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Sample records for oneidensis mr-1 fluxome

  1. 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.

  2. Electroanalysis of Shewanella oneidensis MR-1.

    PubMed

    Shumyantseva, V V; Shebanova, A S; Chalenko, Ya M; Voeikova, T A; Kirpichnikov, M P; Shaitan, K V; Debabov, V G

    2015-01-01

    Electrochemical parameters of bacterial cells Shewanella oneidensis MR-1 were investigated. For registration of the direct electron transfer between S. oneidensis MR-1 and electrode, bacterial cells were pretreated with didodecyldimethylammonium bromide (DDAB), a synthetic membrane-like substance of polycationic nature that exhibits membrane-loosening properties. Such pretreatment of S. oneidensis MR-1 allowed increasing the efficiency of extracellular electron transfer by the proteobacterium due to better availability of electroactive proteins for registration of electron transfer processes. The electroanalysis of bacterial cells S. oneidensis MR-1 under anaerobic conditions allows registering redox-active proteins and biomolecules in the range of potentials of-0.40,-0.16, and-0 V, which corresponds to flavohemoproteins, quinone derivatives, and c-type cytochromes of the external membrane of S. oneidensis MR-1 cells.

  3. Isobutanol production from an engineered Shewanella oneidensis MR-1.

    PubMed

    Jeon, Jong-Min; Park, Hyojung; Seo, Hyung-Min; Kim, Jung-Ho; Bhatia, Shashi Kant; Sathiyanarayanan, Ganesan; Song, Hun-Suk; Park, Sung-Hee; Choi, Kwon-Young; Sang, Byoung-In; Yang, Yung-Hun

    2015-11-01

    Shewanella oneidensis MR-1 is one of the most well-known metal-reducing bacteria and it has been extensively studied for microbial fuel cell and bioremediation aspects. In this study, we have examined S. oneidensis MR-1 as an isobutanol-producing host by assessing three key factors such as isobutanol synthetic genes, carbon sources, and electron supply systems. Heterologous Ehrlich pathway genes, kivD encoding ketoisovalerate decarboxylase and adh encoding alcohol dehydrogenase, were constructed in S. oneidensis MR-1. Among the composition of carbon sources examined, 2% of N-acetylglucosamine, 1.5% of pyruvate and 2% of lactate were found to be the most optimal nutrients and resulted in 10.3 mg/L of isobutanol production with 48 h of microaerobic incubation. Finally, the effects of metal ions (electron acceptor) and direct electron transfer systems on isobutanol production were investigated, and Fe(2+) ions increased the isobutanol production up to 35%. Interestingly, deletion of mtrA and mtrB, genes responsible for membrane transport systems, did not have significant impact on isobutanol production. Finally, we applied engineered S. oneidensis to a bioelectrical reactor system to investigate the effect of a direct electron supply system on isobutanol production, and it resulted in an increased growth and isobutanol production (up to 19.3 mg/L). This report showed the feasibility of S. oneidensis MR-1 as a genetic host to produce valuable biochemicals and combine an electron-supplying system with biotechnological applications.

  4. Isobutanol production from an engineered Shewanella oneidensis MR-1.

    PubMed

    Jeon, Jong-Min; Park, Hyojung; Seo, Hyung-Min; Kim, Jung-Ho; Bhatia, Shashi Kant; Sathiyanarayanan, Ganesan; Song, Hun-Suk; Park, Sung-Hee; Choi, Kwon-Young; Sang, Byoung-In; Yang, Yung-Hun

    2015-11-01

    Shewanella oneidensis MR-1 is one of the most well-known metal-reducing bacteria and it has been extensively studied for microbial fuel cell and bioremediation aspects. In this study, we have examined S. oneidensis MR-1 as an isobutanol-producing host by assessing three key factors such as isobutanol synthetic genes, carbon sources, and electron supply systems. Heterologous Ehrlich pathway genes, kivD encoding ketoisovalerate decarboxylase and adh encoding alcohol dehydrogenase, were constructed in S. oneidensis MR-1. Among the composition of carbon sources examined, 2% of N-acetylglucosamine, 1.5% of pyruvate and 2% of lactate were found to be the most optimal nutrients and resulted in 10.3 mg/L of isobutanol production with 48 h of microaerobic incubation. Finally, the effects of metal ions (electron acceptor) and direct electron transfer systems on isobutanol production were investigated, and Fe(2+) ions increased the isobutanol production up to 35%. Interestingly, deletion of mtrA and mtrB, genes responsible for membrane transport systems, did not have significant impact on isobutanol production. Finally, we applied engineered S. oneidensis to a bioelectrical reactor system to investigate the effect of a direct electron supply system on isobutanol production, and it resulted in an increased growth and isobutanol production (up to 19.3 mg/L). This report showed the feasibility of S. oneidensis MR-1 as a genetic host to produce valuable biochemicals and combine an electron-supplying system with biotechnological applications. PMID:26280214

  5. 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.

  6. 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. PMID:26692509

  7. 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.

  8. Low-Temperature Growth of Shewanella oneidensis MR-1

    PubMed Central

    Abboud, Randa; Popa, Radu; Souza-Egipsy, Virginia; Giometti, Carol S.; Tollaksen, Sandra; Mosher, Jennifer J.; Findlay, Robert H.; Nealson, Kenneth H.

    2005-01-01

    Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of ≈35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (≈22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of ≈67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C. PMID:15691935

  9. Hydrogen Metabolism in Shewanella oneidensis MR-1

    PubMed Central

    Meshulam-Simon, Galit; Behrens, Sebastian; Choo, Alexander D.; Spormann, Alfred M.

    2007-01-01

    Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions. PMID:17189435

  10. Current Production and Metal Oxide Reduction by Shewanella Oneidensis MR-1 Wild Type and Mutants.

    SciTech Connect

    Bretschger, Orianna; Obraztsova, Anna; Sturm, Carter A.; Chang, In Seop; Gorby, Yuri A.; Reed, Samantha B.; Culley, David E.; Reardon, Catherine L.; Barua, Soumitra; Romine, Margaret F.; Zhou, Jizhong; Beliaev, Alex S.; Bouhenni, Rachida; Saffarini, Daad; Mansfeld, Florian; Kim, Byung Hong; Fredrickson, Jim K.; Nealson, Kenneth H.

    2007-11-01

    Shewanella oneidensis MR-1 is a Gram negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides, and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal-oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degree of participation in each process is very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.

  11. 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

  12. Induction of Rapid Detachment in Shewanella oneidensis MR-1 Biofilms

    PubMed Central

    Thormann, Kai M.; Saville, Renée M.; Shukla, Soni; Spormann, Alfred M.

    2005-01-01

    Active detachment of cells from microbial biofilms is a critical yet poorly understood step in biofilm development. We discovered that detachment of cells from biofilms of Shewanella oneidensis MR-1 can be induced by arresting the medium flow in a hydrodynamic biofilm system. Induction of detachment was rapid, and substantial biofilm dispersal started as soon as 5 min after the stop of flow. We developed a confocal laser scanning microscopy-based assay to quantify detachment. The extent of biomass loss was found to be dependent on the time interval of flow stop and on the thickness of the biofilm. Up to 80% of the biomass of 16-h-old biofilms could be induced to detach. High-resolution microscopy studies revealed that detachment was associated with an overall loosening of the biofilm structure and a release of individual cells or small cell clusters. Swimming motility was not required for detachment. Although the loosening of cells from the biofilm structure was observed evenly throughout thin biofilms, the most pronounced detachment in thicker biofilms occurred in regions exposed to the flow of medium, suggesting a metabolic control of detachability. Deconvolution of the factors associated with the stop of medium flow revealed that a sudden decrease in oxygen tension is the predominant trigger for initiating detachment of individual cells. In contrast, carbon limitation did not trigger any substantial detachment, suggesting a physiological link between oxygen sensing or metabolism and detachment. In-frame deletions were introduced into genes encoding the known and putative global transcriptional regulators ArcA, CRP, and EtrA (FNR), which respond to changes in oxygen tension in S. oneidensis MR-1. Biofilms of null mutants in arcA and crp were severely impacted in the stop-of-flow-induced detachment response, suggesting a role for these genes in regulation of detachment. In contrast, an ΔetrA mutant displayed a variable detachment phenotype. From this genetic

  13. The Electrogenic Bacterium Shewanella Oneidensis MR-1 and its Mutants with Increased Reducing Capacity

    NASA Astrophysics Data System (ADS)

    Voeikova, T. A.; Emelyanova, L. K.; Novikova, L. M.; Mordkovich, N. N.; Shakulov, R. S.; Debabov, V. G.

    2013-02-01

    Mutants of Shewanella oneidensis MR-1 resistant to fosfomycin, a toxic analogue of phosphoenolpyruvate, were obtained. The mutants exhibited an increased reducing activity and a higher rate of lactate utilization. A correlation was shown between the rates of metabolism of oxidized substrates and the rate of reduction of methylene blue, a mediator of electron transport. The mutants of S.oneidensis MR-1 will be used in microbial fuel cells (MFC) to enhance energy production from organic compounds. The strain S. oneidensis MR-1 and its mutants with an increased electron production will be used as a good source of bioelectricity in MFC in the experiments on the International Space Station.

  14. Robustness of flux distribution in Shewanella oneidensis MR-1 under environmental stress and genetic perturbation

    NASA Astrophysics Data System (ADS)

    Feng, X.; Martin, H. G.; Keasling, J.; Tang, Y.

    2008-12-01

    The environmental important bacterium, Shewanella oneidensis MR-1, has the significantly different growth rates under normal growth (doubling time=3hrs) in the minimal lactate medium, salt stress (doubling time > 6 hrs), and enhanced growth with amino acids supplementation (doubling time <1.5hrs). 13C based metabolic flux analysis of Shewanella oneidensis MR-1 indicates that the relative central metabolism maintains stable under all growth conditions. Furthermore, screen of MR-1 transposon mutants using high throughput 13C isotopomer analysis method also indicates the robustness of central metabolism under genetic perturbations. These observations reveal a rigid physiology in the level of flux distribution in MR-1, in contrast to the significant change of transcriptomes and metabolite profiles under various growth conditions. This study provides evidence that microbial metabolism maintains metabolic stability under various environmental conditions, rather than being geared towards growth rate maximization.

  15. 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...

  16. Identification of diverse carbon utilization pathways in Shewanella oneidensis MR-1 via expression profiling

    SciTech Connect

    Driscoll, Michael E.; Romine, Margaret F.; Juhn, Frank S.; Serres, Margrethe H.; McCue, Lee Ann; Beliaev, Alex S.; Fredrickson, Jim K.; Gardner, Timothy S.

    2007-07-01

    We have designed and validated the first high-density expression array for Shewanella oneidensis MR-1, consisting of 105 oligonucleotide probes differentially targeted to genes and intergenic regions of its genome. Beyond its use for global gene expression profiling, this microarray represents a platform for the identification of small non-coding RNAs, improved operon inference, and CHIP-chip studies.

  17. Survival of Shewanella Oneidensis MR-1 to GPa pressures

    NASA Astrophysics Data System (ADS)

    Hazael, Rachael; Foglia, Fabrizia; Leighs, James; Appleby-Thomas, Gareth; Daniel, Isabelle; Eakins, Daniel; Meersman, Filip; McMillian, Paul

    2013-06-01

    Most life on Earth is thought to occupy near-surface environments under relatively mild conditions of temperature, pressure, pH, salinity etc. That view is changing following discovery of extremophile organisms that prefer environments based on high or low T, extreme chemistries, or very high pressures. Over the past three decades, geomicrobiologists have discovered an extensive subsurface biosphere, that may account for between 1/10 to 1/3 of Earth's living biomass. We subjected samples of Shewanella oneidensis to several pressure cycles to examine its survival to static high pressures to above 1.5 GPa. Shewanella forms part of a genus that contains several piezophile species like S. violacea and S. benthica. We have obtained growth curves for populations recovered from high P conditions and cultured in the laboratory, before being subjected to even higher pressures. We have also carried out dynamic shock experiments using a specially designed cell to maintain high-P, low-T conditions during shock-recovery experiments and observe colony formation among the survivors. Colony counts, shape and growth curves allow us to compare the static vs dynamic pressure resistance of wild type vs pressure-adapted strains. Leverhulme

  18. 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.

  19. 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. PMID:23558182

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

    NASA Astrophysics Data System (ADS)

    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.

  1. 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.

  2. Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1

    PubMed Central

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

    2013-01-01

    We performed whole-genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli. Furthermore, comparative analyses suggest that many Gammaproteobacteria, including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication. PMID:23995632

  3. 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.

  4. Transcriptome Analysis of Early Surface-Associated Growth of Shewanella oneidensis MR-1

    PubMed Central

    Gödeke, Julia; Binnenkade, Lucas; Thormann, Kai M.

    2012-01-01

    Bacterial biofilm formation starts with single cells attaching to a surface, however, little is known about the initial attachment steps and the adaptation to the surface-associated life style. Here, we describe a hydrodynamic system that allows easy harvest of cells at very early biofilm stages. Using the metal ion-reducing gammaproteobacterium Shewanella oneidensis MR-1 as a model organism, we analyzed the transcriptional changes occurring during surface-associated growth between 15 and 60 minutes after attachment. 230 genes were significantly upregulated and 333 were downregulated by a factor of ≥2. Main functional categories of the corresponding gene products comprise metabolism, uptake and transport, regulation, and hypothetical proteins. Among the genes highly upregulated those implicated in iron uptake are highly overrepresented, strongly indicating that S. oneidensis MR-1 has a high demand for iron during surface attachment and initial biofilm stages. Subsequent microscopic analysis of biofilm formation under hydrodynamic conditions revealed that addition of Fe(II) significantly stimulated biofilm formation of S. oneidensis MR-1 while planktonic growth was not affected. Our approach to harvest cells for transcriptional analysis of early biofilm stages is expected to be easily adapted to other bacterial species. PMID:22860070

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

    PubMed

    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

  6. 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

  7. 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.

  8. Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor.

    PubMed

    Paulick, Anja; Delalez, Nicolas J; Brenzinger, Susanne; Steel, Bradley C; Berry, Richard M; Armitage, Judith P; Thormann, Kai M

    2015-06-01

    The bacterial flagellar motor is an intricate nanomachine which converts ion gradients into rotational movement. Torque is created by ion-dependent stator complexes which surround the rotor in a ring. Shewanella oneidensis MR-1 expresses two distinct types of stator units: the Na(+)-dependent PomA4 B2 and the H(+)-dependent MotA4 B2. Here, we have explored the stator unit dynamics in the MR-1 flagellar system by using mCherry-labeled PomAB and MotAB units. We observed a total of between 7 and 11 stator units in each flagellar motor. Both types of stator units exchanged between motors and a pool of stator complexes in the membrane, and the exchange rate of MotAB, but not of PomAB, units was dependent on the environmental Na(+)-levels. In 200 mM Na(+), the numbers of PomAB and MotAB units in wild-type motors was determined to be about 7:2 (PomAB:MotAB), shifting to about 6:5 without Na(+). Significantly, the average swimming speed of MR-1 cells at low Na(+) conditions was increased in the presence of MotAB. These data strongly indicate that the S. oneidensis flagellar motors simultaneously use H(+) and Na(+) driven stators in a configuration governed by MotAB incorporation efficiency in response to environmental Na(+) levels.

  9. 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.

  10. 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

  11. 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.

  12. 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. PMID

  13. 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.

  14. 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.

  15. 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.

  16. Purification and Characterization of [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

    SciTech Connect

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

    2011-08-02

    The γ-proteobacterium Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that was implicated in both H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned into a protein expression vector. The resulting plasmid was transformed into a MR-1 mutant deficient in H2 formation. Expression of MR-1 [NiFe]-H2ase in trans restored the mutant’s ability to produce H2 at 37% of that for wild type. Following expression, MR-1 [NiFe]-H2ase was purified to near homogeneity. The purified MR-1 [NiFe]-H2ase could couple H2 oxidation to reduction of Tc(VII) and methyl viologen directly. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated Tc(VII) but not methyl viologen reductions. Under the conditions tested, Tc(VII) reduction was complete in Tris buffer but not in HEPES buffer. The reduced Tc(IV) was soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc(IV) precipitates formed in HEPES buffer were packed with crystallites. Although X-ray absorption near-edge spectroscopy measurements confirmed that the reduction products found in both buffers were Tc(IV), extended X-ray adsorption fine-structure measurements revealed that these products were very different. While the product in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O2•nH2O. These results shows for the first time that MR-1 [NiFe]-H2ase is a bidirectional enzyme that catalyzes both H2 formation and oxidation as well as Tc(VII) reduction directly by coupling H2 oxidation.

  17. Purification and Characterization of the [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

    PubMed Central

    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.

    2011-01-01

    Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that has been implicated in H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H2ase in trans restored the mutant's ability to produce H2 at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H2ase coupled H2 oxidation to reduction of Tc(VII)O4− and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated reduction of Tc(VII)O4− but not methyl viologen. Under the conditions tested, all Tc(VII)O4− used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O4− 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)O2·nH2O, which was also the product of Tc(VII)O4− reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H2ase catalyzes Tc(VII)O4− reduction directly by coupling to H2 oxidation. PMID:21724888

  18. Global profiling of Shewanella oneidensis MR-1: expression of hypothetical genes and improved functional annotations.

    PubMed

    Kolker, Eugene; Picone, Alex F; Galperin, Michael Y; Romine, Margaret F; Higdon, Roger; Makarova, Kira S; Kolker, Natali; Anderson, Gordon A; Qiu, Xiaoyun; Auberry, Kenneth J; Babnigg, Gyorgy; Beliaev, Alex S; Edlefsen, Paul; Elias, Dwayne A; Gorby, Yuri A; Holzman, Ted; Klappenbach, Joel A; Konstantinidis, Konstantinos T; Land, Miriam L; Lipton, Mary S; McCue, Lee-Ann; Monroe, Matthew; Pasa-Tolic, Ljiljana; Pinchuk, Grigoriy; Purvine, Samuel; Serres, Margrethe H; Tsapin, Sasha; Zakrajsek, Brian A; Zhu, Wenhong; Zhou, Jizhong; Larimer, Frank W; Lawrence, Charles E; Riley, Monica; Collart, Frank R; Yates, John R; Smith, Richard D; Giometti, Carol S; Nealson, Kenneth H; Fredrickson, James K; Tiedje, James M

    2005-02-01

    The gamma-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome. PMID:15684069

  19. Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and improved functional annotations

    SciTech Connect

    Picone, Alex F.; Galperin, Michael Y.; Romine, Margaret; Higdon, Roger; Makarova, Kira S.; Kolker, Natali; Anderson, Gordon A; Qiu, Xiaoyun; Babnigg, Gyorgy; Beliaev, Alexander S; Edlefsen, Paul; Elias, Dwayne A.; Gorby, Dr. Yuri A.; Holzman, Ted; Klappenbach, Joel; Konstantinidis, Konstantinos T; Land, Miriam L; Lipton, Mary S.; McCue, Lee Ann; Monroe, Matthew; Pasa-Tolic, Ljiljana; Pinchuk, Grigoriy; Purvine, Samuel; Serres, Margrethe H.; Tsapin, Sasha; Zakrajsek, Brian A.; Zhu, Wenguang; Zhou, Jizhong; Larimer, Frank W; Lawrence, Charles E.; Riley, Monica; Collart, Frank; YatesIII, John R.; Smith, Richard D.; Nealson, Kenneth H.; Fredrickson, James K; Tiedje, James M.

    2005-01-01

    The gamma-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, approximate to40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.

  20. 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

  1. Aggrandizing power output from Shewanella oneidensis MR-1 microbial fuel cells using calcium chloride.

    PubMed

    Fitzgerald, Lisa A; Petersen, Emily R; Gross, Benjamin J; Soto, Carissa M; Ringeisen, Bradley R; El-Naggar, Mohamed Y; Biffinger, Justin C

    2012-01-15

    There are several interconnected metabolic pathways in bacteria essential for the conversion of carbon electron sources directly into electrical currents using microbial fuel cells (MFCs). This study establishes a direct exogenous method to increase power output from a Shewanella oneidensis MR-1 containing MFC by adding calcium chloride to the culture medium. The current output from each CaCl(2) concentration tested revealed that the addition of CaCl(2) to 1400 μM increased the current density by >80% (0.95-1.76 μA/cm(2)) using sodium lactate as the sole carbon source. Furthermore, polarization curves showed that the maximum power output could be increased from 157 to 330 μW with the addition of 2080 μM CaCl(2). Since the conductivity of the culture medium did not change after the addition of CaCl(2) (confirmed by EIS and bulk conductivity measurements), this increase in power was primarily biological and not based on ionic effects. Thus, controlling the concentration of CaCl(2) is a pathway to increase the efficiency and performance of S. oneidensis MR-1 MFCs.

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

    PubMed

    Bennett, Brittany D; Brutinel, Evan D; Gralnick, Jeffrey A

    2015-11-01

    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 (Fe(3+)) results in the production of ferrous iron (Fe(2+)) 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 Fe(2+) from the MR-1 cytoplasm. Mutants lacking feoE exhibit an increased sensitivity to Fe(2+). The export function of FeoE is specific for Fe(2+), as an feoE mutant is equally sensitive to other metal ions known to be substrates of other CDF proteins (Cd(2+), Co(2+), Cu(2+), Mn(2+), Ni(2+), or Zn(2+)). The substrate specificity of FeoE differs from that of FieF, the Escherichia coli homolog of FeoE, which has been reported to be a Cd(2+)/Zn(2+) or Fe(2+)/Zn(2+) exporter. A complemented feoE mutant has an increased growth rate in the presence of excess Fe(2+) compared to that of the ΔfeoE mutant complemented with fieF. It is possible that FeoE has evolved to become an efficient and specific Fe(2+) exporter in response to the high levels of iron often present in the types of environmental niches in which Shewanella species can be found.

  3. 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.

  4. 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

  5. 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.

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

    PubMed Central

    Beliaev, A. S.; Klingeman, D. M.; Klappenbach, J. A.; Wu, L.; Romine, M. F.; Tiedje, J. M.; Nealson, K. H.; Fredrickson, J. K.; Zhou, J.

    2005-01-01

    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. PMID:16199584

  7. 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.

  8. Molecular Underpinnings of Fe(III) Oxide Reduction by Shewanella Oneidensis MR-1

    PubMed Central

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

    2012-01-01

    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 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 MtrA either directly or indirectly through other periplasmic proteins. 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 also serve as the terminal reductases for soluble forms of Fe(III). In addition to Fe(III) oxides, Mtr pathway is also involved in reduction of

  9. 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

  10. 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.

  11. Redox and pH Microenvironments within Shewanella oneidensis MR-1 Biofilms Reveal an Electron Transfer Mechanism

    PubMed Central

    Babauta, Jerome T.; Nguyen, Hung Duc; Beyenal, Haluk

    2011-01-01

    The goal of this research was to quantify the variations in redox potential and pH in Shewanella oneidensis MR-1 biofilms respiring on electrodes. We grew S. oneidensis MR-1 on a graphite electrode, which was used to accept electrons for microbial respiration. We modified well-known redox and pH microelectrodes with a built-in reference electrode so that they could operate near polarized surfaces and quantified the redox potential and pH profiles in these biofilms. In addition, we used a ferri-/ferrocyanide redox system in which electrons were only transferred by mediated electron transfer to explain the observed redox potential profiles in biofilms. We found that regardless of the polarization potential of the biofilm electrode, the redox potential decreased toward the bottom of the biofilm. In a fully redox-mediated control system (ferri-/ferrocyanide redox system), the redox potential increased toward the bottom when the electrode was the electron acceptor. The opposite behavior of redox profiles in biofilms and the redox-controlled system is explained by S. oneidensis MR-1 biofilms not being redox-controlled when they respire on electrodes. The lack of a significant variation in pH implies that there is no proton transfer limitation in S. oneidensis MR-1 biofilms and that redox potential profiles are not caused by pH. PMID:21648431

  12. 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.

  13. Mutants Shewanella Oneidensis MR-1 Effective in Generating Bioelectricity in Microbial Fuel Cells

    NASA Astrophysics Data System (ADS)

    Voeikova, T. A.; Emelyanova, L. K.; Novikova, L. M.; Shakulov, R. S.; Sidoruk, K. V.; Smirnov, I. A.; Ilyin, V. K.; Soldatov, P. E.; Tyurin-Kuz'min, A. Y.; Smolenskaya, T. S.; Debabov, V. G.

    2013-02-01

    The capability of Shewanella oneidensis MR-1 mutants FRS1 and FRB1, characterized by a high reducing activity, to generate electricity was measured in microbial fuel cells (MFC) of different types. The reducing activity of these mutants was previously identified with respect to the rate of discoloration (reduction) of methylene blueё, a dye that served as an electron transfer mediator. The present study demonstrated that the rate of MB discoloration was in direct correlation with the level of voltage in MFC of the above strains. In case when the mutants were used, the parameter in MFC proved to be 30-40% greater than in the case of the parent strain. In the course of this investigation MFC characteristics were identified, optimal electrode materials and ion-selective membranes selected, and MFC laboratory mockups manufactured.

