanism by which OmcA interacts with hematite, we purified OmcA and characterized its solution structure by small angle X-ray scattering (SAXS) and its interaction with hematite by neutron reflectometry (NR). SAXS results showed that OmcA is a monomer that adopts a flat ellipsoidal shape with a dimension of 3.4 9.0 6.5 nm3. Changes in redox state affect OmcA conformation. In addition, OmcA interacts with small organic ligands known to act as electron shuttle molecules, such as flavin mononucleotide (FMN), resulting in the formation of high molecular weight assemblies. A model system, developed using NR to study the interaction of OmcA with hematite, shows that OmcA forms a well-defined monomolecular layer on hematite surfaces. This allows OmcA to preferentially interact with hematite in a conformation that maximizes its contact area with the mineral surface. Overall, these results provide experimental and quantitative evidence for OmcA reduction of solid metal oxides involving both direct and indirect mechanisms.

  14. High Performance Heteroatoms Quaternary-doped Carbon Catalysts Derived from Shewanella Bacteria for Oxygen Reduction

    NASA Astrophysics Data System (ADS)

    Guo, Zhaoyan; Ren, Guangyuan; Jiang, Congcong; Lu, Xianyong; Zhu, Ying; Jiang, Lei; Dai, Liming

    2015-11-01

    A novel heteroatoms (N, P, S and Fe) quaternary-doped carbon (HQDC-X, X refers to the pyrolysis temperature) can be fabricated by directly pyrolyzing a gram-negative bacteria, S. oneidensis MR-1 as precursors at 800 °C, 900 °C and 1000 °C under argon atmosphere. These HQDC-X catalysts maintain the cylindrical shape of bacteria after pyrolysis under high temperatures, while heteroatoms including N, P, S and Fe distribute homogeneously on the carbon frameworks. As a result, HQDC-X catalysts exhibit excellent electrocatalytic activity for ORR via a dominant four-electron oxygen reduction pathway in alkaline medium, which is comparable with that of commercial Pt/C. More importantly, HQDC-X catalysts show better tolerance for methanol crossover and CO poisoning effects, long-term durability than commercial Pt/C, which could be promising alternatives to costly Pt-based electrocatalysts for ORR. The method may provide a promising avenue to develop cheap ORR catalysts from inexpensive, scalable and biological recursors.

  15. Empirical evaluation of a new method for calculating signal-to-noise ratio for microarray data analysis.

    PubMed

    He, Zhili; Zhou, Jizhong

    2008-05-01

    Signal-to-noise-ratio (SNR) thresholds for microarray data analysis were experimentally determined with an oligonucleotide array that contained perfect-match (PM) and mismatch (MM) probes based upon four genes from Shewanella oneidensis MR-1. A new SNR calculation, called the signal-to-both-standard-deviations ratio (SSDR), was developed and evaluated, along with other two methods, the signal-to-standard-deviation ratio (SSR) and the signal-to-background ratio (SBR). At a low stringency, the thresholds of the SSR, SBR, and SSDR were 2.5, 1.60, and 0.80 with an oligonucleotide and a PCR amplicon as target templates and 2.0, 1.60, and 0.70 with genomic DNAs as target templates. Slightly higher thresholds were obtained under high-stringency conditions. The thresholds of the SSR and SSDR decreased with an increase in the complexity of targets (e.g., target types) and the presence of background DNA and a decrease in the compositions of targets, while the SBR remained unchanged in all situations. The lowest percentage of false positives and false negatives was observed with the SSDR calculation method, suggesting that it may be a better SNR calculation for more accurate determination of SNR thresholds. Positive spots identified by SNR thresholds were verified by the Student t test, and consistent results were observed. This study provides general guidance for users to select appropriate SNR thresholds for different samples under different hybridization conditions.

  16. Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones

    PubMed Central

    Okamoto, Akihiro; Hashimoto, Kazuhito; Nealson, Kenneth H.; Nakamura, Ryuhei

    2013-01-01

    Extracellular redox-active compounds, flavins and other quinones, have been hypothesized to play a major role in the delivery of electrons from cellular metabolic systems to extracellular insoluble substrates by a diffusion-based shuttling two-electron-transfer mechanism. Here we show that flavin molecules secreted by Shewanella oneidensis MR-1 enhance the ability of its outer-membrane c-type cytochromes (OM c-Cyts) to transport electrons as redox cofactors, but not free-form flavins. Whole-cell differential pulse voltammetry revealed that the redox potential of flavin was reversibly shifted more than 100 mV in a positive direction, in good agreement with increasing microbial current generation. Importantly, this flavin/OM c-Cyts interaction was found to facilitate a one-electron redox reaction via a semiquinone, resulting in a 103- to 105-fold faster reaction rate than that of free flavin. These results are not consistent with previously proposed redox-shuttling mechanisms but suggest that the flavin/OM c-Cyts interaction regulates the extent of extracellular electron transport coupled with intracellular metabolic activity. PMID:23576738

  17. Cometabolic degradation of chloramphenicol via a meta-cleavage pathway in a microbial fuel cell and its microbial community.

