Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants

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 degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.     

厌氧条件下Shewanella oneidensis MR-1对2,4-二硝基甲苯的还原转化

【目的】研究Shewanella oneidensis MR-1厌氧生物转化2,4-二硝基甲苯(2,4-DNT)的能力、转化过程和影响因素。【方法】以乳酸钠为电子供体, 2,4-DNT为电子受体, S. oneidensis MR-1为降解菌, 黄素为胞外电子载体, 设立四个不同的对照体系并监测各体系在转化过程中2,4-DNT及其产物的动态变化。同时研究不同2,4-DNT浓度下细胞的生长情况, 以及不同黄素浓度下2,4-DNT的降解情况。【结果】S. oneidensis MR-1菌能够高效还原转化2,4-DNT为4-氨基-2-硝基甲苯(4A2NT)和2-氨基-4-硝基甲苯(2A4NT), 并将其进一步还原为2,4-二氨基甲苯(2,4-DAT), 黄素能加速转化过程。【结论】S. oneidensis MR-1菌具备高效还原转化2,4-DNT的能力, 为实际环境中硝基苯污染的原位修复提供科学依据。     

Anaerobic regulation by an atypical Arc system in Shewanella oneidensis

Shewanella oneidensis strain MR-1 is well known for its respiratory versatility, yet little is understood about how it regulates genes involved in anaerobic respiration. The Arc two-component system plays an important role in this process in Escherichia coli; therefore, we determined its function in S. oneidensis. arcA from S. oneidensis complements an E. coli arcA mutant, but the Arc regulon in S. oneidensis constitutes a different suite of genes. For example, one of the strongest ArcA-regulated gene clusters in E. coli, sdh, is not regulated by the Arc system in S. oneidensis, and the cyd locus, which is induced by ArcA in E. coli under microaerobic conditions, is repressed by ArcA in S. oneidensis under anaerobic conditions. One locus that we identified as being potentially regulated by ArcA in S. oneidensis contains genes predicted to encode subunits of a dimethyl sulphoxide (DMSO) reductase. We demonstrate that these genes encode a functional DMSO reductase, and that an arcA mutant cannot fully induce their expression and is defective in growing on DMSO under anaerobic conditions. While S. oneidensis lacks a highly conserved full-length ArcB homologue, ArcA is partially activated by a small protein homologous to the histidine phosphotransfer domain of ArcB from E. coli, HptA. This protein alone is unable to compensate for the lack of arcB in E. coli, indicating that another protein is required in addition to HptA to activate ArcA in S. oneidensis.     

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

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

High frequency of glucose-utilizing mutants in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 has conventionally been considered unable to use glucose as a carbon substrate for growth. The genome sequence of S. oneidensis MR-1 however suggests the ability to use glucose. Here, we demonstrate that during initial glucose exposure, S. oneidensis MR-1 quickly and frequently gains the ability to utilize glucose as a sole carbon source, in contrast to wild-type S. oneidensis, which cannot immediately use glucose as a sole carbon substrate. High-performance liquid chromatography and (14)C glucose tracer studies confirm the disappearance in cultures and assimilation and respiration, respectively, of glucose. The relatively short time frame with which S. oneidensis MR-1 gained the ability to use glucose raises interesting ecological implications.     

Roles of two Shewanella oneidensis MR-1 extracellular endonucleases

The dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 is capable of using extracellular DNA (eDNA) as the sole source of carbon, phosphorus, and nitrogen. In addition, we recently demonstrated that S. oneidensis MR-1 requires eDNA as a structural component during all stages of biofilm formation. In this study, we characterize the roles of two Shewanella extracellular endonucleases, ExeS and ExeM. While ExeS is likely secreted into the medium, ExeM is predicted to remain associated with the cell envelope. Both exeM and exeS are highly expressed under phosphate-limited conditions. Mutants lacking exeS and/or exeM exhibit decreased eDNA degradation; however, the capability of S. oneidensis MR-1 to use DNA as the sole source of phosphorus is only affected in mutants lacking exeM. Neither of the two endonucleases alleviates toxic effects of increased eDNA concentrations. The deletion of exeM and/or exeS significantly affects biofilm formation of S. oneidensis MR-1 under static conditions, and expression of exeM and exeS drastically increases during static biofilm formation. Under hydrodynamic conditions, a deletion of exeM leads to altered biofilms that consist of densely packed structures which are covered by a thick layer of eDNA. Based on these results, we hypothesize that a major role of ExeS and, in particular, ExeM of S. oneidensis MR-1, is to degrade eDNA as a matrix component during biofilm formation to improve nutrient supply and to enable detachment.     