  14. Shewregdb: Database and visualization environment for experimental and predicted regulatory information in Shewanella oneidensis mr-1

    SciTech Connect

    Syed, Mustafa; Karpinets, Tatiana V.; Leuze, Mike; Kora, Guruprasad; Romine, Margaret F.; Uberbacher, Edward

    2009-10-15

    Shewanella oneidensis MR-1 is an important model organism for environmental research as it has an exceptional metabolic and respiratory versatility regulated by a complex regulatory network. We have developed a database to collect experimental and computational data relating to regulation of gene and protein expression and a visualization environment that enables integration of these data types. The regulatory information in the database was collected from the published literature and different Internet resources. It includes predictions of DNA regulator binding sites, sigma factor binding sites, transcription units, operons, promoters, and RNA regulators including non-coding RNAs, riboswitches, and different types of terminators. A visualization environment based on GBrowser was developed for accessing the collected information and for its overlaying with experimental data (experimental results from studies employing microarrays, proteomics, and/or gene mutagenesis) and other genome annotations.

  15. 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

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

    PubMed

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

    2008-06-01

    To better understand the interactions among metal contaminants, nutrients, and microorganisms in subsurface fracture-flow systems, biofilms of pure culture of Shewanella oneidensis MR-1 were grown in six fracture-flow reactors (FFRs) of different geometries. The spatial and temporal distribution of uranium and bacteria were examined using a tracer dye (brilliant blue FCF) and microscopy. The results showed that plugging by bacterial cells was dependent on the geometry of the reactor and that biofilms grown in FFRs had a limited U(VI)-reduction capacity. To quantify the U(VI)-reduction capacity of biofilms, batch experiments for U(VI) reduction were performed with repetitive U(VI) additions. U(VI)-reduction rates of stationary phase cultures decreased after each U(VI) addition. After the fourth U(VI) addition, stationary phase cultures treated with U(VI) with and without spent medium yielded gray and black precipitates, respectively. These gray 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 areas of black precipitates showed that reduced U particles (3-6 nm) were crystalline, whereas gray precipitates were a mixture of crystalline and amorphous solids. Results obtained in this study, including a dramatic limitation of S. oneidensis MR-1 and its biofilms to reduce U(VI) and plugging of FFRs, suggest that alternative organisms should be targeted for stimulation for metal immobilization in subsurface fracture-flow systems. PMID:18468655

  17. 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.

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

    SciTech Connect

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

    2008-01-01

    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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. Validation of Shewanella oneidensis MR-1 Small Proteins by AMT Tag-based Proteome Analysis

    SciTech Connect

    Romine, Margaret F.; Elias, Dwayne A.; Monroe, Matthew E.; Auberry, Kenneth J.; Fang, Ruihua; Fredrickson, Jim K.; Anderson, Gordon A.; Smith, Richard D.; Lipton, Mary S.

    2004-09-01

    Using stringent criteria for protein identification by accurate mass and time (AMT) tag mass spectrometric methodology, we detected 36 proteins <101 amino acids in length, including 10 that were annotated as hypothetical proteins, in 172 global tryptic digests of Shewanella oneidensis MR-1 proteins analyzed. Peptides that map to the conserved, but functionally uncharacterized proteins SO4134 and SO2787, were the most frequently detected small proteins in these samples, while hypotheticals SO2669 and SO2063, conserved hypotheticals SO0335 and SO2176, and the SlyX protein (SO1063) were observed at frequencies similar to small expected abundant ribosomal proteins and translation initiation factor IF-1 and consequently, likely to encode important cellular functions. In addition, 30 proteins including three of the small proteins that map to genes predicted to encode frameshifts, point mutations, or recoding signals were detected. Of these 30 genes, peptides that map to positions beyond internal stop codons were detected in 13 genes (SO0101, SO0419, SO0590, SO0738, SO1113, SO1211, SO3079, SO3130, SO3240, SO4231, SO4328, SO4422, and SO4657). While expression of the full-length formate dehydrogenase encoded by SO0101 can be explained by incorporation of selenocysteine at the internal stop codon, the mechanism of translating downstream sequences in the remaining genes remains unknown.

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

    PubMed

    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 10(8) 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 10(2) 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

  6. 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

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

    PubMed

    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.

  8. Structure of Biogenic Uraninite Produced By Shewanella Oneidensis Strain MR-1

    SciTech Connect

    Schofield, E.J.; Veeramani, H.; Sharp, J.O.; Suvorova, E.; Bernier-Latmani, R.; Mehta, A.; Stahlman, J.; Webb, S.M.; Clark, D.L.; Conradson, S.D.; Ilton, E.S.; Bargar, J.R.

    2009-05-27

    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 UO{sub 2} 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. 1.3 nm and an outer region of thickness ca {approx}0.6 nm in which the structure is locally distorted. The lack of nanoparticle strain and structural homology with stoichiometric UO{sub 2} suggests that established thermodynamic parameters for the latter material are an appropriate starting point to model the behavior of nanobiogenic uraninite. The detailed structural analysis in this study provides an essential foundation for subsequent investigations of environmental samples.

  9. 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.

  10. Reduction of jarosite by Shewanella oneidensis MR-1 and secondary mineralization

    NASA Astrophysics Data System (ADS)

    Bingjie, Ouyang; Xiancai, Lu; Huan, Liu; Juan, Li; Tingting, Zhu; Xiangyu, Zhu; Jianjun, Lu; Rucheng, Wang

    2014-01-01

    Jarosite is a common mineral in a variety of environments formed by the oxidation of iron sulfide normally accompanying with the generation of acid mine drainage (AMD) in mining areas or acid rock drainages (ARD) in many localities. Decomposition of jarosite by dissimilatory iron reducing bacteria (DIRB) influences the mobility of many heavy metals generally accommodated in natural jarosite. This study examined the anaerobic reduction of synthesized jarosite by Shewanella oneidensis strain MR-1, a typical facultative bacteria. The release of ferrous and ferric ion, as well as sulfate and potassium, in the inoculated experimental group lasting 80 days is much higher than that in abiotic control groups. The detection of bicarbonate and acetate in experimental solution further confirms the mechanism of microbial reduction of jarosite, in which lactate acts as the electron donor. The produced ferrous iron stimulates the subsequent secondary mineralization, leading to precipitation and transformation of various iron-containing minerals. Green rust and goethite are the intermediate minerals of the microbial reduction process under anoxic conditions, and the end products include magnetite and siderite. In aerobic environments, goethite, magnetite and siderite were also detected, but the contents were relatively lower. While in abiotic experiments, only goethite has been detected as a product. Thus, the microbial reduction and subsequent mineral transformation can remarkably influence the geochemical cycling of iron and sulfur in supergene environments, as well as the mobility of heavy metals commonly accommodated in jarosite.

  11. 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.

  12. 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

  13. Carbon Nanotubes-Mediated Reduction of Hematite by Shewanella oneidensis MR-1

    NASA Astrophysics Data System (ADS)

    Shoji, K.; Xu, S.; Wang, L.; Yang, Y.; Patel, A.

    2015-12-01

    The worldwide carbon nanotubes (CNTs) industry is rapidly expanding with production capacity exceeding several thousand tons per year. After the service lives, CNTs will be ultimately released into the environment with their concentrations in soils increasing by 0.4-147 ng/kg/year and the consequent environmental impacts have received more and more attentions. Effects of CNTs on the redox reactions of Fe have not been studied, although the CNT-bound quinone functional groups can potentially act as an electron shuttle. In this study, we investigated the impact of multi-walled carbon nanotubes (MWCNTs) on the reduction of hematite by metal-respiring bacterium Shewanella oneidensis MR-1. Our results showed that the presence of MWCNT with concentration ranging from 0.05 to 1 g/L inhibited the reduction of hematite (Fe2O3) by up to 35%, likely due to the toxicity effects of MWCNTs. However, the microbially-reduced MWCNTs (0.1 g/L) stimulated the reduction of Fe(III) by 3.6 times, indicating the potential role of MWCNTs as an electron shuttle. These results suggest MWCNTs play a dual role in regulating the microbial reduction of hematite through both toxicity effects and electron shuttle. We plan to further explore the mechanism for the impact of MWCNTs on the redox reactions of iron by studying the influences of carboxyl functionalized MWCNTs, analyzing the impact of MWCNTs on bacterial growth and characterizing the quinone functional groups in MWCNTs. Potential results will provide a novel perception for the impact of CNTs on the biogeochemical cycles of iron.The worldwide carbon nanotubes (CNTs) industry is rapidly expanding with production capacity exceeding several thousand tons per year. After the service lives, CNTs will be ultimately released into the environment with their concentrations in soils increasing by 0.4-147 ng/kg/year and the consequent environmental impacts have received more and more attentions. Effects of CNTs on the redox reactions of Fe have not

  14. 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

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

    PubMed

    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

  16. 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.

  17. 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.

  18. 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.

  19. Effects of the anaerobic respiration of Shewanella oneidensis MR-1 on the stability of extracellular U(VI) nanofibers.

    PubMed

    Jiang, Shenghua; Hur, Hor-Gil

    2013-01-01

    Uranium (VI) is considered to be one of the most widely dispersed and problematic environmental contaminants, due in large part to its high solubility and great mobility in natural aquatic systems. We previously reported that under anaerobic conditions, Shewanella oneidensis MR-1 grown in medium containing uranyl acetate rapidly accumulated long, extracellular, ultrafine U(VI) nanofibers composed of polycrystalline chains of discrete meta-schoepite (UO(3)·2H2O) nanocrystallites. Wild-type MR-1 finally transformed the uranium (VI) nanofibers to uranium (IV) nanoparticles via further reduction. In order to investigate the influence of the respiratory chain in the uranium transformation process, a series of mutant strains lacking a periplasmic cytochrome MtrA, outer membrane (OM) cytochrome MtrC and OmcA, a tetraheme cytochrome CymA anchored to the cytoplasmic membrane, and a trans-OM protein MtrB, were tested in this study. Although all the mutants produced U(VI) nanofibers like the wild type, the transformation rates from U(VI) nanofibers to U(IV) nanoparticles varied; in particular, the mutant with deletion in tetraheme cytochrome CymA stably maintained the uranium (VI) nanofibers, suggesting that the respiratory chain of S. oneidensis MR-1 is probably involved in the stability of extracellular U(VI) nanofibers, which might be easily treated via the physical processes of filtration or flocculation for the remediation of uranium contamination in sediments and aquifers, as well as the recovery of uranium in manufacturing processes.

  20. 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

  1. Cu-doped TiO(2) nanoparticles enhance survival of Shewanella oneidensis MR-1 under ultraviolet light (UV) exposure.

    PubMed

    Wu, Bing; Zhuang, Wei-Qin; Sahu, Manoranjan; Biswas, Pratim; Tang, Yinjie J

    2011-10-01

    It has been shown that photocatalytic TiO(2) nanoparticles (NPs) can be used as an efficient anti-microbial agent under UV light due to generation of reactive oxygen species (ROS), while Shewanella oneidensis MR-1 is a metal-reducing bacterium highly susceptible to UV radiation. Interestingly, we found that the presence of Cu-doped TiO(2) NPs in the cultural medium dramatically increased the survival rates (based on colony-forming unit) of strain MR-1 by over 10,000-fold (incubation without shaking) and ~200 fold (incubation with shaking) after a 2-h exposure to UV light. Gene expression results (via qPCR measurement) indicated that the DNA repair gene recA in MR-1 was significantly induced by UV exposure (indicating cellular damage under UV stress), but the influence of NPs on recA expression was not statistically evident. Plausible explanations to NP attenuation of UV stresses are: 1. TiO(2) based NPs are capable of scattering and absorbing UV light and thus create a shading effect to protect MR-1 from UV radiation; 2. more importantly, Cu-doped TiO(2) NPs can co-agglomerate with MR-1 to form large flocs that improves cells' survival against the environmental stresses. This study improves our understanding of NP ecological impacts under natural solar radiation and provides useful insights to application of photocatalytic-NPs for bacterial disinfection. PMID:21855961

  2. 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.

  3. 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

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

    SciTech Connect

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

    2010-06-24

    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 flexibility of the electron transfer networks as well as central and peripheral carbon metabolism pathways. To understand the factors contributing to the ecophysiological success of Shewanellae, the metabolic network of S. oneidensis MR-1 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 futile cycles, (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

  5. Enhanced performance of hexavalent chromium reducing cathodes in the presence of Shewanella oneidensis MR-1 and lactate.

    PubMed

    Xafenias, Nikolaos; Zhang, Yue; Banks, Charles J

    2013-05-01

    Biocathodes for the reduction of the highly toxic hexavalent chromium (Cr(VI)) were investigated using Shewanella oneidensis MR-1 (MR-1) as a biocatalyst and performance was assessed in terms of current production and Cr(VI) reduction. Potentiostatically controlled experiments (-500 mV vs Ag/AgCl) showed that a mediatorless MR-1 biocathode started up under aerated conditions in the presence of lactate, received 5.5 and 1.7 times more electrons for Cr(VI) reduction over a 4 h operating period than controls without lactate and with lactate but without MR-1, respectively. Cr(VI) reduction was also enhanced, with a decrease in concentration over the 4 h operating period of 9 mg/L Cr(VI), compared to only 1 and 3 mg/L, respectively, in the controls. Riboflavin, an electron shuttle mediator naturally produced by MR-1, was also found to have a positive impact in potentiostatically controlled cathodes. Additionally, a microbial fuel cell (MFC) with MR-1 and lactate present in both anode and cathode produced a maximum current density of 32.5 mA/m(2) (1000 Ω external load) after receiving a 10 mg/L Cr(VI) addition in the cathode, and cathodic efficiency increased steadily over an 8 day operation period with successive Cr(VI) additions. In conclusion, effective and continuous Cr(VI) reduction with associated current production were achieved when MR-1 and lactate were both present in the biocathodes. PMID:23517384

  6. 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

  7. 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.

  8. Estimating the dual-enzyme kinetic parameters for Cr (VI) reduction by Shewanella oneidensis MR-1 from soil column experiments.

    PubMed

    Hossain, Md Akram; Alam, Mahbub; Yonge, David R

    2005-09-01

    Hexavalent chromium (Cr (VI)) contamination of soil and groundwater is considered a major environmental concern. Bioreduction of Cr (VI) to trivalent chromium (Cr (III)) can be considered an effective technology in remediating Cr (VI) contaminated sites. Among the Cr (VI) reducing bacteria, Shewanella oneidensis MR-1 (MR-1) is relatively effective. Reduction of Cr (VI) by MR-1 is defined by the dual-enzyme kinetic model. The feasibility of bioreduction of Cr (VI) is usually evaluated by performing batch experiments which may not accurately represent the subsurface environment. The objective of this paper is to present a rational approach to determine the dual-enzyme reaction kinetics of Cr (VI) reduction by MR-1 from continuous flow soil column experiments which more accurately simulate the subsurface environment. Kinetic parameters obtained from soil column data were found to be much smaller than that obtained from batch data. The stable enzyme induced reaction rate was found to be 127th and the average deactivating enzyme induced reaction rate was 117th of the corresponding values obtained from batch reactor data as reported in literature.

  9. 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

    2014-09-01

    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.

  10. 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.

  11. 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

  12. Involvement of cytochrome c CymA in the anaerobic metabolism of RDX by Shewanella oneidensis MR-1.

    PubMed

    Perreault, Nancy N; Crocker, Fiona H; Indest, Karl J; Hawari, Jalal

    2012-02-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitramine explosive commonly used for military applications that is responsible for severe soil and groundwater contamination. In this study, Shewanella oneidensis MR-1 was shown to efficiently degrade RDX anaerobically (3.5 µmol·h(-1)·(g protein)(-1)) via two initial routes: (1) sequential N-NO(2) reductions to the corresponding nitroso (N-NO) derivatives (94% of initial RDX degradation) and (2) denitration followed by ring cleavage. To identify genes involved in the anaerobic metabolism of RDX, a library of ~2500 mutants of MR-1 was constructed by random transposon mutagenesis and screened for mutants with a reduced ability to degrade RDX compared with the wild type. An RDX-defective mutant (C9) was isolated that had the transposon inserted in the c-type cytochrome gene cymA. C9 transformed RDX at ~10% of the wild-type rate, with degradation occurring mostly via early ring cleavage caused by initial denitration leading to the formation of methylenedinitramine, 4-nitro-2,4-diazabutanal, formaldehyde, nitrous oxide, and ammonia. Genetic complementation of mutant C9 restored the wild-type phenotype, providing evidence that electron transport components have a role in the anaerobic reduction of RDX by MR-1.

  13. 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...

  14. 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.

  15. Hydrogenase- and Outer Membrane c-Type Cytochrome-Facilitated Reduction of Technetium(VII) by Shewanella oneidensis MR-1

    SciTech Connect

    Marshall, Matthew J.; Plymale, Andrew E.; Kennedy, David W.; Shi, Liang; Wang, Zheming; Reed, Samantha B.; Dohnalkova, Alice; Simonson, Cody J.; Liu, Chongxuan; Saffarini, Daad; Romine, Margaret F.; Zachara, John M.; Beliaev, Alex S.; Fredrickson, Jim K.

    2008-01-15

    Pertechnetate, 99Tc(VII)O4-, is a highly mobile radionuclide contaminant at U.S. Department of Energy sites that can be enzymatically reduced by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble Tc(IV)O2(s). In other microorganisms, Tc(VII)O4- reduction is generally considered to be catalyzed by hydrogenase. Here, we provide evidence that although the NiFe hydrogenase of MR-1 was involved in the H2-driven reduction of Tc(VII)O4- (presumably through a direct coupling of H2 oxidation and Tc(VII) reduction), the deletion of both hydrogenase genes did not completely eliminate the ability of MR-1 to reduce Tc(VII). With lactate as the electron donor, mutants lacking the outer membrane c-type cytochromes MtrC and OmcA or the proteins required for the maturation of c-type cytochromes were defective in reducing Tc(VII) to nanoparticulate TcO2·nH2O(s) relative to MR-1 or a NiFe hydrogenase mutant. In addition, reduced MtrC and OmcA were oxidized by Tc(VII)O4-, confirming the capacity for direct electron transfer from these OMCs to TcO4-. c-Type cytochrome-catalyzed Tc(VII) reduction could be a potentially important mechanism in environments where organic electron donor concentrations are sufficient to allow this reaction to dominate.

  16. 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

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

    PubMed

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

    2015-01-23

    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.

  18. 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.

  19. 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.

  20. 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.

  1. Immobilization of Shewanella oneidensis MR-1 in diffusive gradients in thin films for determining metal bioavailability.

    PubMed

    Baker, Paul W; Högstrand, Christer; Lead, Jamie; Pickup, Roger W; Zhang, Hao

    2015-11-01

    Assessing metal bioavailability in soil is important in modeling the effects of metal toxicity on the surrounding ecosystem. Current methods based on diffusive gradient thin films (DGTs) and Gel-Integrated Microelectrode are limited in their availability and sensitivity. To address this, Shewanella oneidensis, an anaerobic iron reducing bacterium, was incorporated into a thin layer of agarose to replace the polyacrylamide gel that is normally present in DGT to form biologically mobilizing DGT (BMDGT). Viability analysis revealed that 16-35% of the cells remained viable within the BMDGTs depending on the culturing conditions over a 20 h period with/without metals. Deployment of BMDGTs in standardized metal solutions showed significant differences to cell-free BMDGTs when cells grown in Luria Broth (LB) were incorporated into BMDGTs and deployed under anaerobic conditions. Deployment of these BMDGTs in hematite revealed no significant differences between BMDGTs and BMDGTs containing heat killed cells. Whether heat killed cells retain the ability to affect bioavailability is uncertain. This is the first study to investigate how a microorganism that was incorporated into a DGT device such as the metal reducing bacteria, S. oneidensis, may affect the mobility of metals.

  2. 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

  3. 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

  4. 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.

  5. Antibody recognition force microscopy shows that outer membrane cytochromes OmcA and MtrC are expressed on the exterior surface of Shewanella oneidensis MR-1

    SciTech Connect

    Lower, Brian H.; Yongsunthon, Ruchirej; Shi, Liang; Wildling, Linda; Gruber, Hermann J.; Wigginton, Nicholas S.; Reardon, Catherine L.; Pinchuk, Grigoriy E.; Droubay, Timothy C.; Boily, Jean F.; Lower, Steven

    2009-05-01

    Antibody-recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells during anaerobic growth, when Fe(III) served as the terminal electron acceptor. OmcA was localized to the interface with hematite, while MtrC was more uniformly displayed on the bacterium’s exterior cell surface. Both cytochromes were also found associated with extracellular material.

  6. 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.

  7. Systematic screening of carbon-based anode materials for microbial fuel cells with Shewanella oneidensis MR-1.

    PubMed

    Kipf, Elena; Koch, Julia; Geiger, Bettina; Erben, Johannes; Richter, Katrin; Gescher, Johannes; Zengerle, Roland; Kerzenmacher, Sven

    2013-10-01

    We present a systematic screening of carbon-based anode materials for microbial fuel cells with Shewanella oneidensis MR-1. Under anoxic conditions nanoporous activated carbon cloth is a superior anode material in terms of current density normalized to the projected anode area and anode volume (24.0±0.3 μA cm(-2) and 482±7 μA cm(-3) at -0.2 vs. SCE, respectively). The good performance can be attributed to the high specific surface area of the material, which is available for mediated electron transfer through self-secreted flavins. Under aerated conditions no influence of the specific surface area is observed, which we attribute to a shift from primary indirect electron transfer by mediators to direct electron transfer via adherent cells. Furthermore, we show that an aerated initial growth phase enhances the current density under subsequent anoxic conditions fivefold when compared to a similar experiment that was conducted under permanently anoxic conditions.

  8. 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.

  9. ArcS, the cognate sensor kinase in an atypical Arc system of Shewanella oneidensis MR-1.

    PubMed

    Lassak, Jürgen; Henche, Anna-Lena; Binnenkade, Lucas; Thormann, Kai M

    2010-05-01

    The availability of oxygen is a major environmental factor for many microbes, in particular for bacteria such as Shewanella species, which thrive in redox-stratified environments. One of the best-studied systems involved in mediating the response to changes in environmental oxygen levels is the Arc two-component system of Escherichia coli, consisting of the sensor kinase ArcB and the cognate response regulator ArcA. An ArcA ortholog was previously identified in Shewanella, and as in Escherichia coli, Shewanella ArcA is involved in regulating the response to shifts in oxygen levels. Here, we identified the hybrid sensor kinase SO_0577, now designated ArcS, as the previously elusive cognate sensor kinase of the Arc system in Shewanella oneidensis MR-1. Phenotypic mutant characterization, transcriptomic analysis, protein-protein interaction, and phosphotransfer studies revealed that the Shewanella Arc system consists of the sensor kinase ArcS, the single phosphotransfer domain protein HptA, and the response regulator ArcA. Phylogenetic analyses suggest that HptA might be a relict of ArcB. Conversely, ArcS is substantially different with respect to overall sequence homologies and domain organizations. Thus, we speculate that ArcS might have adopted the role of ArcB after a loss of the original sensor kinase, perhaps as a consequence of regulatory adaptation to a redox-stratified environment.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Viability and metal reduction of Shewanella oneidensis MR-1 under CO2 stress: implications for ecological effects of CO2 leakage from geologic CO2 sequestration.

    PubMed

    Wu, Bing; Shao, Hongbo; Wang, Zhipeng; Hu, Yandi; Tang, Yinjie J; Jun, Young-Shin

    2010-12-01

    To study potential ecological impacts of CO(2) leakage to shallow groundwater and soil/sediments from geologic CO(2) sequestration (GCS) sites, this work investigated the viability and metal reduction of Shewanella oneidensis MR-1 under CO(2) stress. While MR-1 could grow under high-pressure nitrogen gas (500 psi), the mix of 1% CO(2) with N(2) at total pressures of 15 or 150 psi significantly suppressed the growth of MR-1, compared to the N(2) control. When CO(2) partial pressures were over 15 psi, the growth of MR-1 stopped. The reduced bacterial viability was consistent with the pH decrease and cellular membrane damage under high pressure CO(2). After exposure to 150 psi CO(2) for 5 h, no viable cells survived, the cellular contents were released, and microscopy images confirmed significant cell structure deformation. However, after a relatively short exposure (25 min) to 150 psi CO(2), MR-1 could fully recover their growth within 24 h after the stress was removed, and the reduction of MnO(2) by MR-1 was observed right after the stress was removed. Furthermore, MR-1 survived better if the cells were aggregated rather than suspended, or if pH buffering minerals, such as calcite, were present. To predict the cell viability under different CO(2) pressures and exposure times, a two-parameter mathematical model was developed.

  17. 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.

  18. 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.

  19. 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.

  20. Iron Triggers λSo Prophage Induction and Release of Extracellular DNA in Shewanella oneidensis MR-1 Biofilms

    PubMed Central

    Binnenkade, Lucas; Teichmann, Laura

    2014-01-01

    Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in Shewanella oneidensis MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (ΔoxyR) or iron homeostasis (Δfur) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H2O2). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H2O2 in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA. PMID:24951794

  1. 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...

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

    PubMed

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

    2010-04-01

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

  3. 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.