    PubMed

    Zhang, Qinghua; Zhang, Yanyan; Li, Daping

    2017-04-01

    The performance of a microbial fuel cell (MFC) in terms of degradation of chloramphenicol (CAP) was investigated. Approximately 84% of 50mg/L CAP was degraded within 12h in the MFC. A significant interaction of pH, temperature, and initial CAP concentration was found on removal of CAP, and a maximum degradation rate of 96.53% could theoretically be achieved at 31.48°C, a pH of 7.12, and an initial CAP concentration of 106.37mg/L. Moreover, CAP was further degraded through a ring-cleavage pathway. The antibacterial activity of CAP towards Escherichia coli ATCC 25922 and Shewanella oneidensis MR-1 was largely eliminated by MFC treatment. High-throughput sequencing analysis indicated that Azonexus, Comamonas, Nitrososphaera, Chryseobacterium, Azoarcus, Rhodococcus, and Dysgonomonas were the predominant genera in the MFC anode biofilm. In conclusion, the MFC shows potential for the treatment of antibiotic residue-containing wastewater due to its high rates of CAP removal and energy recovery.

  18. Comparative proteogenomics: combining mass spectrometry and comparative genomics to analyze multiple genomes

    SciTech Connect

    Gupta, Nitin; Benhamida, Jamal; Bhargava, Vipul; Goodman, Daniel; Kain , Elisabeth; Kerman, Ian; Nguyen , Ngan; Ollikainen, Noah; Rodriguez, Jesse; Wang, J.; Lipton, Mary S.; Romine, Margaret F.; Bafna, Vineet; Smith, Richard D.; Pevzner, Pavel A.

    2008-07-30

    While bacterial genome annotations have significantly improved in recent years, techniques for bacterial proteome annotation (including post-translational chemical modifications, signal peptides, proteolytic events, etc.) are still in their infancy. At the same time, the number of sequenced bacterial genomes is rising sharply, far outpacing our ability to validate the predicted genes, let alone annotate bacterial proteomes. In this study, we use tandem mass spectrometry (MS/MS) to annotate the proteome of Shewanella oneidensis MR-1, an important microbe for bioremediation. In particular, we provide the first comprehensive map of post-translational modifications in a bacterial genome, including a large number of chemical modifications, signal peptide cleavages and cleavage of N-terminal methionine residues. We also detect multiple genes that were missed or assigned incorrect start positions by gene prediction programs and suggest corrections to improve the gene annotation. This study demonstrates that complementing every genome sequencing project by an MS/MS project would significantly improve both genome and proteome annotations for a reasonable cost.

  19. Whole proteome analysis of post-translational modifications: applications of mass-spectrometry for proteogenomic annotation

    SciTech Connect

    Gupta, Nitin; Tanner, Stephen; Jaitly, Navdeep; Adkins, Joshua N.; Lipton, Mary S.; Edwards, Robert; Romine, Margaret F.; Osterman, Andrei; Bafna, Vineet; Smith, Richard D.; Pevzner, Pavel A.

    2007-09-04

    While bacterial genome annotations have significantly improved in recent years, techniques for bacterial proteome annotation (including post-translational chemical modifications, signal peptides, proteolytic events, etc.) are still in their infancy. At the same time, the number of sequenced bacterial genomes is rising sharply, far outpacing our ability to validate the predicted genes, let alone annotate bacterial proteomes. In this study, we use tandem mass spectrometry (MS/MS) to annotate the proteome of Shewanella oneidensis MR-1, an important microbe for bioremediation. In particular, we provide the first comprehensive map of post-translational modifications in a bacterial genome, including a large number of chemical modifications, signal peptide cleavages and cleavage of N-terminal methionine residues. We also detect multiple genes that were missed or assigned incorrect start positions by gene prediction programs and suggest corrections to improve the gene annotation. This study demonstrates that complementing every genome sequencing project by an MS/MS project would significantly improve both genome and proteome annotations for a reasonable cost.

  20. Hydrous manganese oxide doped gel probe sampler for measuring in situ reductive dissolution rates. 1. Laboratory development.

    PubMed

    Farnsworth, Claire E; Hering, Janet G

    2010-01-01

    Reductive dissolution of redox-sensitive minerals such as manganese (Mn) oxides in natural sediments is an important mechanism for trace element mobilization into groundwater. A gel probe sampler has been constructed to study in situ reductive dissolution of Mn oxides. The gel consists of a polyacrylamide polymer matrix doped with hydrous Mn oxide (HMO). Gel slabs are mounted into a probe, which is designed to be inserted into the sediments. The amount of Mn released from the gel by reductive dissolution is determined by comparing the amount of Mn initially embedded into the gel with the amount remaining in the gel after exposure to conditions in the sediments or, in laboratory studies, to reducing agents. In this laboratory study, the performance of the gel probes was examined using the model reductant ascorbate and the Mn-reducing bacteria Shewanella oneidensis strain MR-1. In addition, a 1-D model was used to relate the reaction rates observed for HMO embedded in gels to those for HMO in suspension. One limitation of the HMO-doped gels for assessing microbial reduction rates is that the gels prevent direct contact between the microbes and the HMO and hence preclude enzymatic reduction at the cell surface. Nonetheless, the HMO-doped gel probes offer the possibility to establish a lower bound for Mn-reduction capacity in sediments.