The influence of acidity on microbial fuel cells containing Shewanella oneidensis

Microbial fuel cells (MFCs) traditionally operate at pH values between 6 and 8. However, the effect of pH on the growth and electron transfer abilities of Shewanella oneidensis MR-1 (wild-type) and DSP10 (spontaneous mutant), bacteria commonly used in MFCs, to electrodes has not been examined. Miniature MFCs using bare graphite felt electrodes and nanoporous polycarbonate membranes with MR-1 or DSP10 cultures generated >8W/m(3) and approximately 400muA between pH 6-7. The DSP10 strain significantly outperformed MR-1 at neutral pH but underperformed at pH 5. Higher concentrations of DSP10 were sustained at pH 7 relative to that of MR-1, whereas at pH 5 this trend was reversed indicating that cell count was not solely responsible for the observed differences in current. S. oneidensis MR-1 was determined to be more suitable than DSP10 for MFCs with elevated acidity levels. The concentration of riboflavin in the bacterial cultures was reduced significantly at pH 5 for DSP10, as determined by high performance liquid chromatography (HPLC) of the filter sterilized growth media. In addition, these results suggest that mediator biosynthesis and not solely bacterial concentration plays a significant role in current output from S. oneidensis containing MFCs.     

Growth with high planktonic biomass in Shewanella oneidensis fuel cells

Shewanella oneidensis MR-1 grew for over 50 days in microbial fuel cells, incompletely oxidizing lactate to acetate with high recovery of the electrons derived from this reaction as electricity. Electricity was produced with lactate or hydrogen and current was comparable to that of electricigens which completely oxidize organic substrates. However, unlike fuel cells with previously described electricigens, in which cells are primarily attached to the anode, at least as many of the S. oneidensis cells were planktonic as were attached to the anode. These results demonstrate that S. oneidensis may conserve energy for growth with an electrode serving as an electron acceptor and suggest that multiple strategies for electron transfer to fuel cell anodes exist.     

Structural domains, protein modules, and sequence similarities enrich our understanding of the Shewanella oneidensis MR-1 proteome

The protein coding sequences of S. oneidensis MR-1 were analyzed, and new annotations were given to 491 gene products, 306 of which were previously of unknown function. New information was mainly brought in from structural domain predictions for S. oneidensis proteins of the SUPERFAM database (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/) and newly identified and experimentally verified functions of homologous proteins. Proteins encoded by fused genes were identified and separated into modules, protein units of at least 83 aa with independent functions and distinct evolutionary histories. A reannotation of the fused gene products was done to assign functions to the appropriate module within the protein. Groups of sequence-similar proteins of S. oneidensis were assembled. The fused gene products were represented by their modular entities for the grouping process. The protein groups were analyzed for their size and functions, and they were used to indicate activities that are of importance to the environmental adaptation of this organism. Making use of several approaches not commonly used in annotation, we have been able to enrich our understanding of the functions encoded by the S. oneidensis genome.     

Oxygen exposure promotes fuel diversity for Shewanella oneidensis microbial fuel cells

Miniature microbial fuel cells (mini-MFCs) were used to monitor the current generated by Shewanella oneidensis DSP10 under both anaerobic and aerobic conditions when exposed to glucose as a potential electron donor. In addition to glucose, other carbon fuels including fructose, sucrose, acetate, and ascorbic acid were also tested. When the anolyte containing S. oneidensis was grown in the presence of oxygen, power densities of 270+/-10, 350+/-20, and 120+/-10 W/m(3) were recorded from the mini-MFC for glucose, fructose, and ascorbic acid electron donors, respectively, while sucrose and acetate produced no response. The power produced from glucose decreased considerably (相似文献   

A simple, rapid, and highly efficient gene expression system for multiheme cytochromes c

The genes of tetraheme cytochrome c3 and hexadecaheme high-molecular-weight cytochrome c from Desulfovibrio vulgaris could be overexpressed as holoproteins in Shewanella oneidensis TSP-C using pUC-type vectors of E. coli. Surprisingly, S. oneidensis was transformed directly by pUC-type vectors through electroporation. The yields of the recombinant proteins in this expression system were much higher than the previously reported ones.     

Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli

Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is not applicable to C. thermocellum proteins because it did not account for the peculiarities of protein sequences from thermophiles.     

产电微生物基因调控网络的构建和特异性通路分析

研究产电微生物胞外电子传递过程和机制,发现与产电效率相关的关键基因、通路和代谢物,是微生物燃料电池研究中的关键技术。为了发现在胞外电子传递过程中起到关键作用的基因以及通路,首先利用比较基因组学的方法,以模式微生物大肠杆菌和同属希瓦氏菌的其他菌株为参考,构建了Shewanella.onedensis MR-1的全基因组基因转录调控网络,大大扩展了目前已知的基因调控关系。然后以此网络为基础,结合基于蛋白质相互作用分析得到的胞外电子传递通路,构建了与胞外电子传递直接传递密切相关的细胞色素C编码基因及其相关调控基因构成的子网络,结合全基因组基因表达数据,研究了特异性条件下胞外电子传递的可能通路和基因调控过程。