  4. ShewRegDB: a Visualization Environment for Integration of Experimental and In Silico Derived Regulatory Information in Shewanella oneidensis MR-1

    SciTech Connect

    Syed, Mustafa H; Karpinets, Tatiana V; Leuze, Michael Rex; Kora, Guruprasad H; Romine, Margaret; Uberbacher, Edward C

    2009-01-01

    Shewanella oneidensis MR-1 is an important model organism for environmental research as it has an exceptional metabolic and respiratory versatility regulated by a complex regulatory network. We have developed a database to collect experimental and computational data relating to regulation of gene and protein expression, and, a visualization environment that enables integration of these data types. The regulatory information in the database includes predictions of DNA regulator binding sites, sigma factor binding sites, transcription units, operons, promoters, and RNA regulators including non-coding RNAs, riboswitches, and different types of terminators.

  5. Rapid precipitation of amorphous silica in experimental systems with nontronite (NAu-1) and Shewanella oneidensis MR-1

    NASA Astrophysics Data System (ADS)

    Furukawa, Yoko; O'Reilly, S. E.

    2007-01-01

    Nanometer-size (<50 nm) precipitates of amorphous silica globules were observed in laboratory systems containing nontronite NAu-1, Shewanella oneidensis strain MR-1, and lean aqueous media. Their formation was attributed to the release of polysilicic acids at the expense of dissolving NAu-1, and subsequent polymerization and stabilization mediated by biomolecules. Rapid (<24 h) silica globule formation was confirmed in the immediate vicinity of bacterial cells and extracellular polymeric substances in all experimental systems that contained bacteria, whether the bacteria were respiring dissolved O 2 or Fe(III) originating from NAu-1, and whether the bacteria were viable or heat-killed. Silica globules were not observed in bacteria- and biomolecule-free systems. Thermodynamic calculations using disilicic acid, rather than monomeric silica, as the primary aqueous silica species suggest that the systems may have been supersaturated with respect to amorphous silica even though they appeared to be undersaturated if all aqueous silica was assumed to be monomeric H 4SiO 4. The predominant aqueous silica species in the experimental systems was likely polysilicic acids because aqueous silica was continuously supplied from the concurrent dissolution of aluminosilicate. Further polymerization and globule formation may have been driven by the presence of polyamines, a group of biologically produced compounds that are known to drive amorphous silica precipitation in diatom frustules. Globules were likely to be positively charged in our systems due to chemisorption of organic polycations onto silica surfaces that would have been otherwise negatively charged. We propose the following steps for the formation of nanometer-size silica globules in our experimental systems: (i) continuous supply of polysilicic acids due to NAu-1 dissolution; (ii) polysilicic acid polymerization to form <50 nm silica globules and subsequent stabilization mediated by microbially produced polyamines

  6. 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.

  7. 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

  8. 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.

  9. 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}.

  10. 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 ° .

  11. 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. 

  12. 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.

  13. Evidence-Based Annotation of Gene Function in Shewanella oneidensis MR-1 Using Genome-Wide Fitness Profiling across 121 Conditions

    PubMed Central

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Shao, Wenjun; Baumohl, Jason K.; Xu, Zhuchen; Nguyen, Michelle; Tamse, Raquel; Davis, Ronald W.; Arkin, Adam P.

    2011-01-01

    Most genes in bacteria are experimentally uncharacterized and cannot be annotated with a specific function. Given the great diversity of bacteria and the ease of genome sequencing, high-throughput approaches to identify gene function experimentally are needed. Here, we use pools of tagged transposon mutants in the metal-reducing bacterium Shewanella oneidensis MR-1 to probe the mutant fitness of 3,355 genes in 121 diverse conditions including different growth substrates, alternative electron acceptors, stresses, and motility. We find that 2,350 genes have a pattern of fitness that is significantly different from random and 1,230 of these genes (37% of our total assayed genes) have enough signal to show strong biological correlations. We find that genes in all functional categories have phenotypes, including hundreds of hypotheticals, and that potentially redundant genes (over 50% amino acid identity to another gene in the genome) are also likely to have distinct phenotypes. Using fitness patterns, we were able to propose specific molecular functions for 40 genes or operons that lacked specific annotations or had incomplete annotations. In one example, we demonstrate that the previously hypothetical gene SO_3749 encodes a functional acetylornithine deacetylase, thus filling a missing step in S. oneidensis metabolism. Additionally, we demonstrate that the orphan histidine kinase SO_2742 and orphan response regulator SO_2648 form a signal transduction pathway that activates expression of acetyl-CoA synthase and is required for S. oneidensis to grow on acetate as a carbon source. Lastly, we demonstrate that gene expression and mutant fitness are poorly correlated and that mutant fitness generates more confident predictions of gene function than does gene expression. The approach described here can be applied generally to create large-scale gene-phenotype maps for evidence-based annotation of gene function in prokaryotes. PMID:22125499

  14. Comparative proteomics reveal the impact of OmcA/MtrC deletion on Shewanella oneidensis MR-1 in response to hexavalent chromium exposure.

    PubMed

    Wang, Chao; Chen, Juan; Hu, Wen-Jun; Liu, Ji-Yun; Zheng, Hai-Lei; Zhao, Feng

    2014-12-01

    Hexavalent chromium [Cr(VI)] is a priority pollutant causing serious environmental issues. Microbial reduction provides an alternative strategy for Cr(VI) remediation. The dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, was employed to study Cr(VI) reduction and toxicity in this work. To understand the effect of membrane cytochromes on Cr(VI) response, a comparative protein profile analysis from S. oneidensis MR-1 wild type and its mutant of deleting OmcA and MtrC (△omcA/mtrC) was conducted using two-dimensional electrophoresis (2-DE) technology. The 2-DE patterns were compared, and the proteins with abundant changes of up to twofold in the Cr(VI) treatment were detected. Using mass spectrometry, 38 and 45 differentially abundant proteins were identified in the wild type and the mutant, respectively. Among them, 25 proteins were shared by the two strains. The biological functions of these identified proteins were analyzed. Results showed that Cr(VI) exposure decreased the abundance of proteins involved in transcription, translation, pyruvate metabolism, energy production, and function of cellular membrane in both strains. There were also significant differences in protein expressions between the two strains under Cr(VI) treatment. Our results suggest that OmcA/MtrC deletion might result in the Cr(VI) toxicity to outer membrane and decrease assimilation of lactate, vitamin B12, and cystine. When carbohydrate metabolism was inhibited by Cr(VI), leucine and sulfur metabolism may act as the important compensatory mechanisms in the mutant. Furthermore, the mutant may regulate electron transfer in the inner membrane and periplasm to compensate for the deletion of OmcA and MtrC in Cr(VI) reduction.

  15. The conjugated oligoelectrolyte DSSN+ enables exceptional coulombic efficiency via direct electron transfer for anode-respiring Shewanella oneidensis MR-1-a mechanistic study.

    PubMed

    Kirchhofer, Nathan D; Chen, Xiaofen; Marsili, Enrico; Sumner, James J; Dahlquist, Frederick W; Bazan, Guillermo C

    2014-10-14

    Shewanella oneidensis MR-1 was cultivated on lactate with poised graphite electrode acceptors (E = +0.2 V vs. Ag/AgCl) in order to explore the basis for sustained increases in anodic current output following the addition of the lipid-intercalating conjugated oligoelectrolyte (COE), 4,4'-bis(4'-(N,N-bis(6''-(N,N,N-trimethylammonium)hexyl)amino)-styryl)stilbene tetraiodide (DSSN+). Microbial cultures, which were spiked with DSSN+, exhibit a ∼2.2-fold increase in charge collected, a ∼3.1-fold increase in electrode colonization by S. oneidensis, and a ∼1.7-fold increase in coulombic efficiency from 51 ± 10% to an exceptional 84 ± 7% without obvious toxicity effects. Direct microbial biofilm voltammetry reveals that DSSN+ rapidly and sustainably increases cytochrome-based direct electron transfer and subsequently increases flavin-based mediated electron transfer. Control experiments indicate that DSSN+ does not contribute to the current in the absence of bacteria. PMID:25171764

  16. 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

  17. Role of outer-membrane cytochromes MtrC and OmcA in the biomineralization of ferrihydrite by Shewanella oneidensis MR-1.

    PubMed

    Reardon, C L; Dohnalkova, A C; Nachimuthu, P; Kennedy, D W; Saffarini, D A; Arey, B W; Shi, L; Wang, Z; Moore, D; McLean, J S; Moyles, D; Marshall, M J; Zachara, J M; Fredrickson, J K; Beliaev, A 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 cytochromes (OMCs), MtrC and OmcA, were characterized for the ability to reduce ferrihydrite (FH) using a suite of microscopic, spectroscopic, and biochemical techniques. Analysis of purified recombinant proteins demonstrated that both cytochromes undergo rapid electron exchange with FH in vitro with MtrC displaying faster transfer rates than OmcA. Immunomicroscopy with cytochrome-specific antibodies revealed that MtrC co-localizes with iron solids on the cell surface while OmcA exhibits a more diffuse distribution over the cell surface. After 3-day incubation of MR-1 with FH, pronounced reductive transformation mineral products were visible by electron microscopy. Upon further incubation, the predominant phases identified were ferrous phosphates including vivianite [Fe(3)(PO(4))(2)x8H(2)O] and a switzerite-like phase [Mn(3),Fe(3)(PO(4))(2)x7H(2)O] that were heavily colonized by MR-1 cells with surface-exposed outer-membrane cytochromes. In the absence of both MtrC and OmcA, the cells ability to reduce FH was significantly hindered and no mineral transformation products were detected. Collectively, these results highlight the importance of the outer-membrane cytochromes in the reductive transformation of FH and support a role for direct electron transfer from the OMCs at the cell surface to the mineral.

  18. Generation and Validation of a Shewanella oneidensis MR-1 Clone Set for Protein Expression and Phage Display

    PubMed Central

    Wang, Xiaohu; Petrosino, Joseph; Hemphill, Lisa; Wan, Xiufeng; Leaphart, Adam B.; Weinstock, George M.; Palzkill, Timothy; Zhou, Jizhong

    2008-01-01

    A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST- tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro. PMID:18714347

  19. The surface properties of Shewanella putrefaciens 200 and S. oneidensis MR-1: the effect of pH and terminal electron acceptors

    PubMed Central

    2013-01-01

    Background We investigated the surface characteristics of two strains of Shewanella sp., S. oneidensis MR-1 and S. putrefaciens 200, that were grown under aerobic conditions as well as under anaerobic conditions with trimethylamine oxide (TMAO) as the electron acceptor. The investigation focused on the experimental determination of electrophoretic mobility (EPM) under a range of pH and ionic strength, as well as by subsequent modeling in which Shewanella cells were considered to be soft particles with water- and ion-permeable outermost layers. Results The soft layer of p200 is significantly more highly charged (i.e., more negative) than that of MR-1. The effect of electron acceptor on the soft particle characteristics of Shewanella sp. is complex. The fixed charge density, which is a measure of the deionized and deprotonated functional groups in the soft layer polymers, is slightly greater (i.e., more negative) for aerobically grown p200 than for p200 grown with TMAO. On the other hand, the fixed charge density of aerobically grown MR1 is slightly less than that of p200 grown with TMAO. The effect of pH on the soft particle characteristics is also complex, and does not exhibit a clear pH-dependent trend. Conclusions The Shewanella surface characteristics were attributed to the nature of the outermost soft layer, the extracellular polymeric substances (EPS) in case of p200 and lypopolysaccharides (LPS) in case of MR1 which generally lacks EPS. The growth conditions (i.e., aerobic vs. anaerobic TMAO) have an influence on the soft layer characteristics of Shewanella sp. cells. Meanwhile, the clear pH dependency of the mechanical and morphological characteristics of EPS and LPS layers, observed in previous studies through atomic force microscopy, adhesion tests and spectroscopies, cannot be corroborated by the electrohydrodynamics-based soft particle characteristics which does not exhibited a clear pH dependency in this study. While the electrohydrodynamics-based soft

  20. Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT2

    SciTech Connect

    Xiong, Yijia; Chen, Baowei; Shi, Liang; Fredrickson, Jim K.; Bigelow, Diana J.; Squier, Thomas C.

    2011-10-14

    Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) at its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore, carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC which is dependent on the presence of a functional type-2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 hr-1 that is insensitive to O2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 hr-1) that are consistent

  1. 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.

  2. 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.

  3. Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1.

    PubMed

    Qian, Yufeng; Paquete, Catarina M; Louro, Ricardo O; Ross, Daniel E; Labelle, Edward; Bond, Daniel R; Tien, Ming

    2011-07-19

    In the model microbe Shewanella oneidensis, multi-heme proteins are utilized for respiratory metabolism where metals serve as the terminal electron acceptor. Among those is the periplasm-localized small tetraheme cytochrome (STC). STC has been extensively characterized structurally and electrochemically to which electron flow in and out of the protein has been modeled. However, until the present work, no kinetic studies have been performed to probe the route of electron flow or to determine the iron-binding site on STC. Using iron chelated by EDTA, NTA, or citrate, we have used chemical modification, site-directed mutagenesis along with isothermal titration calorimetry (ITC), and stopped-flow measurements to identify the iron binding site of STC. Chemical modifications of STC revealed that carboxyl groups on STC are involved in binding of EDTA-Fe(3+). Scanning mutagenesis was performed on Asp and Glu to probe the putative iron-binding site on STC. Two STC mutants (D21N; D80N) showed ∼70% decrease in observed electron transfer rate constant with EDTA-Fe(3+) from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe(3+) was further confirmed by a significant decrease (>10-fold) in iron binding affinity.

  4. Deciphering the aggregation mechanism of bacteria (Shewanella oneidensis MR1) in the presence of polyethyleneimine: Effects of the exopolymeric superstructure and polymer molecular weight.

    PubMed

    Krapf, Marie-Eve M; Lartiges, Bruno; Merlin, Christophe; Francius, Grégory; Ghanbaja, Jaafar; Duval, Jérôme F L

    2016-03-01

    Aggregation tests between bacteria and Polyethyleneimine (PEI) of low (600g/mol) and high (750,000g/mol) molecular weight were performed in order to address the physico-chemical mechanisms underlying the interactions between cationic polymer and bacterial membranes. The selected strain, Schewanella oneidensis MR-1, produces a lipopolysaccharide (LPS) of various lengths depending on the growth conditions. Optical density, bioaggregate size, electrophoretic mobility measurements, TEM and AFM observations, and cell lysis tests (crystal violet release), were collected to describe the PEI-mediated aggregation of LPS-O-antigen-free and LPS-O-antigen-decorated bacteria. The results show that PEI of low molecular weight (600g/mol) fails to aggregate bacteria, whereas PEIs of higher molecular weight (60,000 and 750,000g/mol) lead to flocculation at low polymer concentrations. In addition, the LPS-O antigen bacterial superstructure is shown to act as a protective barrier, thus delaying the harmful effects of the cationic polymer. Despite this protection, the interaction of bacterial membranes with increasing concentrations of PEI leads to a series of deleterious processes including biosurface modification (peeling, membrane permeabilization and/or lysis), aggregation of bacterial cells, and complexation of PEI with both released biosurface fragments and cytoplasmic residues issued from lysis. PMID:26774052

  5. Deciphering the aggregation mechanism of bacteria (Shewanella oneidensis MR1) in the presence of polyethyleneimine: Effects of the exopolymeric superstructure and polymer molecular weight.

    PubMed

    Krapf, Marie-Eve M; Lartiges, Bruno; Merlin, Christophe; Francius, Grégory; Ghanbaja, Jaafar; Duval, Jérôme F L

    2016-03-01

    Aggregation tests between bacteria and Polyethyleneimine (PEI) of low (600g/mol) and high (750,000g/mol) molecular weight were performed in order to address the physico-chemical mechanisms underlying the interactions between cationic polymer and bacterial membranes. The selected strain, Schewanella oneidensis MR-1, produces a lipopolysaccharide (LPS) of various lengths depending on the growth conditions. Optical density, bioaggregate size, electrophoretic mobility measurements, TEM and AFM observations, and cell lysis tests (crystal violet release), were collected to describe the PEI-mediated aggregation of LPS-O-antigen-free and LPS-O-antigen-decorated bacteria. The results show that PEI of low molecular weight (600g/mol) fails to aggregate bacteria, whereas PEIs of higher molecular weight (60,000 and 750,000g/mol) lead to flocculation at low polymer concentrations. In addition, the LPS-O antigen bacterial superstructure is shown to act as a protective barrier, thus delaying the harmful effects of the cationic polymer. Despite this protection, the interaction of bacterial membranes with increasing concentrations of PEI leads to a series of deleterious processes including biosurface modification (peeling, membrane permeabilization and/or lysis), aggregation of bacterial cells, and complexation of PEI with both released biosurface fragments and cytoplasmic residues issued from lysis.

  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. Uranium reduction by Shewanella oneidensis MR-1 as a function of NaHCO3 concentration: surface complexation control of reduction kinetics.

    PubMed

    Sheng, Ling; Fein, Jeremy B

    2014-04-01

    It is crucial to determine the controls on the kinetics of U(VI) bioreduction in order to understand and model the fate and mobility of U in groundwater systems and also to enhance the effectiveness of U bioremediation strategies. In this study, we measured the rate of U(VI) reduction by Shewanella oneidensis strain MR-1 as function of NaHCO3 concentration. The experiments demonstrate that increasing concentrations of NaHCO3 in the system lead to slower U(VI) reduction kinetics. The NaHCO3 concentration also strongly affects the speciation of U(VI) on the bacterial cell envelope. We used a thermodynamic surface complexation modeling approach to determine the speciation and concentration of U(VI) adsorbed onto the bacteria as a function of the NaHCO3 concentration in the experimental systems. We observed a strong positive correlation between the measured U(VI) reduction rates and the calculated total concentration of U(VI) surface complexes formed on the bacterial cell envelope. This positive correlation indicates that the speciation and concentration of U(VI) adsorbed on the bacterial cell envelope control the kinetics of U(VI) bioreduction under the experimental conditions. The results of this study serve as a basis for developing speciation-based kinetic rate laws for enzymatic reduction of U(VI) by bacteria. PMID:24576101

  8. Comparative c-type cytochrome expression analysis in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C grown with soluble and insoluble oxidised metal electron acceptors

    SciTech Connect

    Nissen, Silke; Liu, Xiaoxin; Chourey, Karuna; Hettich, Robert {Bob} L; Wagner, Darlene D; Pffifner, Susan; Loeffler, Frank E

    2012-01-01

    The genomes of Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C encode 40 and 69 putative c-type cytochrome genes, respectively. Deletion mutant and biochemical studies have assigned specific functions to a few c-type cytochromes involved in electron transfer to oxidised metals in Shewanella oneidensis strain MR-1. Although promising, the genetic approach is limited to gene deletions that produce a distinct phenotype, and organism for which a genetic system is available. To more comprehensively investigate and compare c-type cytochrome expression in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C, proteomic measurements were used to characterise lysates of cells grown with soluble Fe(III) (as ferric citrate) and insoluble Mn(IV) (as MnO2) as electron acceptors. Strain MR-1 expressed 19 and 20, and strain 2CP-C expressed 27 and 25 c-type cytochromes when grown with Fe(III) and Mn(IV), respectively. The majority of c-type cytochromes (77% for strain MR-1 and 63% for strain 2CP-C) were expressed under both growth conditions; however, the analysis also revealed unique c-type cytochromes that were specifically expressed in cells grown with soluble Fe(III) or insoluble Mn(IV). Proteomic characterisation proved to be a promising approach for determining the c-type cytochrome complement expressed under different growth conditions, and will help elucidating the specific functions of more c-type cytochromes that are the basis for Shewanella and Anaeromyxobacter respiratory versatility.

  9. 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.

  10. Kinetic Characterization of OmcA and MtrC, Terminal Reductases Involved in Respiratory Electron Transfer for Dissimilatory Iron Reduction in Shewanella oneidensis MR-1

    PubMed Central

    Ross, Daniel E.; Brantley, Susan L.; Tien, Ming

    2009-01-01

    We have used scaling kinetics and the concept of kinetic competence to elucidate the role of hemeproteins OmcA and MtrC in iron reduction by Shewanella oneidensis MR-1. Second-order rate constants for OmcA and MtrC were determined by single-turnover experiments. For soluble iron species, a stopped-flow apparatus was used, and for the less reactive iron oxide goethite, a conventional spectrophotometer was used to measure rates. Steady-state experiments were performed to obtain molecular rate constants by quantifying the OmcA and MtrC contents of membrane fractions and whole cells by Western blot analysis. For reduction of soluble iron, rates determined from transient-state experiments were able to account for rates obtained from steady-state experiments. However, this was not true with goethite; rate constants determined from transient-state experiments were 100 to 1,000 times slower than those calculated from steady-state experiments with membrane fractions and whole cells. In contrast, addition of flavins to the goethite experiments resulted in rates that were consistent with both transient- and steady-state experiments. Kinetic simulations of steady-state results with kinetic constants obtained from transient-state experiments supported flavin involvement. Therefore, we show for the first time that OmcA and MtrC are kinetically competent to account for catalysis of soluble iron reduction in whole Shewanella cells but are not responsible for electron transfer via direct contact alone with insoluble iron-containing minerals. This work supports the hypothesis that electron shuttles are important participants in the reduction of solid Fe phases by this organism. PMID:19542342

  11. 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

  12. 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

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

    SciTech Connect

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

    2006-07-01

    SUMMARY Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium that is capable of using insoluble oxidized metals, such as manganese [Mn(III, IV)] and iron [Fe(III)] oxides and oxyhydroxides, as terminal electron acceptors during anaerobic respiration. The ability of S. oneidensis MR-1 to reduce oxidized Mn and/or Fe has previously been linked to OmcA and MtrC: two decaheme c-type cytochromes that are localized to the outer membrane. To investigate how the electron transport proteins OmcA and MtrC are organized, we expressed and purified recombinant OmcA and MtrC from wild type S. oneidensis MR-1 as well as a mutant that lacked OmcA and MtrC (ΔomcA/mtrC). After purification to the nearly electrophoretic homogeneity from the ΔomcA/mtrC mutant, the recombinant OmcA and MtrC exhibited the characteristics of c-type cytochromes, and each of their polypeptides was confirmed to contain 10 hemes. When purified from wild type cells, endogenous MtrC or OmcA was always co-purified with recombinant OmcA or MtrC, respectively. Fluorescence polarization experiment showed that recombinant OmcA bound to the FlAsH-labeled MtrC with a dissociation constant of 7 ×10-7 M. The purified recombinant OmcA or MtrC alone displayed intrinsic ferric reductase activity with NADH used as an electron donor. Ferric reductase specific activity increased by 35 to 41% when nearly equimolar concentrations of OmcA and MtrC were assayed relative to the two proteins assayed independently. These results demonstrate that OmcA and MtrC directly interact with each other to form a stable complex with high ferric reductase activity.

  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. 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.

  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. Transcriptional Analysis of Shewanella oneidensis MR-1 with an Electrode Compared to Fe(III)Citrate or Oxygen as Terminal Electron Acceptor

    PubMed Central

    Rosenbaum, Miriam A.; Bar, Haim Y.; Beg, Qasim K.; Segrè, Daniel; Booth, James; Cotta, Michael A.; Angenent, Largus T.

    2012-01-01

    Shewanella oneidensis is a target of extensive research in the fields of bioelectrochemical systems and bioremediation because of its versatile metabolic capabilities, especially with regard to respiration with extracellular electron acceptors. The physiological activity of S. oneidensis to respire at electrodes is of great interest, but the growth conditions in thin-layer biofilms make physiological analyses experimentally challenging. Here, we took a global approach to evaluate physiological activity with an electrode as terminal electron acceptor for the generation of electric current. We performed expression analysis with DNA microarrays to compare the overall gene expression with an electrode to that with soluble iron(III) or oxygen as the electron acceptor and applied new hierarchical model-based statistics for the differential expression analysis. We confirmed the differential expression of many genes that have previously been reported to be involved in electrode respiration, such as the entire mtr operon. We also formulate hypotheses on other possible gene involvements in electrode respiration, for example, a role of ScyA in inter-protein electron transfer and a regulatory role of the cbb3-type cytochrome c oxidase under anaerobic conditions. Further, we hypothesize that electrode respiration imposes a significant stress on S. oneidensis, resulting in higher energetic costs for electrode respiration than for soluble iron(III) respiration, which fosters a higher metabolic turnover to cover energy needs. Our hypotheses now require experimental verification, but this expression analysis provides a fundamental platform for further studies into the molecular mechanisms of S. oneidensis electron transfer and the physiologically special situation of growth on a poised-potential surface. PMID:22319591

  18. Global Molecular Characterization of the Chromate Stress Response in Shewanella oneidensis MR-1: Identification of a Putative DNA-Binding Response Regulator and Azoreductase Involved in Cr(VI) Detoxification

    SciTech Connect

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

    2006-04-05

    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). Effective bioremediation of Cr-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 is focused on the identification and functional analysis of genes/proteins comprising the response pathways for chromate detoxification and/or reduction. Previous transcriptomic profiling and whole-cell proteomic analyses 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. Here we describe a detailed functional analysis of SO2426 and SO3585 in order to begin to understand the role of these proteins in the cellular response to chromate. The protein products encoded by genes so2426 and so3585 were expressed and detected only in chromate-shocked samples as determined by multidimensional high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Both genes were also highly induced (>46-fold) in MR-1 cells actively reducing chromate based on whole-genome microarray analysis. We have created in-frame deletions of the so2426 and so3585 loci in the MR-1 chromosome and have characterized the phenotype of the resulting mutants in the presence of varying concentrations of Cr, Cu, Co, Sr, and H{sub 2}O{sub 2} under aerobic respiratory conditions. Growth studies indicated that the so2426 deletion mutant was more sensitive to heavy metals compared to the WT reference, and chromate reduction by the so2426 mutant was impaired significantly. The growth response of the mutant to H{sub 2}O{sub 2} was similar to that of MR-1. To gain insight into the regulon of this response regulator, MR-1 microarrays were used to

  19. 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.