  1. High Performance Heteroatoms Quaternary-doped Carbon Catalysts Derived from Shewanella Bacteria for Oxygen Reduction

    PubMed Central

    Guo, Zhaoyan; Ren, Guangyuan; Jiang, Congcong; Lu, Xianyong; Zhu, Ying; Jiang, Lei; Dai, Liming

    2015-01-01

    A novel heteroatoms (N, P, S and Fe) quaternary-doped carbon (HQDC-X, X refers to the pyrolysis temperature) can be fabricated by directly pyrolyzing a gram-negative bacteria, S. oneidensis MR-1 as precursors at 800 °C, 900 °C and 1000 °C under argon atmosphere. These HQDC-X catalysts maintain the cylindrical shape of bacteria after pyrolysis under high temperatures, while heteroatoms including N, P, S and Fe distribute homogeneously on the carbon frameworks. As a result, HQDC-X catalysts exhibit excellent electrocatalytic activity for ORR via a dominant four-electron oxygen reduction pathway in alkaline medium, which is comparable with that of commercial Pt/C. More importantly, HQDC-X catalysts show better tolerance for methanol crossover and CO poisoning effects, long-term durability than commercial Pt/C, which could be promising alternatives to costly Pt-based electrocatalysts for ORR. The method may provide a promising avenue to develop cheap ORR catalysts from inexpensive, scalable and biological recursors. PMID:26602287

  2. The use of electrochemical impedance spectroscopy (EIS) in the evaluation of the electrochemical properties of a microbial fuel cell.

    PubMed

    Manohar, Aswin K; Bretschger, Orianna; Nealson, Kenneth H; Mansfeld, Florian

    2008-04-01

    Electrochemical impedance spectroscopy (EIS) has been used to determine several electrochemical properties of the anode and cathode of a mediator-less microbial fuel cell (MFC) under different operational conditions. These operational conditions included a system with and without the bacterial catalyst and EIS measurements at the open-circuit potential of the anode and the cathode or at an applied cell voltage. In all cases the impedance spectra followed a simple one-time-constant model (OTCM) in which the solution resistance is in series with a parallel combination of the polarization resistance and the electrode capacitance. Analysis of the impedance spectra showed that addition of Shewanella oneidensis MR-1 to a solution of buffer and lactate greatly increased the rate of the lactate oxidation at the anode under open-circuit conditions. The large decrease of open-circuit potential of the anode increased the cell voltage of the MFC and its power output. Measurements of impedance spectra for the MFC at different cell voltages resulted in determining the internal resistance (R(int)) of the MFC and it was found that R(int) is a function of cell voltage. Additionally, R(int) was equal to R(ext) at the cell voltage corresponding to maximum power, where R(ext) is the external resistance that must be applied across the circuit to obtain the maximum power output.

  3. Multistep hopping and extracellular charge transfer in microbial redox chains.

    PubMed

    Pirbadian, Sahand; El-Naggar, Mohamed Y

    2012-10-28

    Dissimilatory metal-reducing bacteria are microorganisms that gain energy by transferring respiratory electrons to extracellular solid-phase electron acceptors. In addition to its importance for physiology and natural environmental processes, this form of metabolism is being investigated for energy conversion and fuel production in bioelectrochemical systems, where microbes are used as biocatalysts at electrodes. One proposed strategy to accomplish this extracellular charge transfer involves forming a conductive pathway to electrodes by incorporating redox components on outer cell membranes and along extracellular appendages known as microbial nanowires within biofilms. To describe extracellular charge transfer in microbial redox chains, we employed a model based on incoherent hopping between sites in the chain and an interfacial treatment of electrochemical interactions with the surrounding electrodes. Based on this model, we calculated the current-voltage (I-V) characteristics and found the results to be in good agreement with I-V measurements across and along individual microbial nanowires produced by the bacterium Shewanella oneidensis MR-1. Based on our analysis, we propose that multistep hopping in redox chains constitutes a viable strategy for extracellular charge transfer in microbial biofilms.

  4. Biochar-Facilitated Reduction of Crystalline Fe(III) in Hematite

    NASA Astrophysics Data System (ADS)

    Xu, S.; Yang, Y.; Roden, E. E.; Tang, Y.; Huang, R.; Adhikari, D.