  20. Shewanella oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms

    PubMed Central

    Lies, Douglas P.; Hernandez, Maria E.; Kappler, Andreas; Mielke, Randall E.; Gralnick, Jeffrey A.; Newman, Dianne K.

    2005-01-01

    We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this “Fe-bead” system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance. PMID:16085832

  1. 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.

  2. Contributions of the [NiFe]- and [FeFe]-hydrogenase to H2 production in Shewanella oneidensis MR-1 as revealed by isotope ratio analysis of evolved H(2).

    PubMed

    Kreuzer, Helen W; Hill, Eric A; Moran, James J; Bartholomew, Rachel A; Yang, Hui; 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 are 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 organisms, 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 natural-abundance stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.

  3. 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.

  4. High-syn conformation of uridine and asymmetry of the hexameric molecule revealed in the high-resolution structures of Shewanella oneidensis MR-1 uridine phosphorylase in the free form and in complex with uridine.

    PubMed

    Safonova, Tatyana N; Mikhailov, Sergey N; Veiko, Vladimir P; Mordkovich, Nadezhda N; Manuvera, Valentin A; Alekseev, Cyril S; Kovalchuk, Mikhail V; Popov, Vladimir O; Polyakov, Konstantin M

    2014-12-01

    Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP from Shewanella oneidensis MR-1 (SoUP) was performed in Escherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-syn conformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine.

  5. Concerted action of two subunits of the functional dimer of Shewanella oneidensis MR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme.

    PubMed

    Safonova, T N; Mordkovich, N N; Veiko, V P; Okorokova, N A; Manuvera, V A; Dorovatovskii, P V; Popov, V O; Polyakov, K M

    2016-02-01

    Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacterium Shewanella oneidensis MR-1 (SoUP) was determined at 1.68 Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

  6. 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

  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

    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.

  8. MR1 discovery.

    PubMed

    Hashimoto, Keiichiro

    2016-08-01

    The moment of MR1 discovery is described. The MR1 gene is the first and the last reported human MHC-related gene intentionally isolated from the human genome composed of three billion base pairs. Evolutionary considerations formed the basis of its isolation. Some details surrounding the moment and some retrospective descriptions with various kinds of encounters are also included.

  9. Fluxome analysis using GC-MS

    PubMed Central

    Wittmann, Christoph

    2007-01-01

    Fluxome analysis aims at the quantitative analysis of in vivo carbon fluxes in metabolic networks, i. e. intracellular activities of enzymes and pathways. It allows investigating the effects of genetic or environmental modifications and thus precisely provides a global perspective on the integrated genetic and metabolic regulation within the intact metabolic network. The experimental and computational approaches developed in this area have revealed fascinating insights into metabolic properties of various biological systems. Most of the comprehensive approaches for metabolic flux studies today involve isotopic tracer studies and GC-MS for measurement of the labeling pattern of metabolites. Initially developed and applied mainly in the field of biomedicine these GC-MS based metabolic flux approaches have been substantially extended and optimized during recent years and today display a key technology in metabolic physiology and biotechnology. PMID:17286851

  10. 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. PMID:26632613

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

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

    PubMed

    Burns, Justin L; Ginn, Brian R; Bates, David J; Dublin, Steven N; Taylor, Jeanette V; Apkarian, Robert P; Amaro-Garcia, Samary; Neal, Andrew L; Dichristina, Thomas J

    2010-01-01

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

  17. 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.

  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. AMT Tag Approach to Proteomic Characterization of Deinococcus Radiodurans and Shewanella Oneidensis

    SciTech Connect

    Lipton, Mary S.; Romine, Margaret F.; Monroe, Matthew E.; Elias, Dwayne A.; Pasa-Tolic, Liljiana; Anderson, Gordon A.; Anderson, David J.; Fredrickson, Jim K.; Hixson, Kim K.; Masselon, Christophe D.; Mottaz, Heather M.; Tolic, Nikola; Smith, Richard D.

    2006-09-01

    Biology is transitioning from a largely qualitative, mostly descriptive science to a quantitative and ultimately predictive science. Advances in high throughput DNA sequencing have made increasing numbers of genome sequences available and enabled a “systems” level analysis of complex biological organisms. The ability to quantitatively measure the array of proteins, also termed the proteome, in prokaryotic cells and communities of cells is key to understanding microbial systems. This chapter focuses on the utility of the AMT tag mass spectrometric approach used to characterize the proteomes of two microbes, Deinococcus radiodurans and Shewanella oneidensis MR-1.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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. PMID:26479486

  10. 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. PMID:27092947

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. Adoptive T Cell Therapy Targeting CD1 and MR1

    PubMed Central

    Guo, Tingxi; Chamoto, Kenji; Hirano, Naoto

    2015-01-01

    Adoptive T cell immunotherapy has demonstrated clinically relevant efficacy in treating malignant and infectious diseases. However, much of these therapies have been focused on enhancing, or generating de novo, effector functions of conventional T cells recognizing HLA molecules. Given the heterogeneity of HLA alleles, mismatched patients are ineligible for current HLA-restricted adoptive T cell therapies. CD1 and MR1 are class I-like monomorphic molecules and their restricted T cells possess unique T cell receptor specificity against entirely different classes of antigens. CD1 and MR1 molecules present lipid and vitamin B metabolite antigens, respectively, and offer a new front of targets for T cell therapies. This review will cover the recent progress in the basic research of CD1, MR1, and their restricted T cells that possess translational potential. PMID:26052329

  17. 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

  18. 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

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

    PubMed

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

    2010-07-01

    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 gamma-proteobacterium, Shewanella oneidensis MR-1, upon incubation with aqueous silver nitrate solution. Further characterization of these particles revealed that the crystals consist of small, reasonably monodispersed spheres in the 2-11 nm size range (average of 4 +/- 1.5 nm). The bactericidal effect of these nanoparticles (biogenic-Ag) is compared to chemically synthesized silver nanoparticles (colloidal-Ag and oleate capped silver nanoparticles, oleate-Ag) and assessed using Gram-negative (E. coli and S. oneidensis) and Gram-positive (B. subtilis) bacteria. Relative toxicity was based on the diameter of inhibition zone in disk diffusion tests, minimum inhibitory concentrations, live/dead assays, and atomic force microscopy. From a toxicity perspective, strain-dependent inhibition depended on the synthesis procedure and the surface coat. Biogenic-Ag was found to be of higher toxicity compared to colloidal-Ag for all three strains tested, whereas E. coli and S. oneidensis were found to be more resistant to either of these nanoparticles than B. subtilis. In contrast, oleate-Ag was not toxic to any of the bacteria. These findings have implications for the potential uses of Ag nanomaterials and for their fate in biological and environmental systems.

  20. 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

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

    PubMed

    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

  2. Transcriptomic and Proteomic Characterization of the Fur Modulon in the Metal-Reducing Bacterium Shewanella oneidensis

    PubMed Central

    Wan, Xiu-Feng; VerBerkmoes, Nathan C.; McCue, Lee Ann; Stanek, Dawn; Connelly, Heather; Hauser, Loren J.; Wu, Liyou; Liu, Xueduan; Yan, Tingfen; Leaphart, Adam; Hettich, Robert L.; Zhou, Jizhong; Thompson, Dorothea K.

    2004-01-01

    The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium. We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S. oneidensis Fur regulon. Using this integrated approach, we identified nine probable operons (containing 24 genes) and 15 individual open reading frames (ORFs), either with unknown functions or encoding products annotated as transport or binding proteins, that are predicted to be direct targets of Fur-mediated repression. This study suggested, for the first time, possible roles for four operons and eight ORFs with unknown functions in iron metabolism or iron transport-related functions. Proteomic analysis clearly identified a number of transporters, binding proteins, and receptors related to iron uptake that were up-regulated in response to a fur deletion and verified the expression of nine genes originally annotated as pseudogenes. Comparison of the transcriptome and proteome data revealed strong correlation for genes shown to be undergoing large changes at the transcript level. A number of genes encoding components of the electron transport system were also differentially expressed in a fur deletion mutant. The gene omcA (SO1779), which encodes a decaheme cytochrome c, exhibited significant decreases in both mRNA and protein abundance in the fur mutant and possessed a strong candidate Fur-binding site in its upstream region, thus suggesting that omcA may be a direct target of Fur activation. PMID:15576789

  3. 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.

  4. Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes.

    PubMed

    Meyer, Terry E; Tsapin, Alexandre I; Vandenberghe, Isabel; de Smet, Lina; Frishman, Dmitrij; Nealson, Kenneth H; Cusanovich, Michael A; van Beeumen, Jozef J

    2004-01-01

    Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. The molecular basis for Mucosal-Associated Invariant T cell recognition of MR1 proteins

    PubMed Central

    López-Sagaseta, Jacinto; Dulberger, Charles L.; Crooks, James E.; Parks, Chelsea D.; Luoma, Adrienne M.; McFedries, Amanda; Van Rhijn, Ildiko; Saghatelian, Alan; Adams, Erin J.

    2013-01-01

    Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved αβ T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 Å complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 α1 and α2 helices, similar to classical αβ TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the α-chain, focused on the MR1 α2 helix. TCR β-chain contacts are mostly through the variable CDR3β loop that is positioned proximal to the CDR3α loop directly over the MR1 open groove. The elucidation of the MAIT TCR/MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of αβ T-cell recognition of MHC and MHC-like ligands. PMID:23613577

  10. 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.

  11. 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

  12. Core fluxome and metafluxome of lactic acid bacteria under simulated cocoa pulp fermentation conditions.

    PubMed

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

    2013-09-01

    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 (13)C labeling approach allowed to quantify carbon fluxes during simulated cocoa fermentation by (i) parallel (13)C studies with [(13)C6]glucose, [1,2-(13)C2]glucose, and [(13)C6]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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

    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.

  14. Effects of bio-Au nanoparticles on electrochemical activity of Shewanella oneidensis wild type and ΔomcA/mtrC mutant.

    PubMed

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

    2013-11-22

    Both Shewanella oneidensis MR-1 wild type and its mutant ΔomcA/mtrC are capable of transforming Au(III) into Au nanoparticles (AuNPs). Cyclic voltammetry reveals a decrease in redox current after the wild type is exposed to Au(III) 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 Au(III), 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.

  15. Comparative fluxome and metabolome analysis for overproduction of succinate in Escherichia coli.

    PubMed

    Taymaz-Nikerel, Hilal; De Mey, Marjan; Baart, Gino J E; Maertens, Jo; Foulquié-Moreno, Maria Remedios; Charlier, Daniel; Heijnen, Joseph J; van Gulik, Walter M

    2016-04-01

    An aerobic succinate-producing Escherichia coli mutant was compared to its wild-type by quantitatively analyzing both the metabolome and fluxome, during glucose-limited steady-state and succinate excess dynamic conditions, in order to identify targets for further strain engineering towards more efficient succinate production. The mutant had four functional mutations under the conditions investigated: increased expression of a succinate exporter (DcuC), deletion of a succinate importer (Dct), deletion of succinate dehydrogenase (SUCDH) and expression of a PEP carboxylase (PPC) with increased capacity due to a point mutation. The steady-state and dynamic patterns of the intracellular metabolite levels and fluxes in response to changes were used to locate the quantitative differences in the physiology/metabolism of the mutant strain. Unexpectedly the mutant had a higher energy efficiency, indicated by a much lower rate of oxygen consumption, under glucose-limited conditions, caused by the deletion of the transcription factors IclR and ArcA. Furthermore the mutant had a much lower uptake capacity for succinate (26-fold) and oxygen (17-fold under succinate excess) compared to the wild-type strain. The mutant strain produced 7.9 mmol.CmolX(-1).h(-1) succinate during chemostat cultivation, showing that the choice of the applied genetic modifications was a successful strategy. Furthermore, the applied genetic modifications resulted in multiple large changes in metabolite levels (FBP, pyruvate, 6PG, NAD(+) /NADH ratio, α-ketogluarate) corresponding to large changes in fluxes. Compared to the wild-type a considerable flux shift occurred from the tricarboxylic acid (TCA) cycle to the oxidative part of the pentose phosphate pathway, including an inversion of the pyruvate kinase flux. The mutant responded very differently to excess of succinate, with a remarkable possible reversal of the TCA cycle. The mutant and the wild-type both showed homeostatic behaviour with respect

  16. 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.

  17. 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.

  18. MHC class I-related molecule, MR1, and mucosal-associated invariant T cells.

    PubMed

    Franciszkiewicz, Katarzyna; Salou, Marion; Legoux, Francois; Zhou, Qian; Cui, Yue; Bessoles, Stéphanie; Lantz, Olivier

    2016-07-01

    The MHC-related 1, MR1, molecule presents a new class of microbial antigens (derivatives of the riboflavin [Vitamin B2] biosynthesis pathway) to mucosal-associated invariant T (MAIT) cells. This raises many questions regarding antigens loading and intracellular trafficking of the MR1/ligand complexes. The MR1/MAIT field is also important because MAIT cells are very abundant in humans and their frequency is modified in many infectious and non-infectious diseases. Both MR1 and the invariant TCRα chain expressed by MAIT cells are strikingly conserved among species, indicating important functions. Riboflavin is synthesized by plants and most bacteria and yeasts but not animals, and its precursor derivatives activating MAIT cells are short-lived unless bound to MR1. The recognition of MR1 loaded with these compounds is therefore an exquisite manner to detect invasive bacteria. Herein, we provide an historical perspective of the field before describing the main characteristics of MR1, its ligands, and the few available data regarding its cellular biology. We then summarize the current knowledge of MAIT cell differentiation and discuss the definition of MAIT cells in comparison to related subsets. Finally, we describe the phenotype and effector activities of MAIT cells. PMID:27319347

  19. 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.

  20. Bound Flavin-Cytochrome Model of Extracellular Electron Transfer in Shewanella oneidensis: Analysis by Free Energy Molecular Dynamics Simulations.

    PubMed

    Hong, Gongyi; Pachter, Ruth

    2016-06-30

    Flavins are known to enhance extracellular electron transfer (EET) in Shewanella oneidensis MR-1 bacteria, which reduce electron acceptors through outer-membrane (OM) cytochromes c. Free-shuttle and bound-redox cofactor mechanisms were proposed to explain this enhancement, but recent electrochemical reports favor a flavin-bound model, proposing two one-electron reductions of flavin, namely, oxidized (Ox) to semiquinone (Sq) and semiquinone to hydroquinone (Hq), at anodic and cathodic conditions, respectively. In this work, to provide a mechanistic understanding of riboflavin (RF) binding at the multiheme OM cytochrome OmcA, we explored binding configurations at hemes 2, 5, 7, and 10. Subsequently, on the basis of molecular dynamics (MD) simulations, binding free energies and redox potential shifts upon RF binding for the Ox/Sq and Sq/Hq reductions were analyzed. Our results demonstrated an upshift in the Ox/Sq and a downshift in the Sq/Hq redox potentials, consistent with a bound RF-OmcA model. Furthermore, binding free energy MD simulations indicated an RF binding preference at heme 7. MD simulations of the OmcA-MtrC complex interfacing at hemes 5 revealed a small interprotein redox potential difference with an electron transfer rate of 10(7)-10(8)/s. PMID:27266856

  1. The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1.

    PubMed

    McWilliam, Hamish E G; Eckle, Sidonia B G; Theodossis, Alex; Liu, Ligong; Chen, Zhenjun; Wubben, Jacinta M; Fairlie, David P; Strugnell, Richard A; Mintern, Justine D; McCluskey, James; Rossjohn, Jamie; Villadangos, Jose A

    2016-05-01

    The antigen-presenting molecule MR1 presents vitamin B-related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability. PMID:27043408

  2. Impacts of Nitrate and Nitrite on Physiology of Shewanella oneidensis

    PubMed Central

    Zhang, Haiyan; Fu, Huihui; Wang, Jixuan; Sun, Linlin; Jiang, Yaoming; Zhang, Lili; Gao, Haichun

    2013-01-01

    Shewanella oneidensis exhibits a remarkable versatility in anaerobic respiration, which largely relies on its diverse respiratory pathways. Some of these are expressed in response to the existence of their corresponding electron acceptors (EAs) under aerobic conditions. However, little is known about respiration and the impact of non-oxygen EAs on the physiology of the microorganism when oxygen is present. Here we undertook a study to elucidate the basis for nitrate and nitrite inhibition of growth under aerobic conditions. We discovered that nitrate in the form of NaNO3 exerts its inhibitory effects as a precursor to nitrite at low concentrations and as an osmotic-stress provider (Na+) at high concentrations. In contrast, nitrite is extremely toxic, with 25 mM abolishing growth completely. We subsequently found that oxygen represses utilization of all EAs but nitrate. To order to utilize EAs with less positive redox potential, such as nitrite and fumarate, S. oneidensis must enter the stationary phase, when oxygen respiration becomes unfavorable. In addition, we demonstrated that during aerobic respiration the cytochrome bd oxidase confers S. oneidensis resistance to nitrite, which likely functions via nitric oxide (NO). PMID:23626841

  3. 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...

  4. 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

  5. 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

  6. Convection-enhanced Delivery of Free Gadolinium with the Recombinant Immunotoxin MR1-1

    PubMed Central

    Ding, Dale; Kanaly, Charles W; Bigner, Darrell D; Cummings, Thomas J; Herndon, James E; Pastan, Ira; Raghavan, Raghu

    2011-01-01

    Purpose A major obstacle in glioblastoma (GBM) therapy is the restrictive nature of the blood-brain barrier (BBB). Convection-enhanced delivery (CED) is a novel method of drug administration which allows direct parenchymal infusion of therapeutics, bypassing the BBB. MR1-1 is a novel recombinant immunotoxin that targets the GBM tumor-specific antigen EGFRvIII and can be delivered via CED infusion. However, drug distribution via CED varies dramatically, which necessitates active monitoring. Gadolinium conjugated to diethylenetriamine penta-acetic acid (Gd-DTPA) is a commonly used MRI contrast agent which can be co-infused with therapies using CED and may be useful in monitoring infusion leak and early distribution. Experimental design Forty immunocompetent rats were implanted with intracerebral cannulas that were connected to osmotic pumps and subsequently randomized into four groups that each received 0.2% human serum albumin (HSA) mixed with a different experimental infusion: 1) 25 ng/mL MR1-1; 2) 7 μmol/mL Gd-DTPA; 3) 25 ng/mL MR1-1 and 7 μmol/mL Gd-DTPA; 4) 250 ng/mL MR1-1 and 7μmol/mL Gd-DTPA. The rats were monitored clinically for six weeks then necropsied and histologically assessed for CNS toxicity. Results All rats survived the entirety of the study without clinical or histological toxicity attributable to the study drugs. There was no statistically significant difference in weight change over time among groups (p>0.999). Conclusion MR1-1 co-infused with Gd-DTPA via CED is safe in the long-term setting in a pre-clinical animal model. Our data supports the use of Gd-DTPA, as a surrogate tracer, co-infused with MR1-1 for drug distribution monitoring in patients with GBM. PMID:19898744

  7. 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

  8. 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

    Laugel, Bruno; 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; Sewell, Andrew K

    2016-08-01

    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.

  9. Structure and function of the non-classical major histocompatibility complex molecule MR1.

    PubMed

    Krovi, S Harsha; Gapin, Laurent

    2016-08-01

    Polymorphic major histocompatibility complex (MHC) molecules play a central role in the vertebrate adaptive immune system. By presenting short peptides derived from pathogen-derived proteins, these "classical" MHC molecules can alert the T cell branch of the immune system of infected cells and clear the pathogen. There exist other "non-classical" MHC molecules, which while similar in structure to classical MHC proteins, are contrasted by their limited polymorphism. While the functions of many class Ib MHC molecules have still to be elucidated, the nature and diversity of antigens (if any) that some of them might present to the immune system is expected to be more restricted and might function as another approach to distinguish self from non-self. The MHC-related 1 (MR1) molecule is a member of this family of non-classical MHC proteins. It was recently shown to present unique antigens in the form of vitamin metabolites found in certain microbes. MR1 is strongly conserved genetically, structurally, and functionally through mammalian evolution, indicating its necessity in ensuring an effective immune system for members of this class. Although MR1 will be celebrating 21 years this year since its discovery, most of our understanding of how this molecule functions has only been uncovered in the past decade. Herein, we discuss where MR1 is expressed, how it selectively is able to bind to its appropriate antigens and how it, then, is able to specifically activate a distinct population of T cells.

  10. Structure and function of the non-classical major histocompatibility complex molecule MR1.

    PubMed

    Krovi, S Harsha; Gapin, Laurent

    2016-08-01

    Polymorphic major histocompatibility complex (MHC) molecules play a central role in the vertebrate adaptive immune system. By presenting short peptides derived from pathogen-derived proteins, these "classical" MHC molecules can alert the T cell branch of the immune system of infected cells and clear the pathogen. There exist other "non-classical" MHC molecules, which while similar in structure to classical MHC proteins, are contrasted by their limited polymorphism. While the functions of many class Ib MHC molecules have still to be elucidated, the nature and diversity of antigens (if any) that some of them might present to the immune system is expected to be more restricted and might function as another approach to distinguish self from non-self. The MHC-related 1 (MR1) molecule is a member of this family of non-classical MHC proteins. It was recently shown to present unique antigens in the form of vitamin metabolites found in certain microbes. MR1 is strongly conserved genetically, structurally, and functionally through mammalian evolution, indicating its necessity in ensuring an effective immune system for members of this class. Although MR1 will be celebrating 21 years this year since its discovery, most of our understanding of how this molecule functions has only been uncovered in the past decade. Herein, we discuss where MR1 is expressed, how it selectively is able to bind to its appropriate antigens and how it, then, is able to specifically activate a distinct population of T cells. PMID:27448212

  11. 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

  12. Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells.

    PubMed

    Harriff, Melanie J; Karamooz, Elham; Burr, Ansen; Grant, Wilmon F; Canfield, Elizabeth T; Sorensen, Michelle L; Moita, Luis F; Lewinsohn, David M

    2016-03-01

    Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment. PMID:27031111

  13. Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

    PubMed Central

    Burr, Ansen; Grant, Wilmon F.; Canfield, Elizabeth T.; Sorensen, Michelle L.; Moita, Luis F.; Lewinsohn, David M.

    2016-01-01

    Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment. PMID:27031111

  14. 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

  15. 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.

  16. 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; 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 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

  17. 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.

  18. 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

  19. Involvement and specificity of Shewanella oneidensis outer membrane cytochromes in the reduction of soluble and solid-phase terminal electron acceptors.

    PubMed

    Bücking, Clemens; Popp, Felix; Kerzenmacher, Sven; Gescher, Johannes

    2010-05-01

    The formation of outer membrane (OM) cytochromes seems to be a key step in the evolution of dissimilatory iron-reducing bacteria. They are believed to be the endpoints of an extended respiratory chain to the surface of the cell that establishes the connection to insoluble electron acceptors such as iron or manganese oxides. The gammaproteobacterium Shewanella oneidensis MR-1 contains the genetic information for five putative OM cytochromes. In this study, the role and specificity of these proteins were investigated. All experiments were conducted using a markerless deletion mutant in all five OM cytochromes that was complemented via the expression of single, plasmid-encoded genes. MtrC and MtrF were shown to be potent reductases of chelated ferric iron, birnessite, and a carbon anode in a microbial fuel cell. OmcA-producing cells were unable to catalyze iron and electrode reduction, although the protein was correctly produced and oriented. However, OmcA production resulted in a higher birnessite reduction rate compared with the mutant. The presence of the decaheme cytochrome SO_2931 as well as the diheme cytochrome SO_1659 did not rescue the phenotype of the deletion mutant.

  20. 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.

  1. 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

  2. MR1-Restricted Mucosal-Associated Invariant T Cells and Their Activation during Infectious Diseases

    PubMed Central

    Howson, Lauren J.; Salio, Mariolina; Cerundolo, Vincenzo

    2015-01-01

    MR1-restricted mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites, which are generated by a broad range of bacteria, from Escherichia coli to Mycobacterium tuberculosis and BCG. MAIT cells have been described as innate sensors of infection as they accumulate early in infected tissues. MAIT cells maintain an activated phenotype throughout the course of infections, secrete inflammatory cytokines, and have the potential to directly kill infected cells, playing an important role in shaping the host response. In this review, we will discuss the current knowledge regarding the molecular mechanisms that underline MAIT cells activation in sterile and non-sterile inflammatory conditions. PMID:26136743

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

    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.

  9. Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens

    PubMed Central

    Meermeier, Erin W.; Laugel, Bruno F.; Sewell, Andrew K.; Corbett, Alexandra J.; Rossjohn, Jamie; McCluskey, James; Harriff, Melanie J.; Franks, Tamera; Gold, Marielle C.; Lewinsohn, David M.

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2+ MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2+ clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites. PMID:27527800

  10. Receptor-like protein kinase HvLysMR1 of barley (Hordeum vulgare L.) is induced during leaf senescence and heavy metal stress.

    PubMed

    Ouelhadj, Akli; Kaminski, Marc; Mittag, Maria; Humbeck, Klaus

    2007-01-01

    The Hordeum vulgare cDNA clone HvLysMR1 that encodes a putative receptor-like protein kinase was identified by restriction fragment differential display-polymerase chain reaction (PCR) comparing cDNA populations derived from mRNAs of primary leaves stressed with chromium for 48 h with controls. The full-length sequence codes for a protein with 622 amino acids which includes characteristic domains of lysine motif receptor-like kinases: an N-terminal signal peptide, two lysine motifs, a transmembrane region, and a serine/threonine kinase domain at the C-terminal end. The expression of HvLysMR1 is induced during exposure to different heavy metals and its transcript accumulates during leaf senescence. Addition of the calcium ionophore A23187 induces HvLysMR1 expression, indicating the involvement of Ca2+ in the regulation of HvLysMR1. In vitro phosphorylation of HvLysMR1 was analysed with [32P]ATP. Using the overexpressed and purified HvLysMR1-kinase domain, the phosphorylation of HvLysMR1 could be confirmed by nano-liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) with neutral loss-triggered MS-MS-MS spectra at amino acids localized at the juxtamembrane region. The involvement of HvLysMR1 during heavy metal stress and leaf senescence is discussed. PMID:17322551

  11. Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens.

    PubMed

    Meermeier, Erin W; Laugel, Bruno F; Sewell, Andrew K; Corbett, Alexandra J; Rossjohn, Jamie; McCluskey, James; Harriff, Melanie J; Franks, Tamera; Gold, Marielle C; Lewinsohn, David M

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites. PMID:27527800

  12. Crp-dependent cytochrome bd oxidase confers nitrite resistance to Shewanella oneidensis.

    PubMed

    Fu, Huihui; Chen, Haijiang; Wang, Jixuan; Zhou, Guangqi; Zhang, Haiyan; Zhang, Lili; Gao, Haichun

    2013-08-01

    Shewanella oneidensis is able to respire on a variety of organic and inorganic substrates, including nitrate and nitrite. Conversion of nitrate to nitrite and nitrite to ammonium is catalysed by periplasmic nitrate and nitrite reductases (NAP and NRF) respectively. Global regulator Crp (cyclic AMP receptor protein) is essential for growth of S. oneidensis on both nitrate and nitrite. In this study, we discovered that crp mutants are not only severely deficient in nitrate or nitrite respiration, but are also hypersensitive to nitrite. This hypersusceptibility phenotype is independent of nitrite respiration. Using random transposon mutagenesis, we obtained 73 Δcrp suppressor strains resistant to nitrite. Transposon insertion sites in 24 suppressor strains were exclusively mapped in the region upstream of the cyd operon encoding a cytochrome bd oxidase, resulting in expression of the operon now driven by a Crp-independent promoter. Further investigation indicated that the promoter in suppressor strains comes from the transposon. Mutational analysis of the cydB gene (encoding the essential subunit II of the bd oxidase) confirmed that the cytochrome bd oxidase confers nitrite resistance to S. oneidensis.

  13. 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

  14. 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

  15. 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

  16. Role of the Tetraheme Cytochrome CymA in Anaerobic Electron Transport in Cells of Shewanella putrefaciens MR-1 with Normal Levels of Menaquinone

    PubMed Central

    Myers, Judith M.; Myers, Charles R.

    2000-01-01

    Shewanella putrefaciens MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome c. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the cymA gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a cymA knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the cymA transcript and CymA protein was also restored, by complementation of MR1-CYMA with the cymA gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1. PMID:10613864

  17. 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.

  18. 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.

  19. 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.

  20. Impacts of Shewanella oneidensis c‐type cytochromes on aerobic and anaerobic respiration

    PubMed Central

    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

    2010-01-01

    Summary 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. PMID:21255343

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

    PubMed

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

    2015-11-01

    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.

  2. 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

  3. 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.

  4. An arsenic-specific biosensor with genetically engineered Shewanella oneidensis in a bioelectrochemical system.

    PubMed

    Webster, Dylan P; TerAvest, Michaela A; Doud, Devin F R; Chakravorty, Arun; Holmes, Eric C; Radens, Caleb M; Sureka, Swati; Gralnick, Jeffrey A; Angenent, Largus T

    2014-12-15

    Genetically engineered microbial biosensors have yet to realize commercial success in environmental applications due, in part, to difficulties associated with transducing and transmitting traditional bioluminescent information. Bioelectrochemical systems (BESs) output a direct electric signal that can be incorporated into devices for remote environmental monitoring. Here, we describe a BES-based biosensor with genetically encoded specificity for a toxic metal. By placing an essential component of the metal reduction (Mtr) pathway of Shewanella oneidensis under the control of an arsenic-sensitive promoter, we have genetically engineered a strain that produces increased current in response to arsenic when inoculated into a BES. Our BES-based biosensor has a detection limit of ~40 μM arsenite with a linear range up to 100 μM arsenite. Because our transcriptional circuit relies on the activation of a single promoter, similar sensing systems may be developed to detect other analytes by the swap of a single genetic part.

  5. 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.

  6. Integrative analysis of transcriptomic and proteomic data of Shewanella oneidensis: missing value imputation using temporal datasets

    SciTech Connect

    Torres-García, Wandaliz; Brown, Steven D; Johnson, Roger; Zhang, Weiwen; Runger, George; Meldrum, Deirdre

    2011-01-01

    Despite significant improvements in recent years, proteomic datasets currently available still suffer large number of missing values. Integrative analyses based upon incomplete proteomic and transcriptomic da-tasets could seriously bias the biological interpretation. In this study, we applied a non-linear data-driven stochastic gradient boosted trees (GBT) model to impute missing proteomic values for proteins experi-mentally undetected, using a temporal transcriptomic and proteomic dataset of Shewanella oneidensis. In this dataset, genes expression was measured after the cells were exposed to 1 mM potassium chromate for 5-, 30-, 60-, and 90-min, while protein abundance was measured only for 45- and 90-min samples. With the goal of elucidating the relationship between temporal gene expression and protein abundance data, and then using it to impute missing proteomic values for samples of 45-min (which does not have cognate transcriptomic data) and 90-min, we initially used nonlinear Smoothing Splines Curve Fitting (SSCF) to identify temporal relationships among transcriptomic data at different time points and then imputed missing gene expression measurements for the sample at 45-min. After the imputation was validated by biological constrains (i.e. operons), we used a data-driven Gradient Boosted Trees (GBT) model to uncover possible non-linear relationships between temporal transcriptomic and proteomic data, and to impute protein abundance for the proteins experimentally undetected in the 45- and 90-min sam-ples, based on relevant predictors such as temporal mRNA gene expression data, cellular roles, molecular weight, sequence length, protein length, guanine-cytosine (GC) content and triple codon counts. The imputed protein values were validated using biological constraints such as operon, regulon and pathway information. Finally, we demonstrated that such missing value imputation improved characterization of the temporal response of S. oneidensis to chromate.

  7. 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).

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant

    PubMed Central

    Shi, Miaomiao; Wan, Fen; Mao, Yinting

    2015-01-01

    ABSTRACT Oxidative stresses triggered by reactive oxygen species (ROS) that damage various cellular components are unavoidable for virtually all living organisms. In defense, microorganisms have evolved sophisticated mechanisms to sense, respond to, and battle against ROS. Shewanella oneidensis, an important research model for applied and environmental microbes, employs OxyR to mediate the response to H2O2 by derepressing the production of the major H2O2 scavenger KatB as a major means toward these goals. Surprisingly, despite enhanced H2O2 degradation, the oxyR mutant carries a viability deficiency phenotype (plating defect), which can be suppressed by the addition of exogenous iron species. Experiments showed that the defect was not due to iron starvation. Rather, multiple lines of evidence suggested that H2O2 generated abiotically in lysogeny broth (LB) is responsible for the defect by quickly killing mutant cells. We then showed that the iron species suppressed the plating defect by two distinct mechanisms, either as an H2O2 scavenger without involving living cells or as an environmental cue to stimulate an OxyR-independent response to help cells cope with oxidative stress. Based on the suppression of the plating defect by overproduction of H2O2 scavengers in vivo, we propose that cellular components that are vulnerable to H2O2 and responsible for the defect may reside outside the cytoplasm. IMPORTANCE In bacteria, OxyR is the major regulator controlling the cellular response to H2O2. The loss of OxyR results in reduced viability in many species, but the underlying mechanism is unknown. We showed in S. oneidensis that this defect was due to H2O2 generated abiotically in LB. We then showed that this defect could be corrected by the addition of Fe2+ or catalase to the LB or increased intracellular production of catalase. Further analyses revealed that Fe2+ was able not only to decompose H2O2 directly but also to stimulate the activity of OxyR-independent H2O2

  13. Distinct Roles of Major Peptidoglycan Recycling Enzymes in β-Lactamase Production in Shewanella oneidensis

    PubMed Central

    Yin, Jianhua; Mao, Yinting; Ju, Lili; Jin, Miao; Sun, Yiyang; Jin, Shouguang

    2014-01-01

    β-Lactam antibiotics were the earliest discovered and are the most widely used group of antibiotics that work by inactivating penicillin-binding proteins to inhibit peptidoglycan biosynthesis. As one of the most efficient defense strategies, many bacteria produce β-lactam-degrading enzymes, β-lactamases, whose biochemical functions and regulation have been extensively studied. A signal transduction pathway for β-lactamase induction by β-lactam antibiotics, consisting of the major peptidoglycan recycling enzymes and the LysR-type transcriptional regulator, AmpR, has been recently unveiled in some bacteria. Because inactivation of some of these proteins, especially the permease AmpG and the β-hexosaminidase NagZ, results in substantially elevated susceptibility to the antibiotics, these have been recognized as potential therapeutic targets. Here, we show a contrasting scenario in Shewanella oneidensis, in which the homologue of AmpR is absent. Loss of AmpG or NagZ enhances β-lactam resistance drastically, whereas other identified major peptidoglycan recycling enzymes are dispensable. Moreover, our data indicate that there exists a parallel signal transduction pathway for β-lactamase induction, which is independent of either AmpG or NagZ. PMID:25136029

  14. 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.

  15. 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

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

    NASA Astrophysics Data System (ADS)

    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.

  17. 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.

  18. 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.

  19. Addressing Shewanella oneidensis cytochromome : a first step towards high-throughput expression of cytochromes c.

    SciTech Connect

    Londer, Y. Y.; Giuliani, S.; Peppler, T.; Collart, F.; Biosciences Division

    2008-11-01

    Integrated studies that address proteins structure and function in the new era of systems biology and genomics often require the application of high-throughput approaches for parallel production of many different purified proteins from the same organism. Cytochromes c-electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain--are essential in most organisms. However, they are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. We have addressed this challenge by designing two families of vectors (total of 6 vectors) suitable for ligation-independent cloning and developing a pipeline for expression and solubility analysis of cytochromes c. This system has been validated by expression analysis of thirty genes from Shewanella oneidensis coding for cytochromes c or cytochromes c-type domains predicted to have 1-4 hemes. Out of 30 targets, 26 (87%) were obtained in soluble form in one or more vectors. This work establishes a methodology for high-throughput expression of this class of proteins and provides a clone resource for the microbiological and functional genomics research communities.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. 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.

  2. Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence of interactions across the dimeric interface.

    PubMed

    Judd, Evan T; Youngblut, Matthew; Pacheco, A Andrew; Elliott, Sean J

    2012-12-21

    Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, conducts the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by "boosts" and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to the properties of ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed; e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA [Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) J. Biol. Chem. 277, 23374-23381]. Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which has previously been observed in any cytochrome c nitrite reductase. Collectively, these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

  3. Endogenous generation of hydrogen sulfide and its regulation in Shewanella oneidensis

    PubMed Central

    Wu, Genfu; Li, Ning; Mao, Yinting; Zhou, Guangqi; Gao, Haichun

    2015-01-01

    Hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions across all domains of life. In this study, mechanisms of endogenous H2S generation in Shewanella oneidensis were investigated. As a research model with highly diverse anaerobic respiratory pathways, the microorganism is able to produce H2S by respiring on a variety of sulfur-containing compounds with SirACD and PsrABC enzymatic complexes, as well as through cysteine degradation with three enzymes, MdeA, SO_1095, and SseA. We showed that the SirACD and PsrABC complexes, which are predominantly, if not exclusively, responsible for H2S generation via respiration of sulfur species, do not interplay with each other. Strikingly, a screen for regulators controlling endogenous H2S generation by transposon mutagenesis identified global regulator Crp to be essential to all H2S-generating processes. In contrast, Fnr and Arc, two other global regulators that have a role in respiration, are dispensable in regulating H2S generation via respiration of sulfur species. Interestingly, Arc is involved in the H2S generation through cysteine degradation by repressing expression of the mdeA gene. We further showed that expression of the sirA and psrABC operons is subjected to direct regulation of Crp, but the mechanisms underlying the requirement of Crp for H2S generation through cysteine degradation remain elusive. PMID:25972854

  4. 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

    2016-01-01

    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

  5. Control of Proteobacterial Central Carbon Metabolism by the HexR Transcriptional Regulator. A Case Study in Shewanella oneidensis

    SciTech Connect

    Leyn, Semen; Li, Xiaoqing; Zheng, Qijing; Novichkov, Pavel; Reed, Samantha B.; Romine, Margaret F.; Fredrickson, Jim K.; Yang, Chen; Osterman, Andrei L.; Rodionov, Dmitry A.

    2011-08-17

    Bacteria exploit multiple mechanisms for controlling central carbon metabolism (CCM). Thus, a bioinformatic analysis together with some experimental data implicated HexR transcriptional factor as a global CCM regulator in some lineages of Gammaproteobacteria operating as a functional replacement of Cra regulator characteristic of Enterobacteriales. In this study we combined a large-scale comparative genomic reconstruction of HexRcontrolled regulons in 87 species of Proteobacteria with the detailed experimental analysis of HexR regulatory network in Shewanella oneidensis model system. Although nearly all of the HexR-controlled genes are associated with CCM, remarkable variations were revealed in the scale (from 1-2 target operons in Enterobacteriales up to 20 operons in Aeromonadales) and gene content of HexR regulons between 11 compared lineages. A predicted 17-bp pseudo-palindrome with a consensus tTGTAATwwwATTACa, was confirmed as HexR-binding motif for 15 target operons (comprising 30 genes) by in vitro binding assays. The negative effect of the key CCM intermediate, 2-keto-3-deoxy-6- phosphogluconate, on the DNA-regulator complex formation was verified. A dual mode of HexR action on various target promoters, repression of genes involved in catabolic pathways and activation of gluconeogenic genes, was for the first time predicted by the bioinformatc analysis and experimentally verified by changed gene expression pattern in S. oneidensis AhexR mutant. Phenotypic profiling revealed the inability of this mutant to grow on lactate or pyruvate as a single carbon source. A comparative metabolic flux analysis of wild-type and mutant strains of S. oneidensis using 13Clactate labeling and GC-MS analysis confirmed the hypothesized HexR role as a master regulator of gluconeogenic flux from pyruvate via the transcriptional activation of phosphoenolpyruvate synthase (PpsA).

  6. The effect of metal loading on Cd adsorption onto Shewanella oneidensis bacterial cell envelopes: The role of sulfhydryl sites

    NASA Astrophysics Data System (ADS)

    Yu, Qiang; Fein, Jeremy B.

    2015-10-01

    The adsorption and desorption of Cd onto Shewanella oneidensis bacterial cells with and without blocking of sulfhydryl sites was measured in order to determine the effect of metal loading and to understand the role of sulfhydryl sites in the adsorption reactions. The observed adsorption/desorption behaviors display strong dependence on metal loading. Under a high loading of 40 μmol Cd/g bacterial cells, blocking the sulfhydryl sites within the cell envelope by exposure of the biomass to monobromo(trimethylammonio)bimane bromide (qBBr) does not significantly affect the extent of Cd adsorption, and we observed fully reversible adsorption under this condition. In contrast, under a low metal loading of 1.3 μmol Cd/g bacterial cells, the extent of Cd adsorption onto sulfhydryl-blocked S. oneidensis cells was significantly lower than that onto untreated cells, and only approximately 50-60% of the adsorbed Cd desorbed from the cells upon acidification. In conjunction with previous EXAFS results, our findings demonstrate that Cd adsorption onto S. oneidensis under low metal loading conditions is dominated by sulfhydryl binding, and thus is controlled by a distinct adsorption mechanism from the non-sulfhydryl site binding which controls Cd adsorption under high metal loading conditions. We use the data to develop a surface complexation model that constrains the values of the stability constants for individual Cd-sulfhydryl and Cd-non-sulfhydryl bacterial complexes, and we use this approach to account for the Cd adsorption behavior as a function of both pH and metal loading. This approach is crucial in order to predict metal adsorption onto bacteria under environmentally relevant metal loading conditions where sulfhydryl binding sites can dominate the adsorption reaction.

  7. Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence for interactions across the dimeric interface

    PubMed Central

    Judd, Evan T.; Youngblut, Matthew; Pacheco, A. Andrew; Elliott, Sean J.

    2013-01-01

    Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by ‘boosts’ and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed, e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA (Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase, J Biol Chem 277, 23374-23381). Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which have previously been observed in any cytochrome c nitrite reductase. Collectively these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer. PMID:23210513

  8. 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.

  9. 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.

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

    PubMed Central

    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. PMID:27014246

  11. 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.

  12. Protection from oxidative stress relies mainly on derepression of OxyR-dependent KatB and Dps in Shewanella oneidensis.

    PubMed

    Jiang, Yaoming; Dong, Yangyang; Luo, Qixia; Li, Ning; Wu, Genfu; Gao, Haichun

    2014-01-01

    Shewanella thrives in redox-stratified environments where accumulation of H2O2 becomes inevitable because of the chemical oxidation of reduced metals, sulfur species, or organic molecules. As a research model, the representative species Shewanella oneidensis has been extensively studied for its response to various stresses. However, little progress has been made toward an understanding of the physiological and genetic responses of this bacterium to oxidative stress, which is critically relevant to its application as a dissimilatory metal-reducing bacterium. In this study, we systematically investigated the mechanism underlying the response to H2O2 at cellular, genomic, and molecular levels. Using transcriptional profiling, we found that S. oneidensis is hypersensitive to H2O2 in comparison with Escherichia coli, and well-conserved defense genes such as ahpCF, katB, katG, and dps appear to form the first line of defense, whereas iron-sulfur-protecting proteins may not play a significant role. Subsequent identification and characterization of an analogue of the E. coli oxyR gene revealed that S. oneidensis OxyR is the master regulator that mediates the bacterial response to H2O2-induced oxidative stress by directly repressing or activating the defense genes. The sensitivity of S. oneidensis to H2O2 is likely attributable to the lack of an inducible manganese import mechanism during stress. To cope with stress, major strategies that S. oneidensis adopts include rapid removal of the oxidant and restriction of intracellular iron concentrations, both of which are achieved predominantly by derepression of the katB and dps genes.

  13. Protection from Oxidative Stress Relies Mainly on Derepression of OxyR-Dependent KatB and Dps in Shewanella oneidensis

    PubMed Central

    Jiang, Yaoming; Dong, Yangyang; Luo, Qixia; Li, Ning; Wu, Genfu

    2014-01-01

    Shewanella thrives in redox-stratified environments where accumulation of H2O2 becomes inevitable because of the chemical oxidation of reduced metals, sulfur species, or organic molecules. As a research model, the representative species Shewanella oneidensis has been extensively studied for its response to various stresses. However, little progress has been made toward an understanding of the physiological and genetic responses of this bacterium to oxidative stress, which is critically relevant to its application as a dissimilatory metal-reducing bacterium. In this study, we systematically investigated the mechanism underlying the response to H2O2 at cellular, genomic, and molecular levels. Using transcriptional profiling, we found that S. oneidensis is hypersensitive to H2O2 in comparison with Escherichia coli, and well-conserved defense genes such as ahpCF, katB, katG, and dps appear to form the first line of defense, whereas iron-sulfur-protecting proteins may not play a significant role. Subsequent identification and characterization of an analogue of the E. coli oxyR gene revealed that S. oneidensis OxyR is the master regulator that mediates the bacterial response to H2O2-induced oxidative stress by directly repressing or activating the defense genes. The sensitivity of S. oneidensis to H2O2 is likely attributable to the lack of an inducible manganese import mechanism during stress. To cope with stress, major strategies that S. oneidensis adopts include rapid removal of the oxidant and restriction of intracellular iron concentrations, both of which are achieved predominantly by derepression of the katB and dps genes. PMID:24214945

  14. The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

    PubMed Central

    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, James K.

    2007-01-01

    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 largely 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 Shewanellaoneidensis 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 variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research. PMID:18030449

  15. A Matter of Timing: Contrasting Effects of Hydrogen Sulfide on Oxidative Stress Response in Shewanella oneidensis

    PubMed Central

    Wu, Genfu; Wan, Fen; Fu, Huihui; Li, Ning

    2015-01-01

    ABSTRACT Hydrogen sulfide (H2S), well known for its toxic properties, has recently become a research focus in bacteria, in part because it has been found to prevent oxidative stress caused by treatment with some antibiotics. H2S has the ability to scavenge reactive oxygen species (ROS), thus preventing oxidative stress, but it is also toxic, leading to conflicting reports of its effects in different organisms. Here, with Shewanella oneidensis as a model, we report that the effects of H2S on the response to oxidative stress are time dependent. When added simultaneously with H2O2, H2S promoted H2O2 toxicity by inactivating catalase, KatB, a heme-containing enzyme involved in H2O2 degradation. Such an inhibitory effect may apply to other heme-containing proteins, such as cytochrome cbb3 oxidase. When H2O2 was supplied 20 min or later after the addition of H2S, the oxidative-stress-responding regulator OxyR was activated, resulting in increased resistance to H2O2. The activation of OxyR was likely triggered by the influx of iron, a response to lowered intracellular iron due to the iron-sequestering property of H2S. Given that Shewanella bacteria thrive in redox-stratified environments that have abundant sulfur and iron species, our results imply that H2S is more important for bacterial survival in such environmental niches than previously believed. IMPORTANCE Previous studies have demonstrated that H2S is either detrimental or beneficial to bacterial cells. While it can act as a growth-inhibiting molecule by damaging DNA and denaturing proteins, it helps cells to combat oxidative stress. Here we report that H2S indeed has these contrasting biological functions and that its effects are time dependent. Immediately after H2S treatment, there is growth inhibition due to damage of heme-containing proteins, at least to catalase and cytochrome c oxidase. In contrast, when added a certain time later, H2S confers an enhanced ability to combat oxidative stress by activating the

  16. MAIT, MR1, microbes and riboflavin: a paradigm for the co-evolution of invariant TCRs and restricting MHCI-like molecules?

    PubMed

    Mondot, Stanislas; Boudinot, Pierre; Lantz, Olivier

    2016-08-01

    MAIT cells express an invariant TCR that recognizes non-peptidic microbial antigens presented by the non-polymorphic MHCI-like molecule, MR1. We briefly describe how the antigens recognized by MAIT cells are generated from an unstable precursor of the riboflavin (Vitamin B2) biosynthesis pathway, as well as the main features of MAIT cells in comparison with other related T cell subsets. In silico analysis of bacterial genomes shows that the riboflavin biosynthesis pathway is highly prevalent in all groups of Prokaryotes with, however, notable exceptions. We discuss the putative functions and the evolution of the MAIT/MR1 couple: it appeared in the ancestors of mammals and is highly conserved across this group, but was independently lost in three orders. We describe the four instances of known invariant TCR and MHC-I-like molecules encountered in Vertebrates. Both T cells bearing semi-invariant TCR and the associated, evolutionarily conserved MHC-I related molecules have been found in mammals or in amphibians, which suggests that other MHC1-like/invariant TCR couples might be present in other classes of Vertebrates to detect generic microbial compounds. This allows us to discuss how the recognition of riboflavin precursor derivatives by the MAIT TCR may be a way to detect invasive microbes in specific organs, and may epitomize other invariant T cell systems across vertebrates. PMID:27393664

  17. Depositional style of mass-flow lobes in Ulleung Basin (East Sea, Korea) interpreted from MR1 sonar image, chirp profiles, and piston cores

    NASA Astrophysics Data System (ADS)

    Lee, S. H.; Jung, W.; Bahk, J.; Gardner, J. M.