    2015-12-01

    Pyrogenic organic matter is a significant component of soil organic matter, the transformation of which may play a crucial role in the coupled redox cycles of carbon and iron. However, scant information is available for the role of pyrogenic carbon in the redox cycle of iron. Herein, we studied the influences of wheat straw-derived biochar on the microbial reduction of hematite by Shewanella oneidensis MR-1. In the presence of 10 mg/L biochar, microbial reduction of hematite was substantially accelerated by 41% to 142%. Reduction of hematite was enhanced to similar degrees by aqueous biochar with the concentration of 1-3 mg C/L. Importance of the aqueous biochar was also supported by the response of enhancement of Fe reduction to the dose of biochar particles, closely linked to the change in aqueous biochar concentration rather than the amount of total biochar particles. Microbiologically pre-reduced biochar reduced hematite abiotically, demonstrating the electron shuttling capacity of aqueous biochar for hematite reduction. On the other side, biochar particles sorbed Fe(II) and consequently decreased the accumulation of Fe(II) in solution to facilitate the reduction of hematite further. We reported for the first time the biochar-facilitated microbial reduction of crystalline Fe(III), through electron shuttling processes mediated by aqueous biochar and complexation of Fe(II) by biochar particles. Such impacted redox cycles of Fe would be important for the soil environment with relatively high content of indigenous pyrogenic carbon or substantial application of biochar.

  5. Metal Cycling by Bacteria: Moving Electrons Around

    ScienceCinema

    Nealson, Ken

    2016-07-12

    About 20 years ago, Shewanella oneidensis MR-1 was isolated from a manganese-rich lack in upstate New York, and subsequently shown to utilize solid forms of oxidized manganese or iron as an electron acceptor. Recent studies of metal-reducing bacterial have unveiled a number of unexpected properties of microbes that have enlarged our view of microbes and their role(s) in natural ecosystems. For example, the processes of metal reduction themselves are fundamental to the carbon cycle in many lakes and sediments, where iron and manganese account for the major portion of organic carbon oxidation in many sediments. On more modest spatial scales, iron and manganese reduction can be linked to the oxidation of a wide variety of carbon compounds, many of them recalcitrant and/or toxic. One remarkable property of metal reducers is their ability to reduce solid, often highly crystalline substrates such as iron and manganese oxides and oxyhydroxides. It is now clear that this is done via the utilization of enzymes located on the outer wall of the bacteria - enzymes that apparently interact directly with these solid substrates. Molecular and genomic studies combined have revealed the genes and protoeins responsible for these activities, and many facets of the regulation. This talk focuses on the general features and properties of these remarkable organisms that seem to communicate via electron transfer across a wide variety of soluable, insoluable, and even "inert" substrates, and the way that these processes may be mechanistically linked.

  6. Indirect and suboptimal control of gene expression is widespread in bacteria

    PubMed Central

    Price, Morgan N; Deutschbauer, Adam M; Skerker, Jeffrey M; Wetmore, Kelly M; Ruths, Troy; Mar, Jordan S; Kuehl, Jennifer V; Shao, Wenjun; Arkin, Adam P

    2013-01-01

    Gene regulation in bacteria is usually described as an adaptive response to an environmental change so that genes are expressed when they are required. We instead propose that most genes are under indirect control: their expression responds to signal(s) that are not directly related to the genes' function. Indirect control should perform poorly in artificial conditions, and we show that gene regulation is often maladaptive in the laboratory. In Shewanella oneidensis MR-1, 24% of genes are detrimental to fitness in some conditions, and detrimental genes tend to be highly expressed instead of being repressed when not needed. In diverse bacteria, there is little correlation between when genes are important for optimal growth or fitness and when those genes are upregulated. Two common types of indirect control are constitutive expression and regulation by growth rate; these occur for genes with diverse functions and often seem to be suboptimal. Because genes that have closely related functions can have dissimilar expression patterns, regulation may be suboptimal in the wild as well as in the laboratory. PMID:23591776

  7. Correlated Atomic Force Microscopy and Flourescence Lifetime Imaging of Live Bacterial Cells

    SciTech Connect

    Micic, Miodrag; Hu, Dehong; Suh, Yung D.; Newton, Greg J.; Romine, Margaret F.; Lu, H PETER.

    2004-04-01

    We report on the imaging of living bacterial cells by using a new correlated tapping-mode atomic force microscopy (AFM) and confocal al fluorescence lifetime imaging microscopy (FLIM). Different methods of preparing the bacterial sample were explored for optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells on poly-1-lysine coated surfaces and agarose gel coated surfaces. We have found that the agarose gel containing 99% buffer can provide a local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and single-to-noise ration of the AFM images. Near-field AFM-tip enhanced fluorescence lifetime imaging (AFM-FLIM) holds great promise for obtaining fluorescence images beyond the optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond the diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging living bacterial cells, we demonstrate a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging to characterize cell polarity.

  8. Impact of iron-reducing bacteria on the corrosion rate of carbon steel under simulated geological disposal conditions.