    2011-12-01

    A detailed analysis of MR1 sonar and chirp (2-7 kHz) seismic data with 5 (2.9-9.4 m long) piston cores and 17 AMS 14C ages reveals spatial and temporal variations in depositional style of mass-flow lobes in the Ulleung Basin. On MR1 sonar image, eight mass-flow lobes are identified in the western basin plain (>2100 m). They are covered by ca. 2-m thick Holocene pelagic sediments, and show the northward flow direction. Lobes 1-4 have large dimensions (>27 km long and 15-25 km wide) and occupy in the lower stratigraphic position. On the other hand, lobes 5-8, deposited in the more proximal area, have small dimensions (8.8-31.5 km long and 1.2-12 km wide) and occur in the upper stratigraphic position. Lobes 1-4 deposited retrogressively. Lobes 1 and 2 are characterized by relatively strong back-scattering intensity with smooth surfaces on MR1 image, and show flat, sharp bottom echo and several distinct to diffuse internal reflectors in chirp profiles. Sediments near their edges consist of fine-grained turbidites (laminated sand/mud and homogeneous mud) with minor massive clay-rich sand. However, they change to mud-matrix disorganized gravel and massive sand with the overlying fine-grained turbidites toward the proximal part. Lobes 1 and 2 deposited between ca. 20 and 18 cal. ka B.P. Lobes 3 and 4, overlying lobe 2, show weak to medium back-scattering intensity on MR1 image with slightly irregular to hummocky surfaces corresponding to small-scale (<100 m wide and <5 m high) hyperbolic bottom echoes in chirp profiles. Upper surfaces of the lobes are nearly flat to convex-up in cross section. Lobes 5-8 deposited on the upper surfaces of lobes 3 and 4. Their widths abruptly decrease to the proximal part, forming a bottle-neck morphology. They generally show weak to medium back-scattering intensity with relatively strong back-scattering intensity along the margins on MR1 image. They exhibit convex-up upper surfaces with distinct lateral margins in chirp profiles as their

  18. 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.

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

    PubMed

    Beliaev, Alex S; Thompson, Dorothea K; Khare, Tripti; Lim, Hanjo; Brandt, Craig C; Li, Guangshan; Murray, Alison E; Heidelberg, John F; Giometti, Carol S; Yates, John; Nealson, Kenneth H; Tiedje, James M; Zhoui, Jizhong

    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 c552, 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. PMID:11881834

  20. 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.

  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. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. Shewanella oneidensis in a lactate-fed pure-culture and a glucose-fed co-culture with Lactococcus lactis with an electrode as electron acceptor.

    PubMed

    Rosenbaum, Miriam A; Bar, Haim Y; Beg, Qasim K; Segrè, Daniel; Booth, James; Cotta, Michael A; Angenent, Largus T

    2011-02-01

    Bioelectrochemical systems (BESs) employing mixed microbial communities as biocatalysts are gaining importance as potential renewable energy, bioremediation, or biosensing devices. While we are beginning to understand how individual microbial species interact with an electrode as electron donor, little is known about the interactions between different microbial species in a community: sugar fermenting bacteria can interact with current producing microbes in a fashion that is either neutral, positively enhancing, or even negatively affecting. Here, we compare the bioelectrochemical performance of Shewanella oneidensis in a pure-culture and in a co-culture with the homolactic acid fermenter Lactococcus lactis at conditions that are pertinent to conventional BES operation. While S. oneidensis alone can only use lactate as electron donor for current production, the co-culture is able to convert glucose into current with a comparable coulombic efficiency of ∼17%. With (electro)-chemical analysis and transcription profiling, we found that the BES performance and S. oneidensis physiology were not significantly different whether grown as a pure- or co-culture. Thus, the microbes worked together in a purely substrate based (neutral) relationship. These co-culture experiments represent an important step in understanding microbial interactions in BES communities with the goal to design complex microbial communities, which specifically convert target substrates into electricity. PMID:21036604

  8. Transcription factors FabR and FadR regulate both aerobic and anaerobic pathways for unsaturated fatty acid biosynthesis in Shewanella oneidensis

    PubMed Central

    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. PMID:25566241

  9. 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

  10. 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.

  11. 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.

  12. Two residues predominantly dictate functional difference in motility between Shewanella oneidensis flagellins FlaA and FlaB.

    PubMed

    Sun, Linlin; Dong, Yangyang; Shi, Miaomiao; Jin, Miao; Zhou, Qing; Luo, Zhao-Qing; Gao, Haichun

    2014-05-23

    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.

  13. Regulation of nitrite resistance of the cytochrome cbb3 oxidase by cytochrome c ScyA in Shewanella oneidensis.

    PubMed

    Yin, Jianhua; Jin, Miao; Zhang, Haiyan; Ju, Lili; Zhang, Lili; Gao, Haichun

    2015-02-01

    Cytochrome c proteins, as enzymes to exchange electrons with substrates or as pure electron carriers to shuttle electrons, play vital roles in bacterial respiration and photosynthesis. In Shewanella oneidensis, a research model for the respiratory diversity, at least 42 c-type cytochromes are predicted to be encoded in the genome and are regarded to be the foundation of its highly branched electron transport pathways. However, only a small number of c-type cytochromes have been extensively studied. In this study, we identify soluble cytochrome c ScyA as an important factor influencing the nitrite resistance of a strain devoid of the bd oxidase by utilizing a newly developed transposon mutagenesis vector, which enables overexpression of the gene(s) downstream of the insertion site. We show that when in overabundance ScyA facilitates growth against nitrite inhibition by enhancing nitrite resistance of the cbb3 oxidase. Based on the data presented in this study, we suggest two possible mechanisms underlying the observed effect of ScyA: (1) ScyA increases electron flow to the cbb3 oxidase; (2) ScyA promotes nitrite resistance of the cbb3 oxidase, possibly by direct interaction.

  14. Investigation into FlhFG reveals distinct features of FlhF in regulating flagellum polarity in Shewanella oneidensis.

    PubMed

    Gao, Tong; Shi, Miaomiao; Ju, Lili; Gao, Haichun

    2015-10-01

    Rod-shaped bacterial cells are polarized, with many organelles confined to a polar cellular site. In polar flagellates, FlhF and FlhG, a multiple-domain (B-N-G) GTPase and a MinD-like ATPase respectively, function as a cognate pair to regulate flagellar localization and number as revealed in Vibrio and Pseudomonas species. In this study, we show that FlhFG of Shewanella oneidensis (SoFlhFG), a monotrichous γ-proteobacterium renowned for respiratory diversity, also play an important role in the flagellar polar placement and number control. Despite this, SoFlhFG exhibit distinct features that are not observed in the characterized counterparts. Most strikingly, the G domain of SoFlhF determines the polar placement, contrasting the N domain of the Vibrio cholerae FlhF. The SoFlhF N domain in fact counteracts the function of the G domain with respect to the terminal targeting in the absence of the B domain. We further show that GTPase activity of SoFlhF is essential for motility but not positioning. Overall, our results suggest that mechanisms underlying the polar placement of organelles appear to be diverse, even for evolutionally relatively conserved flagellum.

  15. 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

  16. Reduced expression of cytochrome oxidases largely explains cAMP inhibition of aerobic growth in Shewanella oneidensis.

    PubMed

    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

  17. 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.

  18. The X-ray crystal structure of Shewanella oneidensis OmcA reveals new insight at the microbe-mineral interface.

    PubMed

    Edwards, Marcus J; Baiden, Nanakow A; Johs, Alexander; Tomanicek, Stephen J; Liang, Liyuan; Shi, Liang; Fredrickson, Jim K; Zachara, John M; Gates, Andrew J; Butt, Julea N; Richardson, David J; 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 are arranged so the minimum distance between heme 5 on adjacent OmcA monomers is 9 Å, 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 ∼10 Å of a mineral surface.

  19. 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.

  20. The Origin of 8-Amino-3,8-dideoxy-d-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of Shewanella oneidensis*

    PubMed Central

    Gattis, Samuel G.; Chung, Hak Suk; Trent, M. Stephen; Raetz, Christian R. H.

    2013-01-01

    Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-d-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-d-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus Shewanella. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in Shewanella oneidensis. Expression of these genes recombinantly in Escherichia coli resulted in lipid A containing Kdo8N, and in vitro assays confirmed their proposed enzymatic function. Both the in vivo and in vitro data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)+. Creation of an S. oneidensis in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity. PMID:23413030

  1. Digital Image Processing Techniques for Enhancement and Classification of MR1 Side Scan Sonar Imagery and Preliminary Results of Manganese Nodule Occurrence between the Clarion and Clipperton Fracture Zones, NE Equatorial Pacific

    NASA Astrophysics Data System (ADS)

    Kim, H.; Park, C.; Park, J.; Kim, K.

    2004-12-01

    The recent growth in the production rate of digital side scan sonar images, coupled with the rapid expansion of systematic seafloor exploration programs, has created a need for fast and quantitative means of processing seafloor imagery. A number of numerical techniques used to enhance and classify imagery produced by long range side scan sonar (MR1) in the Clarion and Clippertion Fracture Zones, NE equatorial Pacific. Side scan sonar imagery is traditionally interpreted visually and qualitatively by experts. Textural Analysis enables a more objective approach, supplementing the interpreter with reliable quantitative results. Grey-level co-occurrence matrices describe numerically textual information and detect subtle details invisible to the human eyes. The area between the Clarion and Clipperton fracture zones (NE equatorial Pacific) is one of the highest manganese nodule abundance in the world oceans. A detailed analysis of MR1 sonar images and ground truth - free-fall grab (FFG) data in the area, reveals a close relationship between sonar characters of seafloor and manganese nodule occurrence. The close relationship between distribution of sonar imagery and manganese nodule abundance implies that seafloor topography and sediment thickness are important controlling factors for occurrence of manganese nodules.

  2. 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

  3. Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) does not disproportionate hydroxylamine to ammonia and nitrite, despite a strongly favorable driving force.

    PubMed

    Youngblut, Matthew; Pauly, Daniel J; Stein, Natalia; Walters, Daniel; Conrad, John A; Moran, Graham R; Bennett, Brian; Pacheco, A Andrew

    2014-04-01

    Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

  4. Functional expression of Carassius auratus cytochrome P4501A in a novel Shewanella oneidensis expression system and application for the degradation of benzo[a]pyrene.

    PubMed

    Chang, Ziwei; Lu, Ming; Shon, Kwang-Jae; Park, Jang-Su

    2014-06-10

    Cytochrome P4501A (CYP1A) in fish has attracted an increasing interest because of its important roles in the metabolic activation of certain xenobiotics such as aromatic hydrocarbons. CYPs are reported to be expressed in yeast, insect cells and Escherichia coli, but are critical for high-level expression. Besides, this study found that the purification of recombinant goldfish CYP1A would result in a loss of enzyme activity. Because large quantities of functional CYP1A are required, it is necessary to find a suitable host with high-level expression. In the present study, a novel expression system using Shewanella oneidensis was established successfully for the production of goldfish CYP1A. A signal peptide in the expression vector leads to the high-level expression in the periplasmic space of Shewanella. The recombinant CYP1A in Shewanella reached up to 1μmol per liter of culture, and showed the typical P450 hemoprotein spectra. An ethoxyresorufin O-deethylase assay revealed the amount of functional proteins in Shewanella to be almost ten times more than those in other expression systems. Furthermore, the CYP1A-mediated degradation of benzo[a]pyrene was also observed by (1)H NMR spectroscopy. These results indicate the possible application of periplasmic fractions to obtain sufficient quantities of functional CYP1A for further studies.

  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. PBP1a/LpoA but not PBP1b/LpoB are involved in regulation of the major β-lactamase gene blaA in Shewanella oneidensis.

    PubMed

    Yin, Jianhua; Sun, Yiyang; Mao, Yinting; Jin, Miao; Gao, Haichun

    2015-01-01

    β-Lactamase production is one of the most important strategies for Gram-negative bacteria to combat β-lactam antibiotics. Studies of the regulation of β-lactamase expression have largely been focused on the class C β-lactamase AmpC, whose induction by β-lactams requires LysR-type regulator AmpR and permease AmpG-dependent peptidoglycan recycling intermediates. In Shewanella, which is ubiquitous in aquatic environments and is a reservoir for antibiotic resistance, production of the class D β-lactamase BlaA confers bacteria with natural resistance to many β-lactams. Expression of the blaA gene in the genus representative Shewanella oneidensis is distinct from the AmpC paradigm because of the lack of an AmpR homologue and the presence of an additional AmpG-independent regulatory pathway. In this study, using transposon mutagenesis, we identify proteins that are involved in blaA regulation. Inactivation of mrcA and lpoA, which encode penicillin binding protein 1a (PBP1a) and its lipoprotein cofactor, LpoA, respectively, drastically enhances blaA expression in the absence of β-lactams. Although PBP1b and its cognate, LpoB, also exist in S. oneidensis, their roles in blaA induction are dispensable. We further show that the mrcA-mediated blaA expression is independent of AmpG.

  7. Structures of KdnB and KdnA from Shewanella oneidensis: Key Enzymes in the Formation of 8-Amino-3,8-Dideoxy-d-Manno-Octulosonic Acid.

    PubMed

    Zachman-Brockmeyer, Trevor R; Thoden, James B; Holden, Hazel M

    2016-08-16

    8-Amino-3,8-dideoxy-d-manno-octulosonic acid (Kdo8N) is a unique amino sugar that has thus far only been observed on the lipopolysaccharides of marine bacteria belonging to the genus Shewanella. Although its biological function is still unclear, it is thought that the sugar is important for the integrity of the bacterial cell outer membrane. A three-gene cluster required for the biosynthesis of Kdo8N was first identified in Shewanella oneidensis. Here we describe the three-dimensional structures of two of the enzymes required for Kdo8N biosynthesis in S. oneidensis, namely, KdnB and KdnA. The structure of KdnB was solved to 1.85-Å resolution, and its overall three-dimensional architecture places it into the Group III alcohol dehydrogenase superfamily. A previous study suggested that KdnB did not require NAD(P) for activity. Strikingly, although the protein was crystallized in the absence of any cofactors, the electron density map clearly revealed the presence of a tightly bound NAD(H). In addition, a bound metal was observed, which was shown via X-ray fluorescence to be a zinc ion. Unlike other members of the Group III alcohol dehydrogenases, the dinucleotide cofactor in KdnB is tightly bound and cannot be removed without leading to protein precipitation. With respect to KdnA, it is a pyridoxal 5'-phosphate or (PLP)-dependent aminotransferase. For this analysis, the structure of KdnA, trapped in the presence of the external aldimine with PLP and glutamate, was determined to 2.15-Å resolution. The model of KdnA represents the first structure of a sugar aminotransferase that functions on an 8-oxo sugar. Taken together the results reported herein provide new molecular insight into the biosynthesis of Kdo8N.

  8. 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.

  9. 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

  10. Piezo-adapted 3-isopropylmalate dehydrogenase of the obligate piezophile Shewanella benthica DB21MT-2 isolated from the 11,000-m depth of the Mariana Trench.

    PubMed

    Kasahara, Ryota; Sato, Takako; Tamegai, Hideyuki; Kato, Chiaki

    2009-11-01

    3-isopropylmalate dehydrogenase (IPMDH)-encoding leuB genes were obtained from the obligate piezophile Shewanella benthica DB21MT-2 and non-piezophile Shewanella oneidensis MR-1. The genes were expressed in Escherichia coli and the proteins were purified using His-tag. The estimated kinetic parameters of these enzymes indicated that IPMDH of S. benthica DB21MT-2 is more tolerant of high pressure than that of S. oneidensis MR-1. Thus such an adaptation is one of the mechanisms bacteria utilize for survival at high pressures. PMID:19897891

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Employing a Flexible and Low-Cost Polypyrrole Nanotube Membrane as an Anode to Enhance Current Generation in Microbial Fuel Cells.

    PubMed

    Zhao, Cui-e; Wu, Jiansheng; Kjelleberg, Staffan; Loo, Joachim Say Chey; Zhang, Qichun

    2015-07-01

    The flexible and low-cost polypyrrole nanotube membrane is demonstrated as a promising anode in microbial fuel cells, which significantly enhances the extracellular electron transfer between Shewanella oneidensis MR-1 and the electrode, owing to the large active surface area and high electrical conductivity.

  16. 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.

  17. Bacterial responses to Cu-doped TiO(2) nanoparticles.

    PubMed

    Wu, Bing; Huang, Rick; Sahu, Manoranjan; Feng, Xueyang; Biswas, Pratim; Tang, Yinjie J

    2010-03-01

    The toxicity of Cu-doped TiO(2) nanoparticles (NPs, 20nm), synthesized by a flame aerosol reactor, to Mycobacterium smegmatis and Shewanella oneidensis MR-1, is the primary focus of this study. Both doped and non-doped TiO(2) NPs (20nm) tended to agglomerate in the medium solution, and therefore did not penetrate into the cell and damage cellular structures. TiO(2) particles (<100mg/L) did not apparently interfere with the growth of the two species in aqueous cultures. Cu-doped TiO(2) NPs (20mg/L) significantly reduced the M. smegmatis growth rate by three fold, but did not affect S. oneidensis MR-1 growth. The toxicity of Cu-doped TiO(2) NPs was driven by the release of Cu(2+) from the parent NPs. Compared to equivalent amounts of Cu(2+), Cu-doped TiO(2) NPs exhibited higher levels of toxicity to M. smegmatis (P-value<0.1). Addition of EDTA in the culture appeared to significantly decrease the anti-mycobacterium activity of Cu-doped TiO(2) NPs. S. oneidensis MR-1 produced a large amount of extracellular polymeric substances (EPS) under NP stress, especially extracellular protein. Therefore, S. oneidensis MR-1 was able to tolerate a much higher concentration of Cu(2+) or Cu-doped TiO(2) NPs. S. oneidensis MR-1 also adsorbed NPs on cell surface and enzymatically reduced ionic copper in culture medium with a remediating rate of 61microg/(liter x OD(600) x hour) during its early exponential growth phase. Since the metal reducing Shewanella species can efficiently "clean" metal-oxide NPs, the activities of such environmentally relevant bacteria may be an important consideration for evaluating the ecological risk of metal-oxide NPs. PMID:19931887

  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. 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

  1. 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.

  2. [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.

  3. [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.

  4. Identification of c-Type Heme-Containing Peptides Using Non-Activated Immobilized Metal Affinity Cchromatography Resin Enrichment and Higher-Energy Collisional Dissociation

    SciTech Connect

    Zhang, Haizhen; Yang, Feng; Qian, Weijun; Brown, Roslyn N.; Wang, Yuexi; Merkley, Eric D.; Park, Jea H.; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Shi, Liang; Fredrickson, Jim K.; Pasa-Tolic, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2011-10-01

    c-type cytochromes play essential roles in many biological activities of both prokaryotic and eukaryotic cells, including electron transfer, enzyme catalysis and induction of apoptosis. We report a novel enrichment strategy for identifying c-type heme-containing peptides that uses non-activated IMAC resin. The strategy demonstrated at least seven-fold enrichment for heme-containing peptides digested from a cytochrome c protein standard, and quantitative linear performance was also assessed for heme-containing peptide enrichment. Heme-containing peptides extracted from the periplasmic fraction of Shewanella oneidensis MR-1 were further identified using higher-energy collisional dissociation tandem mass spectrometry. The results demonstrated the applicability of this enrichment strategy to identify c-type heme-containing peptides from a highly complex biological sample, and at the same time, confirmed the periplasmic localization of heme-containing proteins during suboxic respiration activities of S. oneidensis MR-1.

  5. Nitrogen doped carbon nanoparticles enhanced extracellular electron transfer for high-performance microbial fuel cells anode.

    PubMed

    Yu, Yang-Yang; Guo, Chun Xian; Yong, Yang-Chun; Li, Chang Ming; Song, Hao

    2015-12-01

    Nitrogen doped carbon nanoparticles (NDCN) were applied to modify the carbon cloth anodes of microbial fuel cells (MFCs) inoculated with Shewanella oneidensis MR-1, one of the most well-studied exoelectrogens. Experimental results demonstrated that the use of NDCN increased anodic absorption of flavins (i.e., the soluble electron mediator secreted by S. oneidensis MR-1), facilitating shuttle-mediated extracellular electron transfer. In addition, we also found that NDCN enabled enhanced contact-based direct electron transfer via outer-membrane c-type cytochromes. Taken together, the performance of MFCs with the NDCN-modified anode was enormously enhanced, delivering a maximum power density 3.5 times' higher than that of the MFCs without the modification of carbon cloth anodes.

  6. 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

  7. 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.

  8. 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.

  9. Charge-associated effects of fullerene derivatives on microbialstructural integrity and central metabolism

    SciTech Connect

    Tang, Yinjie J.; Ashcroft, Jared M.; Chen, Ding; Min, Guangwei; Kim, Chul; Murkhejee, Bipasha; Larabell, Carolyn; Keasling, Jay D.; Chen,Fanqing Frank

    2007-01-23

    The effects of four types of fullerene compounds (C60,C60-OH, C60-COOH, C60-NH2) were examined on two model microorganisms(Escherichia coli W3110 and Shewanella oneidensis MR-1). Positivelycharged C60-NH2 at concentrations as low as 10 mg/L inhibited growth andreduced substrate uptake for both microorganisms. Scanning ElectronMicroscopy (SEM) revealed damage to cellular structures.Neutrally-charged C60 and C60-OH had mild negative effects on S.oneidensis MR-1, whereas the negatively-charged C60-COOH did not affecteither microorganism s growth. The effect of fullerene compounds onglobal metabolism was further investigated using [3-13C]L-lactateisotopic labeling, which tracks perturbations to metabolic reaction ratesin bacteria by examining the change in the isotopic labeling pattern inthe resulting metabolites (often amino acids).1-3 The 13C isotopomeranalysis from all fullerene-exposed cultures revealed no significantdifferences in isotopomer distributions from unstressed cells. Thisresult indicates that microbial central metabolism is robust toenvironmental stress inflicted by fullerene nanoparticles. In addition,although C60-NH2 compounds caused mechanical stress on the cell wall ormembrane, both S. oneidensis MR-1 and E. coli W3110 can efficientlyalleviate such stress by cell aggregation and precipitation of the toxicnanoparticles. The results presented here favor the hypothesis thatfullerenes cause more membrane stress4, 5, 6 than perturbation to energymetabolism7

  10. Metabolic profiling directly from the Petri dish using nanoDESI imaging mass spectrometry

    PubMed Central

    Watrous, Jeramie; Roach, Patrick; Heath, Brandi; Alexandrov, Theodore; Laskin, Julia; Dorrestein, Pieter C.

    2014-01-01

    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, 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. PMID:24047514

  11. 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.

  12. 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. PMID:23377928

  13. 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

  14. 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.

  15. 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

  16. 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) ). PMID:25899863

  17. 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) ).

  18. 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.

  19. 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].

  20. 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.

  1. Single-Cell Imaging and Spectroscopic Analyses of Cr(VI) Reduction on the Surface of Bacterial Cells

    PubMed Central

    Wang, Yuanmin; Sevinc, Papatya C.; Balchik, Sara M.; Fridrickson, Jim; Shi, Liang; Lu, H. Peter

    2013-01-01

    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 ΔmtrC or mutant ΔomcA > double mutant (ΔomcA-ΔmtrC). 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. PMID:23249294

  2. Characterisation of the dissimilatory reduction of Fe(III)-oxyhydroxide at the microbe-mineral interface: the application of STXM-XMCD.

    PubMed

    Coker, V S; Byrne, J M; Telling, N D; VAN DER Laan, G; Lloyd, J R; Hitchcock, A P; Wang, J; Pattrick, R A D

    2012-07-01

    A combination of scanning transmission X-ray microscopy and X-ray magnetic circular dichroism was used to spatially resolve the distribution of different carbon and iron species associated with Shewanella oneidensis MR-1 cells. S. oneidensis MR-1 couples the reduction of Fe(III)-oxyhydroxides to the oxidation of organic matter in order to conserve energy for growth. Several potential mechanisms may be used by S. oneidensis MR-1 to facilitate Fe(III)-reduction. These include direct contact between the cell and mineral surface, secretion of either exogenous electron shuttles or Fe-chelating agents and the production of conductive 'nanowires'. In this study, the protein/lipid signature of the bacterial cells was associated with areas of magnetite (Fe₃O₄), the product of dissimilatory Fe(III) reduction, which was oversaturated with Fe(II) (compared to stoichiometric magnetite). However, areas of the sample rich in polysaccharides, most likely associated with extracellular polymeric matrix and not in direct contact with the cell surface, were undersaturated with Fe(II), forming maghemite-like (γ-Fe₂O₃) phases compared to stoichiometric magnetite. The reduced form of magnetite will be much more effective in environmental remediation such as the immobilisation of toxic metals. These findings suggest a dominant role for surface contact-mediated electron transfer in this study and also the inhomogeneity of magnetite species on the submicron scale present in microbial reactions. This study also illustrates the applicability of this new synchrotron-based technique for high-resolution characterisation of the microbe-mineral interface, which is pivotal in controlling the chemistry of the Earth's critical zone.

  3. 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.