    PubMed

    Schütz, Marta K; Schlegel, Michel L; Libert, Marie; Bildstein, Olivier

    2015-06-16

    The current projects for the disposal of high-level radioactive waste rely on underground burial and confinement by metallic envelopes that are susceptible to corrosion processes. The impact of microbial activity must be fully clarified in order to provide biological parameters for predictive reactive transport models. This study investigates the impact of hydrogenotrophic iron-reducing bacteria (Shewanella oneidensis strain MR-1) on the corrosion rate of carbon steel under simulated geological disposal conditions by using a geochemical approach. It was found that corrosion damage changes mostly according to the experimental solution (i.e., chemical composition). Magnetite and vivianite were identified as the main corrosion products. In the presence of bacteria, the corrosion rate increased by a factor of 1.3 (according to weight loss analysis) to 1.8 (according to H2 measurements), and the detected amount of magnetite diminished. The mechanism likely to enhance corrosion is the destabilization and dissolution of the passivating magnetite layer by reduction of structural Fe(III) coupled to H2 oxidation.

  9. Characterization of lead nanoparticles formed by Shewanella sp. KR-12

    NASA Astrophysics Data System (ADS)

    Liu, Chien-Liang; Yen, Jui-Hung

    2016-01-01

    The bacterial strain KR-12 was isolated from river sediment in northeast Taiwan. 16S rRNA gene sequencing revealed that it belongs to the genus Shewanella. The strain can accumulate lead (Pb) and form Pb nanoparticles (PbNPs) on exposure to Pb(NO3)2 and sodium formate in HEPES buffer. On transmission electron microscopy (TEM), the KR-12-formed PbNPs were spherical in shape and ranged from 3 to 8 nm. The PbNPs formed a line or curved pattern on bacteria. In addition, one or more pilus-like structures elongated from the bacteria. In contrast, Shewanella oneidensis MR-1 and other bacteria could not form PbNPs pattern or pilus-like structure under the same conditions. High-resolution TEM combined with energy-dispersive X-ray spectroscopy demonstrated that these PbNPs primarily contained Pb and had an amorphous structure. This is the first report of the biosynthesis of PbNPs by a Shewanella species.

  10. Kinetic Monte Carlo Simulations and Molecular Conductance Measurements of the Bacterial Decaheme Cytochrome MtrF

    SciTech Connect

    Byun, H. S.; Pirbadian, S.; Nakano, Aiichiro; Shi, Liang; El-Naggar, Mohamed Y.

    2014-09-05

    Microorganisms overcome the considerable hurdle of respiring extracellular solid substrates by deploying large multiheme cytochrome complexes that form 20 nanometer conduits to traffic electrons through the periplasm and across the cellular outer membrane. Here we report the first kinetic Monte Carlo simulations and single-molecule scanning tunneling microscopy (STM) measurements of the Shewanella oneidensis MR-1 outer membrane decaheme cytochrome MtrF, which can perform the final electron transfer step from cells to minerals and microbial fuel cell anodes. We find that the calculated electron transport rate through MtrF is consistent with previously reported in vitro measurements of the Shewanella Mtr complex, as well as in vivo respiration rates on electrode surfaces assuming a reasonable (experimentally verified) coverage of cytochromes on the cell surface. The simulations also reveal a rich phase diagram in the overall electron occupation density of the hemes as a function of electron injection and ejection rates. Single molecule tunneling spectroscopy confirms MtrF's ability to mediate electron transport between an STM tip and an underlying Au(111) surface, but at rates higher than expected from previously calculated heme-heme electron transfer rates for solvated molecules.

  11. Microbial fuel cells using Cellulomonas spp. with cellulose as fuel.

    PubMed

    Takeuchi, Yuya; Khawdas, Wichean; Aso, Yuji; Ohara, Hitomi

    2017-03-01

    Cellulomonas fimi, Cellulomonas biazotea, and Cellulomonas flavigena are cellulose-degrading microorganisms chosen to compare the degradation of cellulose. C. fimi degraded 2.5 g/L of cellulose within 4 days, which was the highest quantity among the three microorganisms. The electric current generation by the microbial fuel cell (MFC) using the cellulose-containing medium with C. fimi was measured over 7 days. The medium in the MFC was sampled every 24 h to quantify the degradation of cellulose, and the results showed that the electric current increased with the degradation of cellulose. The maximum electric power generated by the MFC was 38.7 mW/m(2), and this numeric value was 63% of the electric power generated by an MFC with Shewanella oneidensis MR-1, a well-known current-generating microorganism. Our results showed that C. fimi was an excellent candidate to produce the electric current from cellulose via MFCs.

  12. mRNA-targeted Fluorescent in Situ Hybridization (FISH) of Gram-negative Bacteria Without Template Amplification or Tyramide Signal Amplification

    SciTech Connect

    Coleman, James R.; Culley, David E.; Chrisler, William B.; Brockman, Fred J.

    2007-12-01

    Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ-RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multicopy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.

  13. Empirical Evaluation of a New Method for Calculating Signal to Noise Ratio (SNR) for Microarray Data Analysis

    SciTech Connect

    Zhou, Jizhong; He, Zhili; Zhou, Jizhong

    2008-03-06

    Signal-to-noise-ratio (SNR) thresholds for microarray data analysis were experimentally determined with an oligonucleotide array that contained perfect match (PM) and mismatch (MM) probes based upon four genes from Shewanella oneidensis MR-1. A new SNR calculation, called signal to both standard deviations ratio (SSDR) was developed, and evaluated along with other two methods, signal to standard deviation ratio (SSR), and signal to background ratio (SBR). At a low stringency, the thresholds of SSR, SBR, and SSDR were 2.5, 1.60 and 0.80 with oligonucleotide and PCR amplicon as target templates, and 2.0, 1.60 and 0.70 with genomic DNA as target templates. Slightly higher thresholds were obtained at the high stringency condition. The thresholds of SSR and SSDR decreased with an increase in the complexity of targets (e.g., target types), and the presence of background DNA, and a decrease in the composition of targets, while SBR remained unchanged under all situations. The lowest percentage of false positives (FP) and false negatives (FN) was observed with the SSDR calculation method, suggesting that it may be a better SNR calculation for more accurate determination of SNR thresholds. Positive spots identified by SNR thresholds were verified by the Student t-test, and consistent results were observed. This study provides general guidance for users to select appropriate SNR thresholds for different samples under different hybridization conditions.

  14. In vitro evolution of a peptide with a hematite binding motif that may constitute a natural metal-oxide binding archetype.

    PubMed

    Lower, Brian H; Lins, Roberto D; Oestreicher, Zachery; Straatsma, Tjerk P; Hochella, Michael F; Shi, Liang; Lower, Steven K

    2008-05-15

    Phage-display technology was used to evolve peptides that selectively bind to the metal-oxide hematite (Fe2O3) from a library of approximately 3 billion different polypeptides. The sequences of these peptides contained the highly conserved amino acid motif, Ser/Thr-hydrophobic/aromatic-Ser/Thr-Pro-Ser/Thr. To better understand the nature of the peptide-metal oxide binding demonstrated by these experiments, molecular dynamics simulations were carried out for Ser-Pro-Ser at a hematite surface. These simulations show that hydrogen bonding occurs between the two serine amino acids and the hydroxylated hematite surface and that the presence of proline between the hydroxide residues restricts the peptide flexibility, thereby inducing a structural-binding motif. A search of published sequence data revealed that the binding motif (Ser/Thr-Pro-Ser/Thr) is adjacent to the terminal heme-binding domain of both OmcA and MtrC, which are outer membrane cytochromes from the metal-reducing bacterium Shewanella oneidensis MR-1. The entire five amino acid consensus sequence (Ser/Thr-hydrophobic/ aromatic-Ser/Thr-Pro-Ser/Thr) was also found as multiple copies in the primary sequences of metal-oxide binding proteins Sil1 and Sil2 from Thalassiosira pseudonana. We suggest that this motif constitutes a natural metal-oxide binding archetype that could be exploited in enzyme-based biofuel cell design and approaches to synthesize tailored metal-oxide nanostructures.

  15. A General System for Studying Protein-Protein Interactions in Gram-Negative Bacteria

    SciTech Connect

    Pelletier, Dale A; Auberry, Deanna L; Buchanan, Michelle V; Cannon, Bill; Daly, Don S.; Doktycz, Mitchel John; Foote, Linda J; Hervey, IV, William Judson; Hooker, Brian; Hurst, Gregory {Greg} B; Kennel, Steve J; Lankford, Patricia K; Larimer, Frank W; Lu, Tse-Yuan S; McDonald, W Hayes; McKeown, Catherine K; Morrell-Falvey, Jennifer L; Owens, Elizabeth T; Schmoyer, Denise D; Shah, Manesh B; Wiley, Steven; Wang, Yisong; Gilmore, Jason

    2008-01-01

    Abstract One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged bait proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

  16. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling.

    PubMed

    Zhang, Haizhen; Brown, Roslyn N; Qian, Wei-Jun; Monroe, Matthew E; Purvine, Samuel O; Moore, Ronald J; Gritsenko, Marina A; Shi, Liang; Romine, Margaret F; Fredrickson, James K; Pasa-Tolić, Ljiljana; Smith, Richard D; Lipton, Mary S

    2010-05-07

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope (18)O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a Gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level (16)O and (18)O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in Delta gspD mutant cells of many outer membrane proteins including the outer membrane c-type cytochromes OmcA and MtrC, in agreement with a previous report that these proteins are substrates of the type II secretion system.

  17. Microbial mass-dependent fractionation of chromium isotopes

    USGS Publications Warehouse

    Sikora, E.R.; Johnson, T.M.; Bullen, T.D.