  4. Biogenic formation of As-S nanotubes by diverse Shewanella strains

    SciTech Connect

    Jiang, Shenghua; Lee, Ji-Hoon; Kim, Min-Gyu; Myung, Nosang V.; Fredrickson, Jim K.; Sadowsky, Michael J.; Hur, Hor-Gil

    2009-11-01

    Shewanella sp. strain HN-41 was previously shown to produce novel, photoactive, As-S nanotubes via the reduction of As(V) and S2O3 2* under anaerobic conditions. To determine if this ability was unique to this bacterium, 10 different Shewanella strains, including Shewanella sp. strain HN-41, Shewanella sp. strain PV-4, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were examined for production of As-S nanotubes under standardized conditions. Of the 10 strains examined, three formed As-S nanotubes like those of strain HN-41. While Shewanella sp. strain HN-41 and S.putrefaciens CN-32 rapidly formed As-S precipitates in 7 days, strains S. alga BrY and S. oneidensis MR-1 reduced As(V) at a much lower rate and formed yellow As-S after 30 days. Electron microscopy, energy-dispersive X-ray spectroscopy, and extended X-ray absorption fine-structure spectroscopy analyses showed that the morphological and chemical properties of As-S formed by strains S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 were similar to those previously determined for Shewanella sp. strain HN-41 As-S nanotubes. These studies indicated that the formation of As-S nanotubes is widespread among Shewanella strains and is closely related to bacterial growth and the reduction rate of As(V) and thiosulfate.

  5. Characterisation of the dissimilatory reduction of Fe(III)-oxyhydroxide at the microbe-mineral interface: the application of STXM-XMCD.

    PubMed

    Coker, V S; Byrne, J M; Telling, N D; VAN DER Laan, G; Lloyd, J R; Hitchcock, A P; Wang, J; Pattrick, R A D

    2012-07-01

    A combination of scanning transmission X-ray microscopy and X-ray magnetic circular dichroism was used to spatially resolve the distribution of different carbon and iron species associated with Shewanella oneidensis MR-1 cells. S. oneidensis MR-1 couples the reduction of Fe(III)-oxyhydroxides to the oxidation of organic matter in order to conserve energy for growth. Several potential mechanisms may be used by S. oneidensis MR-1 to facilitate Fe(III)-reduction. These include direct contact between the cell and mineral surface, secretion of either exogenous electron shuttles or Fe-chelating agents and the production of conductive 'nanowires'. In this study, the protein/lipid signature of the bacterial cells was associated with areas of magnetite (Fe₃O₄), the product of dissimilatory Fe(III) reduction, which was oversaturated with Fe(II) (compared to stoichiometric magnetite). However, areas of the sample rich in polysaccharides, most likely associated with extracellular polymeric matrix and not in direct contact with the cell surface, were undersaturated with Fe(II), forming maghemite-like (γ-Fe₂O₃) phases compared to stoichiometric magnetite. The reduced form of magnetite will be much more effective in environmental remediation such as the immobilisation of toxic metals. These findings suggest a dominant role for surface contact-mediated electron transfer in this study and also the inhomogeneity of magnetite species on the submicron scale present in microbial reactions. This study also illustrates the applicability of this new synchrotron-based technique for high-resolution characterisation of the microbe-mineral interface, which is pivotal in controlling the chemistry of the Earth's critical zone. PMID:22515480

  6. Microbial activity at gigapascal pressures.

    PubMed

    Sharma, Anurag; Scott, James H; Cody, George D; Fogel, Marilyn L; Hazen, Robert M; Hemley, Russell J; Huntress, Wesley T

    2002-02-22

    We observed physiological and metabolic activity of Shewanella oneidensis strain MR1 and Escherichia coli strain MG1655 at pressures of 68 to 1680 megapascals (MPa) in diamond anvil cells. We measured biological formate oxidation at high pressures (68 to 1060 MPa). At pressures of 1200 to 1600 MPa, living bacteria resided in fluid inclusions in ice-VI crystals and continued to be viable upon subsequent release to ambient pressures (0.1 MPa). Evidence of microbial viability and activity at these extreme pressures expands by an order of magnitude the range of conditions representing the habitable zone in the solar system. PMID:11859192

  7. Development and application of microelectrodes in biofilms

    NASA Astrophysics Data System (ADS)

    Nguyen, Hung Duc

    The objective of this dissertation was to develop novel microelectrode techniques and to use microelectrodes for microscale chemistry characterization in biofilms. The first part of the dissertation focused on the development of microelectrodes operated in biofilms respiring on electrodes. The novel improvement required that microelectrodes be compatible within a three-electrode electrochemical cell. Redox potential, pH and hydrogen peroxide microelectrodes were successfully constructed with built-in silver/silver chloride reference electrodes that allowed for such measurements. By using these novel redox potential and pH microelectrodes we found that the decreasing redox potential in Shewanella oneidensis MR-1 biofilms from the bulk phase was not due to pH changes. Shewanella oneidensis MR-1 biofilms were not redox-controlled when they respired on electrodes. The second part of the dissertation focused on the development of a flavin microelectrode to detect flavins, known chemicals playing a role as electron mediators in Shewanella oneidensis MR-1. We successfully developed the novel microelectrode and operated it using a unique voltammetric technique, square wave voltammetry. It was found that the flavin concentration reached 0.7 µM, increasing near the bottom of the Shewanella oneidensis MR-1 biofilm, where no oxygen was present. The third part of the dissertation focused on microelectrode mapping techniques to quantify microscale geochemical gradients in Hanford 300A subsurface sediment biofilms. We found that there were significant differences in geochemical parameters across the sediment biofilm/water interface in the presence and absence of U(VI) under oxic and anoxic conditions. The results also revealed the presence of geochemical hotspots, i.e., microenvironments with relatively high or low biogeochemical activities in sediment biofilms, which had important practical implications for

  8. 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.

  9. 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.

  10. 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.

  11. Towards environmental systems biology of Shewanella

    SciTech Connect

    Beliaev, Alexander S; Nealson, Kenneth H.; Pinchuk, Grigoriy; Rodrigues, Jorge L.M.; Saffarini, Daad; Serres, Margrethe H.; Zhulin, Igor B; Tiedje, James M

    2008-01-01

    Bacteria of the genus Shewanella are known for their versatile electron-accepting capacities, which allow them to couple the decomposition of organic matter to the reduction of the various terminal electron acceptors that they encounter in their stratified environments. Owing to their diverse metabolic capabilities, shewanellae are important for carbon cycling and have considerable potential for the remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species Shewanella oneidensis MR-1 and other members of this genus has provided new insights into the signal-transduction proteins, regulators, and metabolic and respiratory subsystems that govern the remarkable versatility of the shewanellae.

  12. Modeling of Sustainable Base Production by Microbial Electrolysis Cell.

    PubMed

    Blatter, Maxime; Sugnaux, Marc; Comninellis, Christos; Nealson, Kenneth; Fischer, Fabian

    2016-07-01

    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.

  13. Microbial activity at gigapascal pressures.

    PubMed

    Sharma, Anurag; Scott, James H; Cody, George D; Fogel, Marilyn L; Hazen, Robert M; Hemley, Russell J; Huntress, Wesley T

    2002-02-22

    We observed physiological and metabolic activity of Shewanella oneidensis strain MR1 and Escherichia coli strain MG1655 at pressures of 68 to 1680 megapascals (MPa) in diamond anvil cells. We measured biological formate oxidation at high pressures (68 to 1060 MPa). At pressures of 1200 to 1600 MPa, living bacteria resided in fluid inclusions in ice-VI crystals and continued to be viable upon subsequent release to ambient pressures (0.1 MPa). Evidence of microbial viability and activity at these extreme pressures expands by an order of magnitude the range of conditions representing the habitable zone in the solar system.

  14. 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.

  15. Graphical estimation of the dual-enzyme kinetic parameters for Cr(VI) reduction.

    PubMed

    Hossain, Md Akram

    2006-03-01

    Chromium(VI) (Cr(VI)) contamination of soil and groundwater is a major environmental concern. Bioreduction of Cr(VI) by Shewanella oneidensis MR-1 (MR-1) can be considered a feasible option to reduce the toxic and mobile Cr(VI) to the less toxic and less mobile chromium(III) (Cr(III)). The reaction rate expression for Cr(VI) reduction is nonlinear and the rate constants are evaluated by employing nonlinear optimization techniques. The outcome of the optimization techniques, in general, depends on the initial estimate of the kinetic parameters which is not always available. A graphical approach based on sound mathematical reasoning has been developed which is accurate, simpler to use, and can provide the best initial estimate for nonlinear optimization.

  16. Utilization of DNA as a Sole Source of Phosphorus, Carbon, and Energy by Shewanella spp.: Ecological and Physiological Implications for Dissimilatory Metal Reduction▿

    PubMed Central

    Pinchuk, Grigoriy E.; Ammons, Christine; Culley, David E.; Li, Shu-Mei W.; McLean, Jeff S.; Romine, Margaret F.; Nealson, Kenneth H.; Fredrickson, Jim K.; Beliaev, Alexander S.

    2008-01-01

    The solubility of orthophosphate (PO43−) in iron-rich sediments can be exceedingly low, limiting the bioavailability of this essential nutrient to microbial populations that catalyze critical biogeochemical reactions. Here we demonstrate that dissolved extracellular DNA can serve as a sole source of phosphorus, as well as carbon and energy, for metal-reducing bacteria of the genus Shewanella. Shewanella oneidensis MR-1, Shewanella putrefaciens CN32, and Shewanella sp. strain W3-18-1 all grew with DNA but displayed different growth rates. W3-18-1 exhibited the highest growth rate with DNA. While strain W3-18-1 displayed Ca2+-independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca2+ for growth with DNA. For S. oneidensis MR-1, the utilization of DNA as a sole source of phosphorus is linked to the activities of extracellular phosphatase(s) and a Ca2+-dependent nuclease(s), which are regulated by phosphorus availability. Mass spectrometry analysis of the extracellular proteome of MR-1 identified one putative endonuclease (SO1844), a predicted UshA (bifunctional UDP-sugar hydrolase/5′ nucleotidase), a predicted PhoX (calcium-activated alkaline phosphatase), and a predicted CpdB (bifunctional 2′,3′ cyclic nucleotide 2′ phosphodiesterase/3′ nucleotidase), all of which could play important roles in the extracellular degradation of DNA under phosphorus-limiting conditions. Overall, the results of this study suggest that the ability to use exogenous DNA as the sole source of phosphorus is widespread among the shewanellae, and perhaps among all prokaryotes, and may be especially important for nutrient cycling in metal-reducing environments. PMID:18156329

  17. CeCaFDB: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13C-fluxomics

    PubMed Central

    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. PMID:25392417

  18. Mtr Extracellular Electron Transfer Pathways in Fe(III)-reducing or Fe(II)-oxidizing Bacteria: A Genomic Perspective

    SciTech Connect

    Shi, Liang; Rosso, Kevin M.; Zachara, John M.; Fredrickson, Jim K.

    2012-12-01

    Originally discovered in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), the Mtr (i.e., metal-reducing) pathway exists in all characterized strains of metal-reducing Shewanella. The protein components identified to date for the Mtr pathway of MR-1 include four multi-heme c-type cytochromes (c-Cyts), CymA, MtrA, MtrC and OmcA, and a porin-like, outer membrane protein MtrB. They are strategically positioned along the width of the MR-1 cell envelope to mediate electron transfer from the quinone/quinol pool in the inner-membrane to the Fe(III)-containing minerals external to the bacterial cells. A survey of microbial genomes revealed homologues of the Mtr pathway in other dissimilatory Fe(III)-reducing bacteria, including Aeromonas hydrophila, Ferrimonas balearica and Rhodoferax ferrireducens, and in the Fe(II)-oxidizing bacteria Dechloromonas aromatica RCB, Gallionella capsiferriformans ES-2 and Sideroxydans lithotrophicus ES-1. The widespread distribution of Mtr pathways in Fe(III)-reducing or Fe(II)-oxidizing bacteria emphasizes the importance of this type of extracellular electron transfer pathway in microbial redox transformation of Fe. Their distribution in these two different functional groups of bacteria also emphasizes the bi-directional nature of electron transfer reactions carried out by the Mtr pathways. The characteristics of the Mtr pathways may be shared by other pathways used by microorganisms for exchanging electrons with their extracellular environments.

  19. 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.

  20. 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.

  1. 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

  2. ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry.

    PubMed

    Wen, Yurong; Sobott, Frank; Devreese, Bart

    2016-08-01

    Toxin-antitoxin systems are genetic modules involved in a broad range of bacterial cellular processes including persistence, multidrug resistance and tolerance, biofilm formation, and pathogenesis. In type II toxin-antitoxin systems, both the toxin and antitoxin are proteins. In the prototypic Escherichia coli HipA-HipB module, the antitoxin HipB forms a complex with the protein kinase HipA and sequesters it in the nucleoid. HipA is then no longer able to phosphorylate glutamyl-tRNA-synthetase and this prevents the initiation of the forthcoming stringent response. Here we investigated the assembly of the Shewanella oneidensis MR-1 HipA-HipB complex using native electrospray ion mobility-mass spectrometry and chemical crosslinking combined with mass spectrometry. We revealed that the HipA autophosphorylation was accompanied by a large conformational change, and confirmed structural evidence that S. oneidensis MR-1 HipA-HipB assembly was distinct from the prototypic E. coli HipA-HipB complex. Graphical abstract Ion mobility mass spectrometry shows a two phase transition from unstructured HipA to a compact folded phosphorylated protein. PMID:27325463

  3. Anodic biofilms as the interphase for electroactive bacterial growth on carbon veil.

    PubMed

    Artyushkova, Kateryna; Roizman, Dan; Santoro, Carlo; Doyle, Lucinda Elizabeth; Fatima Mohidin, Abeed; Atanassov, Plamen; Marsili, Enrico

    2016-01-01

    The structure and activity of electrochemically active biofilms (EABs) are usually investigated on flat electrodes. However, real world applications such as wastewater treatment and bioelectrosynthesis require tridimensional electrodes to increase surface area and facilitate EAB attachment. The structure and activity of thick EABs grown on high surface area electrodes are difficult to characterize with electrochemical and microscopy methods. Here, the authors adopt a stacked electrode configuration to simulate the high surface and the tridimensional structure of an electrode for large-scale EAB applications. Each layer of the stacked electrode is independently characterized using confocal laser scanning microscopy (CLSM) and digital image processing. Shewanella oneidensis MR-1 biofilm on stacked carbon veil electrodes is grown under constant oxidative potentials (0, +200, and +400 mV versus Ag/AgCl) until a stable current output is obtained. The textural, aerial, and volumetric parameters extracted from CLSM images allow tracking of the evolution of morphological properties within the stacked electrodes. The electrode layers facing the bulk liquid show higher biovolumes compared with the inner layer of the stack. The electrochemical performance of S. oneidensis MR-1 is directly linked to the overall biofilm volume as well as connectivity between cell clusters. PMID:27609094

  4. 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.

  5. 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.

  6. Shewanella frigidimarina microbial fuel cells and the influence of divalent cations on current output.

    PubMed

    Fitzgerald, Lisa A; Petersen, Emily R; Leary, Dagmar H; Nadeau, Lloyd J; Soto, Carissa M; Ray, Richard I; Little, Brenda J; Ringeisen, Bradley R; Johnson, Glenn R; Vora, Gary J; Biffinger, Justin C

    2013-02-15

    The genes involved in the proposed pathway for Shewanella extracellular electron transfer (EET) are highly conserved. While extensive studies involving EET from a fresh water Shewanella microbe (S. oneidensis MR-1) to soluble and insoluble electron acceptors have been published, only a few reports have examined EET from marine strains of Shewanella. Thus, Shewanella frigidimarina (an isolate from Antarctic Sea ice) was used within miniature microbial fuel cells (mini-MFC) to evaluate potential power output. During the course of this study several distinct differences were observed between S. oneidensis MR-1 and S. frigidimarina under comparable conditions. The maximum power density with S. frigidimarina was observed when the anolyte was half-strength marine broth (1/2 MB) (0.28 μW/cm(2)) compared to Luria-Bertani (LB) (0.07 μW/cm(2)) or a defined growth minimal medium (MM) (0.02 μW/cm(2)). The systematic modification of S. frigidimarina cultured in 1/2 MB and LB with divalent cations shows that a maximum current output can be generated independent of internal ionic ohmic losses and the presence of external mediators.

  7. The Roles of Outer Membrane Cytochromes of Shewanella and Geobacter in Extracellular Electron Transfer

    SciTech Connect

    Shi, Liang; Richardson, David; Wang, Zheming; Kerisit, Sebastien N.; Rosso, Kevin M.; Zachara, John M.; Fredrickson, Jim K.

    2009-08-01

    As key components of the electron transfer (ET) pathways used for dissimilatory reduction of solid iron [Fe(III)] and manganese [Mn(IV)] (hydr)oxides, outer membrane cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 and OmcE and OmcS of Geobacter sulfurreducens mediate ET reactions extracellularly. Cell surface-exposed MtrC and OmcA can transfer electrons directly to the metal oxides. S. oneidensis MR-1 cells also secrete flavins that can facilitate ET to the oxides. The secreted flavins are thought to serve either as chelators that form soluble Fe(III)/Mn(IV)-flavin complexes or as electron shuttles that ferry the electrons from cell-associated ET proteins to the metal oxides. Cell-surface localization may also permit MtrC and OmcA to transfer electrons extracellularly to either flavin-chelated Fe(III)/Mn(IV) or oxidized flavins. OmcE and OmcS are proposed to be located on the Geobacter cell surface where they are believed to function as the intermediates to relay electrons to type IV pili, which are then hypothesized to transfer electrons directly to the metal oxides. Thus, cell surface-localization positions these outer membrane cytochromes to transfer electrons to Fe(III)/Mn(IV) oxides external to the bacterial cells either directly, indirectly, or both, demonstrating a common strategy shared by Shewanella and Geobacter for extracellular reduction of the oxides.

  8. Relationship between surface chemistry, biofilm structure, and electron transfer in Shewanella anodes.

    PubMed

    Artyushkova, Kateryna; Cornejo, Jose A; Ista, Linnea K; Babanova, Sofia; Santoro, Carlo; Atanassov, Plamen; Schuler, Andrew J

    2015-03-05

    A better understanding of how anode surface properties affect growth, development, and activity of electrogenic biofilms has great potential to improve the performance of bioelectrochemical systems such as microbial fuel cells. The aim of this paper was to determine how anodes with specific exposed functional groups (-N(CH3)3 (+), -COOH, -OH, and -CH3), created using ω-substituted alkanethiolates self-assembled monolayers attached to gold, affect the surface properties and functional performance of electrogenic Shewanella oneidensis MR-1 biofilms. A combination of spectroscopic, microscopic, and electrochemical techniques was used to evaluate how electrode surface chemistry influences morphological, chemical, and functional properties of S. oneidensis MR-1 biofilms, in an effort to develop improved electrode materials and structures. Positively charged, highly functionalized, hydrophilic surfaces were beneficial for growth of uniform biofilms with the smallest cluster sizes and intercluster diffusion distances, and yielding the most efficient electron transfer. The authors derived these parameters based on 3D morphological features of biofilms that were directly linked to functional properties of the biofilm during growth and that, during polarization, were directly connected to the efficiency of electron transfer to the anode. Our results indicate that substratum chemistry affects not only primary attachment, but subsequent biofilm development and bacterial physiology.

  9. 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

    2013-08-01

    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.

  10. Mechanisms of Bacterial Extracellular Electron Exchange.

    PubMed

    White, G F; Edwards, M J; Gomez-Perez, L; Richardson, D J; Butt, J N; Clarke, T A

    2016-01-01

    The biochemical mechanisms by which microbes interact with extracellular soluble metal ions and insoluble redox-active minerals have been the focus of intense research over the last three decades. The process presents two challenges to the microorganism. Firstly, electrons have to be transported at the cell surface, which in Gram-negative bacteria presents an additional problem of electron transfer across the ~6nm of the outer membrane. Secondly, the electrons must be transferred to or from the terminal electron acceptors or donors. This review covers the known mechanisms that bacteria use to transport electrons across the cell envelope to external electron donors/acceptors. In Gram-negative bacteria, electron transfer across the outer membrane involves the use of an outer membrane β-barrel and cytochrome. These can be in the form of a porin-cytochrome protein, such as Cyc2 of Acidithiobacillus ferrooxidans, or a multiprotein porin-cytochrome complex like MtrCAB of Shewanella oneidensis MR-1. For mineral-respiring organisms, there is the additional challenge of transferring the electrons from the cell to mineral surface. For the strict anaerobe Geobacter sulfurreducens this requires electron transfer through conductive pili to associated cytochrome OmcS that directly reduces Fe(III)oxides, while the facultative anaerobe S. oneidensis MR-1 accomplishes mineral reduction through direct membrane contact, contact through filamentous extensions and soluble flavin shuttles, all of which require the outer membrane cytochromes MtrC and OmcA in addition to secreted flavin. PMID:27134022

  11. 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

  12. 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

  13. Comparative Genomics and Experimental Characterization of N-Acetylglucosamine Utilization Pathway of Shewanella oneidensis

    SciTech Connect

    Yang, Chen; Rodionov, Dmitry A.; Li, Xiaoqing; Laikova, Olga N.; Gelfand, Mikhail S.; Zagnitko, Olga P.; Romine, Margaret F.; Obraztova, Anna; Nealson, Kenneth H.; Osterman, Andrei

    2006-10-06

    Members of the genus Shewanella are widespread in aquatic and sedimentary environments and are, in general, noted both for their respiratory versatility and for their limited repertoire of substrates that they can utilize as electron donors. This study expands the range of carbon substrates utilized by Shewanella spp., unambiguously identifies several genes involved in chitin metabolism, and describes a novel variant of the classical three-step biochemical conversion of GlcNAc to fructose-6-phosphate first described in Escherichia coli.

  14. Metabolically engineered glucose-utilizing Shewanella strains under anaerobic conditions.

    PubMed

    Choi, Donggeon; Lee, Sae Bom; Kim, Sohyun; Min, Byoungnam; Choi, In-Geol; Chang, In Seop

    2014-02-01

    Comparative genome analysis of Shewanella strains predicted that the strains metabolize preferably two- and three-carbon carbohydrates as carbon/electron source because many Shewanella genomes are deficient of the key enzymes in glycolysis (e.g., glucokinase). In addition, all Shewanella genomes are known to have only one set of genes associated with the phosphotransferase system required to uptake sugars. To engineer Shewanella strains that can utilize five- and six-carbon carbohydrates, we constructed glucose-utilizing Shewanella oneidensis MR-1 by introducing the glucose facilitator (glf; ZMO0366) and glucokinase (glk; ZMO0369) genes of Zymomonas mobilis. The engineered MR-1 strain was able to grow on glucose as a sole carbon/electron source under anaerobic conditions. The glucose affinity (Ks) and glucokinase activity in the engineered MR-1 strain were 299.46 mM and 0.259 ± 0.034 U/g proteins. The engineered strain was successfully applied to a microbial fuel cell system and exhibited current generation using glucose as the electron source.

  15. A VOLTAMMETRIC FLAVIN MICROELECTRODE FOR USE IN BIOFILMS

    PubMed Central

    Nguyen, Hung Duc; Renslow, Ryan; Babauta, Jerome; Ahmed, Bulbul; Beyenal, Haluk

    2011-01-01

    Biofilms used in bioelectrochemical systems are expected to transfer electrons using electron transfer mediators. One mediator type, flavins, which includes flavin mononucleotide, riboflavin, and flavin adenine dinucleotide, has been found to be endogenously produced by Shewanella oneidensis MR-1. However, the presence and concentration of flavins inside a S. oneidensis MR-1 biofilm have never been reported. The goal of this study was to develop a flavin microelectrode capable of measuring flavins inside a living biofilm and apply it to a biofilm which produces flavins. Because flavins are electrochemically active molecules, the flavin microelectrode was based on detection via square-wave voltammetry. The microelectrode consisted of a carbon working electrode with a 10–30 μm tip diameter, a built-in platinum counter electrode, and a Ag/AgCl reference electrode, all enclosed in a glass outer case. The microelectrode was calibrated between 0.1 μM and 10 μM flavins and showed a linear correlation between flavin concentration and peak currents located at −424 mVAg/AgCl on a square-wave voltammogram. We also developed a model to explain the electrochemical mechanism of flavin detection, and to determine the effective surface area of the microelectrode, the standard reduction potential, and the transfer coefficient. We found that the effective surface area of the microelectrode was close to 100 times the projected surface area. The model predicted a standard reduction potential for RF/RFH2 of −419 mVAg/AgCl at 20 °C and a transfer coefficient of 0.45. Lastly, we measured flavin concentration inside a S. oneidensis MR-1 biofilm grown on a glass surface using oxygen as the electron acceptor. The flavin concentration reached 0.7 μM, increasing near the bottom of the biofilm, where no oxygen was present. This shows the possibility that flavins are produced in the anaerobic zone to act as intermediate electron acceptors in the deeper parts of the biofilm, where

  16. 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

  17. 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

  18. The influence of high hydrostatic pressure on bacterial dissimilatory iron reduction

    NASA Astrophysics Data System (ADS)

    Picard, Aude; Testemale, Denis; Hazemann, Jean-Louis; Daniel, Isabelle

    2012-07-01

    The impact of deep-subsurface pressure conditions on microbial activity is still poorly constrained. In particular it is unknown how pressure of deep environments affects microbial transformations of iron. We investigated the effects of high hydrostatic pressure (HHP) on the rate and the extent of bacterial dissimilatory iron reduction (DIR). We employed a novel experimental setup that enables in situ monitoring of Fe oxidation state and speciation in bacterial cultures in an optimized HHP incubation system using X-ray Absorption Near-Edge Structure (XANES) spectroscopy. The iron-reducing bacterium Shewanella oneidensis MR-1 was incubated at 30 °C with Fe(III) citrate and tryptone at pressures between 0.1 and 100 MPa. For pressures up to 70 MPa strain MR-1 (108 cells ml-1) was able to reduce all 5 mM Fe(III) provided. Above 70 MPa, the final amount of Fe(III) that MR-1 could reduce decreased linearly and DIR was estimated to stop at 109 ± 7 MPa. The decrease in the reduction yield was correlated with the dramatic decrease in survival (as determined by CFU counts) above 70 MPa. The initial rate of DIR increased with pressure up to 40 MPa, then decreased to reach zero at about 110 MPa. Increased rates of DIR activity and relatively high growth rates for pressures below 40 MPa would potentially ensure the maintenance of MR-1 in most of deep subsurface environments where moderate pressures occur, i.e. deep-sea environments. This study not only provides the first in situ quantitative results for microbial iron metabolism under HHP conditions but also sets the stage for future investigations of deep-sea pressure-adapted iron reducers. Moreover it demonstrates for the first time that XANES at the Fe K-edge is a powerful probe for in vivo monitoring of iron transformations in living microbial cultures.