    2008-01-01

    Mass-dependent fractionation of Cr isotopes occurs during dissimilatory Cr(VI) reduction by Shewanella oneidensis strain MR-1. Cells suspended in a simple buffer solution, with various concentrations of lactate or formate added as electron donor, reduced 5 or 10 ??M Cr(VI) to Cr(III) over days to weeks. In all nine batch experiments, 53Cr/52Cr ratios of the unreacted Cr(VI) increased as reduction proceeded. In eight experiments covering a range of added donor concentrations up to 100 ??M, isotopic fractionation factors were nearly invariant, ranging from 1.0040 to 1.0045, with a mean value somewhat larger than that previously reported for abiotic Cr(VI) reduction (1.0034). One experiment containing much greater donor concentration (10 mM lactate) reduced Cr(VI) much faster and exhibited a lesser fractionation factor (1.0018). These results indicate that 53Cr/52Cr measurements should be effective as indicators of Cr(VI) reduction, either bacterial or abiotic. However, variability in the fractionation factor is poorly constrained and should be studied for a variety of microbial and abiotic reduction pathways. ?? 2008 Elsevier Ltd.

  18. Rapid degradation of sulphamethoxazole and the further transformation of 3-amino-5-methylisoxazole in a microbial fuel cell.

    PubMed

    Wang, Lu; Liu, Yulei; Ma, Jun; Zhao, Feng

    2016-01-01

    Sulphamethoxazole (SMX) is extensively used in humans and livestock, but its appearance in natural water raises environmental concerns. This study demonstrated that SMX and its degradation product, 3-amino-5-methylisoxazole (3A5MI), could be effectively degraded in microbial fuel cell (MFC) reactors. Approximately 85% of 20 ppm SMX was degraded within 12 h, and this was a more rapid biodegradation rate than has been previously shown in the literature. In addition, 3A5MI, a toxic chemical that forms in the SMX degradation process, can be further mineralized. The degradation products of SMX were detected by mass spectrometry, and three speculated by-products were confirmed with chemical standards. It was observed that nitrogen atoms of SMX were progressively eliminated during the degradation process, which may relate with the degradation of SMX and 3A5MI. An antibacterial activity test showed that the biotoxicity of SMX towards Shewanella oneidensis MR-1 and Escherichia coli DH5α was greatly reduced after MFC treatment. Moreover, the ATP level of the MFC microbe was nearly threefold higher than that in open-circuit controls, which may be related to the rapid degradation of SMX in MFCs. This study can facilitate further investigations about the biodegradation of SMX.

  19. Enhanced Photovoltage Response of Hematite-X-Ferrite Interfaces (X = Cr, Mn, Co, or Ni)

    NASA Astrophysics Data System (ADS)

    Bian, Liang; Li, Hai-long; Li, Yu-jin; Nie, Jia-nan; Dong, Fa-qin; Dong, Hai-liang; Song, Mian-xin; Wang, Li-sheng; Zhou, Tian-liang; Zhang, Xiao-yan; Li, Xin-xi; Xie, Lei

    2017-02-01

    High-fluorescent p-X-ferrites (XFe2O4; XFO; X = Fe, Cr, Mn, Co, or Ni) embedded in n-hematite (Fe2O3) surfaces were successfully fabricated via a facile bio-approach using Shewanella oneidensis MR-1. The results revealed that the X ions with high/low work functions modify the unpaired spin Fe2+-O2- orbitals in the XFe2O4 lattices to become localized paired spin orbitals at the bottom of conduction band, separating the photovoltage response signals (73.36 455.16/-72.63 -32.43 meV). These (Fe2O3)-O-O-(XFe2O4) interfacial coupling behaviors at two fluorescence emission peaks (785/795 nm) are explained via calculating electron-hole effective masses (Fe2O3-FeFe2O4 17.23 × 10-31 kg; Fe2O3-CoFe2O4 3.93 × 10-31 kg; Fe2O3-NiFe2O4 11.59 × 10-31 kg; Fe2O3-CrFe2O4 -4.2 × 10-31 kg; Fe2O3-MnFe2O4 -11.73 × 10-31 kg). Such a system could open up a new idea in the design of photovoltage response biosensors.

  20. Decolorization of textile azo dye and Congo red by an isolated strain of the dissimilatory manganese-reducing bacterium Shewanella xiamenensis BC01.