  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. An innovative miniature microbial fuel cell fabricated using photolithography.

    PubMed

    Chen, You-Peng; Zhao, Yue; Qiu, Ke-Qiang; Chu, Jian; Lu, Rui; Sun, Min; Liu, Xian-Wei; Sheng, Guo-Ping; Yu, Han-Qing; Chen, Jie; Li, Wen-Jie; Liu, Gang; Tian, Yang-Chao; Xiong, Ying

    2011-02-15

    Recently microbial fuel cells (MFCs) have attracted increasing interests in both environmental and energy fields. Among the various MFC configurations, miniature microbial fuel cell (mini-MFC) has a great potential for the application in medical, communication and other areas because of its miniature volume and high output power density. In this work, a 25-μL single-chamber mini-MFC was fabricated using the photolithography technique. The plate-shaped gold anodic electrode in the mini-MFC showed a higher electrochemical activity than the stripe-shaped one. A biofilm of Shewanella oneidensis MR-1 was formed on the surface of gold electrode in this micro-liter-scale MFCs. As a result, a maximum power density of 29 mW/m(2) and a maximum current density of 2148 mA/m(2) were achieved by this single-chamber mini-MFC.

  1. Phenothiazine Derivative-Accelerated Microbial Extracellular Electron Transfer in Bioelectrochemical System

    PubMed Central

    Liu, Xian-Wei; Sun, Xue-Fei; Chen, Jie-Jie; Huang, Yu-Xi; Xie, Jia-Fang; Li, Wen-Wei; Sheng, Guo-Ping; Zhang, Yuan-Yuan; Zhao, Feng; Lu, Rui; Yu, Han-Qing

    2013-01-01

    In bioelectrochemical system (BES) the extracellular electron transfer (EET) from bacteria to anode electrode is recognized as a crucial step that governs the anodic reaction efficiency. Here, we report a novel approach to substantially enhance the microbial EET by immobilization of a small active phenothiazine derivative, methylene blue, on electrode surface. A comparison of the currents generated by Shewanella oneidensis MR-1 and its mutants as well as the electrochemical analytical results reveal that the accelerated EET was attributed to enhanced interactions between the bacterial outer-membrane cytochromes and the immobilized methylene blue. A further investigation into the process using in situ Raman spectro-electrochemical method coupled with density functional theory calculations demonstrates that the electron shuttling was achieved through the change of the molecule conformation of phenothiazine in the redox process. These results offer useful information for engineering BES. PMID:23563590

  2. 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.

  3. 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.

  4. 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.

  5. Phenothiazine Derivative-Accelerated Microbial Extracellular Electron Transfer in Bioelectrochemical System

    NASA Astrophysics Data System (ADS)

    Liu, Xian-Wei; Sun, Xue-Fei; Chen, Jie-Jie; Huang, Yu-Xi; Xie, Jia-Fang; Li, Wen-Wei; Sheng, Guo-Ping; Zhang, Yuan-Yuan; Zhao, Feng; Lu, Rui; Yu, Han-Qing

    2013-04-01

    In bioelectrochemical system (BES) the extracellular electron transfer (EET) from bacteria to anode electrode is recognized as a crucial step that governs the anodic reaction efficiency. Here, we report a novel approach to substantially enhance the microbial EET by immobilization of a small active phenothiazine derivative, methylene blue, on electrode surface. A comparison of the currents generated by Shewanella oneidensis MR-1 and its mutants as well as the electrochemical analytical results reveal that the accelerated EET was attributed to enhanced interactions between the bacterial outer-membrane cytochromes and the immobilized methylene blue. A further investigation into the process using in situ Raman spectro-electrochemical method coupled with density functional theory calculations demonstrates that the electron shuttling was achieved through the change of the molecule conformation of phenothiazine in the redox process. These results offer useful information for engineering BES.

  6. 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.

  7. Efficient and selective isotopic labeling of hemes to facilitate the study of multiheme proteins

    SciTech Connect

    Fonseca, Bruno M.; Tien, Ming; Rivera, Mario; Shi, Liang; Louro, Ricardo O.

    2012-04-02

    Specific isotopic labeling of hemes provides a unique opportunity to characterize the structure and function of heme-proteins. Unfortunately, present day methods do not allow efficient labeling in high yields of multiheme cytochromes c, which are of great biotechnological interest. Here, a method for production of recombinant multiheme cytochromes c in Escherichia coli with isotopically labeled hemes is reported. A small tetraheme cytochrome of 12 kDa from Shewanella oneidensis MR-1 was used to demonstrate the method, achieving a production of 4 mg of pure protein per liter. This method achieves, in a single step, efficient expression and incorporation of hemes isotopically labeled in specific atom positions adequate for spectroscopic characterization of these complex heme proteins. It is, furthermore, of general application to heme proteins opening new possibilities in the characterization of this important class of proteins.

  8. 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.

  9. 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

  10. Highly sensitive detection of proteins and bacteria in aqueous solution using surface-enhanced Raman scattering and optical fibers.

    PubMed

    Yang, Xuan; Gu, Claire; Qian, Fang; Li, Yat; Zhang, Jin Z

    2011-08-01

    We report the detection of the proteins lysozyme and cytochrome c as well as the live bacterial cells of Shewanella oneidensis MR-1 in aqueous solutions with sensitivities order(s) of magnitude higher than those previously reported. Two highly sensitive surface-enhanced Raman scattering (SERS)-based biosensors using optical fibers have been employed for such label-free macromolecule detections. The first sensor is based on a tip-coated multimode fiber (TCMMF) with a double-substrate "sandwich" structure, and a detection limit of 0.2 μg/mL is achieved in protein detections. The second sensor is based on a liquid core photonic crystal fiber (LCPCF) with a better confinement of light inside the fiber core, and a detection limit of 10(6) cells/mL is achieved for the bacteria detection. Both SERS biosensors show great potential for highly sensitive and molecule-specific detection and identification of biomolecules. PMID:21692506

  11. The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes

    PubMed Central

    McMillan, Duncan G. G.; Marritt, Sophie J.; Kemp, Gemma L.; Gordon-Brown, Piers; Butt, Julea N.; Jeuken, Lars J. C.

    2014-01-01

    It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes. PMID:24634538

  12. The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes.

    PubMed

    McMillan, Duncan G G; Marritt, Sophie J; Kemp, Gemma L; Gordon-Brown, Piers; Butt, Julea N; Jeuken, Lars J C

    2013-11-01

    It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes.

  13. Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration.

    PubMed

    Costa, N L; Carlson, H K; Coates, J D; Louro, R O; Paquete, C M

    2015-07-01

    Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled.

  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. Microbial iron respiration can protect steel from corrosion.

    PubMed

    Dubiel, M; Hsu, C H; Chien, C C; Mansfeld, F; Newman, D K

    2002-03-01

    Microbiologically influenced corrosion (MC) of steel has been attributed to the activity of biofilms that include anaerobic microorganisms such as iron-respiring bacteria, yet the mechanisms by which these organisms influence corrosion have been unclear. To study this process, we generated mutants of the iron-respiring bacterium Shewanella oneidensis strain MR-1 that were defective in biofilm formation and/or iron reduction. Electrochemical impedance spectroscopy was used to determine changes in the corrosion rate and corrosion potential as a function of time for these mutants in comparison to the wild type. Counter to prevailing theories of MC, our results indicate that biofilms comprising iron-respiring bacteria may reduce rather than accelerate the corrosion rate of steel. Corrosion inhibition appears to be due to reduction of ferric ions to ferrous ions and increased consumption of oxygen, both of which are direct consequences of microbial respiration.

  16. Manipulating bacteria with opto-electrokinetic methods

    NASA Astrophysics Data System (ADS)

    Wereley, Steve; Kwon, Jae-Sung; Ravindranath, Sandeep; Irudayaraj, Joseph

    2010-11-01

    Recently we developed an opto-electrokinetic method for manipulating particles and cells called Rapid Electrokinetic Patterning (REP). REP is a very fast method for manipulating thousands of particles simultaneously and controllably owing to the creation of an electrothermal vortex that transports particles rapidly and in parallel to a site determined by the focal point of a laser beam. Whether particles are trapped at the center of the vortex or not is determined by their electrical properties (conductivity and permittivity). In this talk we demonstrate that REP can be used to manipulate the bacterium Shewanella oneidensis MR-1. The bacteria are assembled into large planar arrays of organisms. The dependence of this assembly process on voltage and frequency is quantified. REP can even be used to selectively manipulate and collect live or dead bacteria.

  17. 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.

  18. 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.

  19. 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

  20. Bacterial decolorization of textile dyes is an extracellular process requiring a multicomponent electron transfer pathway.

    PubMed

    Brigé, Ann; Motte, Bart; Borloo, Jimmy; Buysschaert, Géraldine; Devreese, Bart; Van Beeumen, Jozef J

    2008-01-01

    Many studies have reported microorganisms as efficient biocatalysts for colour removal of dye-containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR-1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox-active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. The key proteins of this network are MtrA and OmcB in the periplasm and outer membrane respectively. A model of the complete dye reduction pathway is proposed in which the dye molecules are reduced by the outer membrane cytochromes either directly or indirectly via melanin.

  1. 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.

  2. 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.

  3. In Situ Analysis of a Silver Nanoparticle-Precipitating Shewanella Biofilm by Surface Enhanced Confocal Raman Microscopy.

    PubMed

    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.

  4. Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping▿ †

    PubMed Central

    Johnson, Ethan T.; Baron, Daniel B.; Naranjo, Belén; Bond, Daniel R.; Schmidt-Dannert, Claudia; Gralnick, Jeffrey A.

    2010-01-01

    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. PMID:20453141

  5. 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

  6. Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry

    SciTech Connect

    Sharma, Seema; Simpson, David C.; Tolic, Nikola; Jaitly, Navdeep; Mayampurath, Anoop M.; Smith, Richard D.; Pasa-Tolic, Liljiana

    2007-02-01

    We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. 715 intact proteins were detected and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post translational modifications were assigned for ~10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction. Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C, 15N depleted media under aerobic and sub-oxic conditions. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. The strategy can be readily applied for measuring differential protein abundances, and provides a platform for high-throughput selection of biologically relevant targets for further characterization.

  7. Bacterial Nanowires: Is the Subsurface Hardwired?

    NASA Astrophysics Data System (ADS)

    Gorby, Y. A.; Davis, C. A.; Atekwana, E.

    2006-05-01

    Bacteria, ranging from oxygenic photosynthetic cyanobacteria to heterotrophic sulfate reducing bacteria, produce electrically-conductive appendages referred to as bacterial nanowires. Dissimilatory metal reducing bacteria, including Shewanella oneidensis and Geobacter sulfurreducens, produce electrically conductive nanowires in direct response to electron acceptor limitation and facilitate electron transfer to solid phase iron oxides. Nanowires produced by S. oneidensis strain MR-1, which served as our primary model organism, are functionalized by decaheme cytochromes MtrC and OmcA that are distributed along the length of the nanowires. Mutants deficient in MtrC and OmcA produce nanowires that were poorly conductive. These mutants also differ from wild type cells in their ability to reduce solid phase iron oxides, to produce electrical current in a mediator less microbial fuel cell, and to form complex biofilms at air liquid interfaces. Recent results obtained using direct cell counts and low frequency electrical measurements demonstrate that microbial growth correlated with real and imaginary electrical conductivity response in uncoated silica sand columns. Direct observation of packing material with environmental scanning electron microscopy (ESEM) revealed a fine network of extracellular structures that were morphologically similar to nanowires observed in metal reducing bacteria. No such structures were observed in control columns. We hypothesize that microbial nanowires may in part be responsible for the electrical response observed in the biostimulated columns.

  8. 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

    ...; Malta/McConnelsville Lock and Dam Project, P-13406-002; Lowell Lock and Dam Project, P-13407-002; Philo....... Washington Near the city of Devola. P-13406 Malta/McConnelsville Morgan Southern shore of the Lock and Dam... facilities. The proposed Malta/McConnelsville Lock and Dam Project would be located at the existing...

  9. 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:...

  10. 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

  11. Roles of UndA and MtrC of Shewanella putrefaciens W3-18-1 in iron reduction

    PubMed Central

    2013-01-01

    Background The completion of genome sequencing in a number of Shewanella species, which are most renowned for their metal reduction capacity, offers a basis for comparative studies. Previous work in Shewanella oneidensis MR-1 has indicated that some genes within a cluster (mtrBAC-omcA-mtrFED) were involved in iron reduction. To explore new features of iron reduction pathways, we experimentally analyzed Shewanella putrefaciens W3-18-1 since its gene cluster is considerably different from that of MR-1 in that the gene cluster encodes only four ORFs. Results Among the gene cluster, two genes (mtrC and undA) were shown to encode c-type cytochromes. The ΔmtrC deletion mutant revealed significant deficiencies in reducing metals of Fe2O3, α-FeO(OH), β-FeO(OH), ferric citrate, Mn(IV) and Co(III), but not organic compounds. In contrast, no deficiency of metal reduction was observed in the ΔundA deletion mutant. Nonetheless, undA deletion resulted in progressively slower iron reduction in the absence of mtrC and fitness loss under the iron-using condition, which was indicative of a functional role of UndA in iron reduction. Conclusions These results provide physiological and biochemical evidences that UndA and MtrC of Shewanella putrefaciens W3-18-1 are involved in iron reduction. PMID:24274142

  12. Growth of iron(III)-reducing bacteria on clay minerals as the sole electron acceptor and comparison of growth yields on a variety of oxidized iron forms.

    PubMed

    Kostka, Joel E; Dalton, Dava D; Skelton, Hayley; Dollhopf, Sherry; Stucki, Joseph W

    2002-12-01

    Smectite clay minerals are abundant in soils and sediments worldwide and are typically rich in Fe. While recent investigations have shown that the structural Fe(III) bound in clay minerals is reduced by microorganisms, previous studies have not tested growth with clay minerals as the sole electron acceptor. Here we have demonstrated that a pure culture of Shewanella oneidensis strain MR-1 as well as enrichment cultures of Fe(III)-reducing bacteria from rice paddy soil and subsurface sediments are capable of conserving energy for growth with the structural Fe(III) bound in smectite clay as the sole electron acceptor. Pure cultures of S. oneidensis were used for more detailed growth rate and yield experiments on various solid- and soluble-phase electron acceptors [smectite, Fe(III) oxyhydroxide FeOOH, Fe(III) citrate, and oxygen] in the same minimal medium. Growth was assessed as direct cell counts or as an increase in cell carbon (measured as particulate organic carbon). Cell counts showed that similar growth of S. oneidensis (10(8) cells ml(-1)) occurred with smectitic Fe(III) and on other Fe forms [amorphous Fe(III) oxyhydroxide, and Fe citrate] or oxygen as the electron acceptor. In contrast, cell yields of S. oneidensis measured as the increase in cell carbon were similar on all Fe forms tested while yields on oxygen were five times higher, in agreement with thermodynamic predictions. Over a range of particle loadings (0.5 to 4 g liter(-1)), the increase in cell number was highly correlated to the amount of structural Fe in smectite reduced. From phylogenetic analysis of the complete 16S rRNA gene sequences, a predominance of clones retrieved from the clay mineral-reducing enrichment cultures were most closely related to the low-G+C gram-positive members of the Bacteria (Clostridium and Desulfitobacterium) and the delta-Proteobacteria (members of the Geobacteraceae). Results indicate that growth with smectitic Fe(III) is similar in magnitude to that with Fe

  13. Manganese sulfide formation via concomitant microbial manganese oxide and thiosulfate reduction.

    SciTech Connect

    Lee, Ji-Hoon; Kennedy, David W.; Dohnalkova, Alice; Moore, Dean A.; Nachimuthu, Ponnusamy; Reed, Samantha B.; Fredrickson, Jim K.

    2011-09-27

    The dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1 produced γ-MnS (rambergite) nanoparticles under the concurrent reduction of synthetic MnO2 and thiosulfate coupled to H2 oxidation. Using two MR-1 mutants defective in outer membrane c-type cytochromes (ΔmtrC/ΔomcA and ΔmtrC/ΔomcA/ΔmtrF) to eliminate the direct reduction pathway for solid electron acceptors, it was determined that respiratory reduction of MnO2 was dominant relative to chemical reduction by biogenic sulfide generated from bacterial thiosulfate reduction. Although bicarbonate was excluded from the medium, incubations of MR-1 using lactate as the sole electron donor produced MnCO3 (rhodochrosite) as well as MnS in nearly equivalent amounts as estimated by micro X-ray diffraction (micro-XRD) analysis. It was concluded that carbonate released from lactate metabolism promoted MnCO3 formation and that Mn(II) mineralogy was strongly affected by carbonate ions even in the presence of abundant sulfide and weakly alkaline conditions that favor the precipitation of MnS. Formation of the biogenic MnS, as determined by a combination of micro-XRD, transmission electron microscopy, energy dispersive X-ray spectroscopy, and selected area electron diffraction analyses was consistent with equilibrium speciation modeling predictions. Although biogenic MnS likely only forms and is stable over a relatively narrow range of conditions, it may be a significant sink for Mn in anoxic marine basins and terrestrial subsurface sediments where Mn and sulfur compounds are undergoing concurrent reduction.

  14. Biogeochemical Coupling of Fe and Tc Speciation in Subsurface Sediments: Implications to Long-Term Tc Immobilization

    SciTech Connect

    Zachara, John M.; Kukkadapu, Ravi K.; Heald, Steve M.; McKinley, James P.; Dohnalkova, Alice C.; Fredrickson, James K.; Byong-Hun Jeon

    2006-04-05

    The overall project has been investigating the reactivity of pertechnetate [Tc(VII)] with Fe(II) forms in model mineral and mineral-microbe systems, and with sediments from the Oak Ridge FRC and the Hanford site. Past project results with Hanford and Oak Ridge sediments have been published in Fredrickson et al., (2004) and Kukkadapu et al., (2006). This poster summarizes a series of model system experiments that investigates whether microbes or biogenic Fe(II) were more important in the reduction of Tc(VII) in an anoxic suspension of ferrihydrite, Shewanella oneidensis MR-1, Tc(VII), and electron donor. Ferrihydrite is used to represent a bioavailable Fe(III) oxide present in small amounts in Oak Ridge and Hanford sediments. In order to address this overall goal, Tc(VII) reduction rates and redox products were studied in less complex systems where individual abiotic and biotic reactions were isolated for rigorous characterization. The specific objectives of the individual experiments in the series were as follows: (1) Identify the rates and products of the reaction of Tc(VII) with aqueous Fe(II) at circumneutral pH values (homogeneous reduction). (2) Identify the rates and products of the reaction of Tc(VII) with surface complexed Fe(II) on goethite and hematite in the circumneutral pH range (heterogeneous reduction). (3) Identify the rates and products of the reaction of Tc(VII) with MR-1 under anoxic conditions individually with hydrogen and lactate as electron donors (biologic reduction). (4) Use insights from the above experiments to determine which of the three above, potentially parallel reactions determine the final speciation of Tc in a mixture of ferrihydrite, respiring MR-1, and Tc(VII).

  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. 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

  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. 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.

  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. 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

  2. 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

  3. 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

  4. 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

  5. The Role of Clonal Interference in the Evolutionary Dynamics of Plasmid-Host Adaptation

    PubMed Central

    Hughes, Julie M.; Lohman, Brian K.; Deckert, Gail E.; Nichols, Eric P.; Settles, Matt; Abdo, Zaid; Top, Eva M.

    2012-01-01

    ABSTRACT Promiscuous plasmids replicate in a wide range of bacteria and therefore play a key role in the dissemination of various host-beneficial traits, including antibiotic resistance. Despite the medical relevance, little is known about the evolutionary dynamics through which drug resistance plasmids adapt to new hosts and thereby persist in the absence of antibiotics. We previously showed that the incompatibility group P-1 (IncP-1) minireplicon pMS0506 drastically improved its stability in novel host Shewanella oneidensis MR-1 after 1,000 generations under antibiotic selection for the plasmid. The only mutations found were those affecting the N terminus of the plasmid replication initiation protein TrfA1. Our aim in this study was to gain insight into the dynamics of plasmid evolution. Changes in stability and genotype frequencies of pMS0506 were monitored in evolving populations of MR-1 (pMS0506). Genotypes were determined by sequencing trfA1 amplicons from individual clones and by 454 pyrosequencing of whole plasmids from entire populations. Stability of pMS0506 drastically improved by generation 200. Many evolved plasmid genotypes with point mutations as well as in-frame and frameshift deletions and duplications in trfA1 were observed in all lineages with both sequencing methods. Strikingly, multiple genotypes were simultaneously present at high frequencies (>10%) in each population. Their relative abundances changed over time, but after 1,000 generations only one or two genotypes dominated the populations. This suggests that hosts with different plasmid genotypes were competing with each other, thus affecting the evolutionary trajectory. Plasmids can thus rapidly improve their stability, and clonal interference plays a significant role in plasmid-host adaptation dynamics. PMID:22761390

  6. 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.

  7. High-pressure-induced water penetration into 3-­isopropylmalate dehydrogenase

    PubMed Central

    Nagae, Takayuki; Kawamura, Takashi; Chavas, Leonard M. G.; Niwa, Ken; Hasegawa, Masashi; Kato, Chiaki; Watanabe, Nobuhisa

    2012-01-01

    Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH. PMID:22349232

  8. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    PubMed Central

    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.; Paša-Tolić, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-01-01

    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 18O 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 16O and 18O 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 ΔgspD mutant cells of many outer membrane proteins including the outer membrane c-cype cytochromes OmcA and MtrC, in agreement with previously investigation demonstrating that these proteins are substrates of the type II secretion system. PMID:20380418

  9. In Vitro Evolution of a Peptide with a Hematite Binding Motif That May Constitute a Natural Metal-Oxide Binding Archetype

    SciTech Connect

    Lower, Brian H.; Lins, Roberto D.; Oestreicher, Zachery W.; Straatsma, T. P.; Hochella Jr., Michael F.; Shi, Liang; Lower, Steven

    2008-04-18

    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.

  10. 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

  11. A whole-cell electrochemical biosensing system based on bacterial inward electron flow for fumarate quantification.

    PubMed

    Si, Rong-Wei; Zhai, Dan-Dan; Liao, Zhi-Hong; Gao, Lu; Yong, Yang-Chun

    2015-06-15

    Fumarate is of great importance as it is an oncometabolite as well as food spoilage indicator. However, cost-effective and fast quantification method for fumarate is lacking although it is urgently required. This work developed an electrochemical whole-cell biosensing system for fumarate quantification. A sensitive inwards electric output (electron flow from electrode into bacteria) responded to fumarate in Shewanella oneidensis MR-1 was characterized, and an electrochemical fumarate biosensing system was developed without genetic engineering. The biosensing system delivered symmetric current peak immediately upon fumarate addition, where the peak area increased in proportion to the increasing fumarate concentration with a wide range of 2 μM-10 mM (R(2)=0.9997). The limit of detection (LOD) and the limit of quantification (LOQ) are 0.83 μM and 1.2 μM, respectively. This biosensing system displayed remarkable specificity to fumarate against other possible interferences. It was also successfully applied to samples of apple juice and kidney tissue. This study added new dimension to electrochemical biosensor design, and provide a simple, cost-effective, fast and robust tool for fumarate quantification.

  12. Biofilm shows spatially stratified metabolic responses to contaminant exposure

    PubMed Central

    Cao, Bin; Majors, Paul D.; Ahmed, Bulbul; Renslow, Ryan S.; Silvia, Crystal P.; Shi, Liang; Kjelleberg, Staffan; Fredrickson, Jim K.; Beyenal, Haluk

    2012-01-01

    Summary Biofilms are core to a range of biological processes, including the bioremediation of environmental contaminants. Within a biofilm population, cells with diverse genotypes and phenotypes coexist, suggesting that distinct metabolic pathways may be expressed based on the local environmental conditions in a biofilm. However, metabolic responses to local environmental conditions in a metabolically active biofilm interacting with environmental contaminants have never been quantitatively elucidated. In this study, we monitored the spatiotemporal metabolic