    PubMed

    Ng, I-Son; Chen, Tingting; Lin, Rong; Zhang, Xia; Ni, Chao; Sun, Dongzhe

    2014-03-01

    Shewanella xiamenensis BC01 (SXM) was isolated from sediment collected off Xiamen, China and was identified based on the phylogenetic tree of 16S rRNA sequences and the gyrB gene. This strain showed high activity in the decolorization of textile azo dyes, especially methyl orange, reactive red 198, and recalcitrant dye Congo red, decolorizing at rates of 96.2, 93.0, and 87.5%, respectively. SXM had the best performance for the specific decolorization rate (SDR) of azo dyes compared to Proteus hauseri ZMd44 and Aeromonas hydrophila NIU01 strains and had an SDR similar to Shewanella oneidensis MR-1 in Congo red decolorization. Luria-Bertani medium was the optimal culture medium for SXM, as it reached a density of 4.69 g-DCW L(-1) at 16 h. A mediator (manganese) significantly enhanced the biodegradation and flocculation of Congo red. Further analysis with UV-VIS, Fourier Transform Infrared spectroscopy, and Gas chromatography-mass spectrometry demonstrated that Congo red was cleaved at the azo bond, producing 4,4'-diamino-1,1'-biphenyl and 1,2'-diamino naphthalene 4-sulfonic acid. Finally, SEM results revealed that nanowires exist between the bacteria, indicating that SXM degradation of the azo dyes was coupled with electron transfer through the nanowires. The purpose of this work is to explore the utilization of a novel, dissimilatory manganese-reducing bacterium in the treatment of wastewater containing azo dyes.

  1. Microbial synthesis of bimetallic PdPt nanoparticles for catalytic reduction of 4-nitrophenol.

    PubMed

    Tuo, Ya; Liu, Guangfei; Dong, Bin; Yu, Huali; Zhou, Jiti; Wang, Jing; Jin, Ruofei

    2017-02-01

    Bimetallic nanoparticles are generally believed to have improved catalytic activity and stability due to geometric and electronic changes. In this work, biogenic-Pd (bio-Pd), biogenic-Pt (bio-Pt), and biogenic-PdPt (bio-PdPt) nanoparticles were synthesized by Shewanella oneidensis MR-1 in the absence or presence of quinone. Compared with direct microbial reduction process, the addition of anthraquinone-2,6-disulfonate (AQDS) could promote the reduction efficiency of Pd(II) or/and Pt(IV) and result in decrease of particles size. All kinds of nanoparticles could catalyze 4-nitrophenol reduction by NaBH4 and their catalytic activities took the following order: bio-PdPt (AQDS) ∼ bio-PdPt > bio-Pd (AQDS) > bio-Pd > bio-Pt (AQDS) ∼ bio-Pt. Moreover, the bio-PdPt (AQDS) nanoparticles could be reused for 6 cycles. We believe that this simple and efficient biosynthesis approach for synthesizing bimetallic bio-PdPt nanocatalysts is important for preparing active and stable catalysts.

  2. The periplasmic nitrate reductase in Shewanella: the resolution, distribution and functional implications of two NAP isoforms, NapEDABC and NapDAGHB.

    PubMed

    Simpson, Philippa J L; Richardson, David J; Codd, Rachel

    2010-02-01

    In the bacterial periplasm, the reduction of nitrate to nitrite is catalysed by a periplasmic nitrate reductase (NAP) system, which is a species-dependent assembly of protein subunits encoded by the nap operon. The reduction of nitrate catalysed by NAP takes place in the 90 kDa NapA subunit, which contains a Mo-bis-molybdopterin guanine dinucleotide cofactor and one [4Fe-4S] iron-sulfur cluster. A review of the nap operons in the genomes of 19 strains of Shewanella shows that most genomes contain two nap operons. This is an unusual feature of this genus. The two NAP isoforms each comprise three isoform-specific subunits - NapA, a di-haem cytochrome NapB, and a maturation chaperone NapD - but have different membrane-intrinsic subunits, and have been named NAP-alpha (NapEDABC) and NAP-beta (NapDAGHB). Sixteen Shewanella genomes encode both NAP-alpha and NAP-beta. The genome of the vigorous denitrifier Shewanella denitrificans OS217 encodes only NAP-alpha and the genome of the respiratory nitrate ammonifier Shewanella oneidensis MR-1 encodes only NAP-beta. This raises the possibility that NAP-alpha and NAP-beta are associated with physiologically distinct processes in the environmentally adaptable genus Shewanella.

  3. Integrated Genome-Based Studies of Shewanella Ecophysiology

    SciTech Connect

    Spormann, Alfred

    2011-07-12

    We have constructed in-frame deletions of 7 of the 10 PAS-GGDEF-EAL proteins in Shewanella oneidensis MR-1. We are currently in the process of characterizing the deletion mutants under a wide range of growth conditions. In addition to characterizing growth, we will also examine the biofilm formation of the deletion mutants. In addition to the genetic analyses of the mutants, we are also interested in comparing the activities of the various PAS-GGDEF-EAL proteins. Proteins containing PAS, GGDEF and EAL amino acid sequence motifs may play an important role in regulating c-di-GMP signaling in response to environmental conditions. A genetic and biochemical analysis into the roles of these proteins is underway. PDE activity was observed for several PAS-GGDEF-EAL proteins. One of these proteins, SO0427, also demonstrates possible DGC activity in vitro. Currently, we are studying the growth, motility and biofilm formation characteristics of deletion mutants, as well as the activity of the pu