Chemotactic responses to metals and anaerobic electron acceptors in Shewanella oneidensis MR-1

Although a previous study indicated that the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 lacks chemotactic responses to metals that can be used as anaerobic electron acceptors, new results show that this bacterium responds to both Mn(III) and Fe(III). Cells were also shown to respond to another unusual electron acceptor, the humic acid analog anthraquinone-2,6-disulfonate. These results indicate that S. oneidensis is capable of moving towards a number of unusual anaerobic electron acceptors, including some that would normally be insoluble in the environment. Additionally, S. oneidensis was shown to migrate in gradients of several divalent cations under anaerobic conditions. Although responses to the reduced forms of redox-active metals, such as Mn(II) and Fe(II), might indicate that S. oneidensis uses gradients of these metals to locate the insoluble electron acceptors Mn(III/IV) and Fe(III) for dissimilatory purposes, responses to non-redox-active metals, such as Zn(II), suggest that movement towards divalent cations might serve other, potentially assimilatory, purposes.     

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

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 low-spin 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.     

Soluble electron acceptors affect bioluminescence from Shewanella woodyi

Shewanella woodyi cultures were used to correlate bioluminescence intensity with changes in the electrochemical potential of a saltwater medium using soluble electron acceptors. A relationship between the concentration of NaNO₃ or CoCl₂ to bioluminescence intensity was confirmed using aerobic cultures of S. woodyi at 20°C with glucose as the sole carbon source. In general, increasing the concentration of nitrate or Co(II) reduced the bioluminescence per cell, with complete luminescence being repressed at ≥5 mM nitrate and ≥0.5 mM Co(II). Results from cell viability fluorescent staining concluded that increasing the concentration of Co(II) or nitrate did not affect the overall viability of the cells when compared with cultures lacking Co(II) or nitrate. These data show that potentials of <0.2 V vs Normal Hydrogen Electrode (NHE) repress the luminescence from the cells, but the exact mechanism is unclear. Our results indicated that the luminescence intensity from S. woodyi could be systematically reduced using these two soluble electron acceptors, making S. woodyi a potential model bacterium for whole‐cell luminescence bioelectrochemical sensor applications.     

NADH dehydrogenases drive inward electron transfer in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a promising chassis organism for microbial electrosynthesis because it has a well-defined biochemical pathway (the Mtr pathway) that can connect extracellular electrodes to respiratory electron carriers inside the cell. We previously found that the Mtr pathway can be used to transfer electrons from a cathode to intracellular electron carriers and drive reduction reactions. In this work, we hypothesized that native NADH dehydrogenases form an essential link between the Mtr pathway and NADH in the cytoplasm. To test this hypothesis, we compared the ability of various mutant strains to accept electrons from a cathode and transfer them to an NADH-dependent reaction in the cytoplasm, reduction of acetoin to 2,3-butanediol. We found that deletion of genes encoding NADH dehydrogenases from the genome blocked electron transfer from a cathode to NADH in the cytoplasm, preventing the conversion of acetoin to 2,3-butanediol. However, electron transfer to fumarate was not blocked by the gene deletions, indicating that NADH dehydrogenase deletion specifically impacted NADH generation and did not cause a general defect in extracellular electron transfer. Proton motive force (PMF) is linked to the function of the NADH dehydrogenases. We added a protonophore to collapse PMF and observed that it blocked inward electron transfer to acetoin but not fumarate. Together these results indicate a link between the Mtr pathway and intracellular NADH. Future work to optimize microbial electrosynthesis in S. oneidensis MR-1 should focus on optimizing flux through NADH dehydrogenases.     

Hydrogen Metabolism in Shewanella oneidensis MR-1

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.     

Hydrogen metabolism in Shewanella oneidensis MR-1

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 DeltahydA, DeltahyaB, and DeltahydA DeltahyaB 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.     

Metabolomic analyses show that electron donor and acceptor ratios control anaerobic electron transfer pathways in Shewanella oneidensis

This study investigated the physiological impact of changing electron donor–acceptor ratios on electron transfer pathways in the metabolically flexible subsurface bacterium Shewanella oneidensis, using batch and chemostat cultures, with an azo dye (ramazol black B) as the model electron acceptor. Altering the growth rate did result in changes in biomass yield, but not in other key physiological parameters including the total cytochrome content of the cells, the production of extracellular flavin redox shuttles or the potential of the organism to reduce the azo dye. Dramatic increases in the ability to reduce the dye were noted when cells were grown under conditions of electron acceptor (fumarate) limitation, although the yields of extracellular redox mediators (flavins) were similar under conditions of electron donor (lactate) or acceptor limitation. FT-IR spectroscopy confirmed shifts in the metabolic fingerprints of cells grown under these contrasting conditions, while spectrophotometric analyses supported a critical role for c-type cytochromes, expressed at maximal concentrations under conditions of electron acceptor limitation. Finally, key intracellular metabolites were quantified in batch experiments at various electron donor and acceptor ratios and analysed using discriminant analysis and a Bayesian network to construct a central metabolic pathway model for cells grown under conditions of electron donor or acceptor limitation. These results have identified key mechanisms involved in controlling electron transfer in Shewanella species, and have highlighted strategies to maximise reductive activity for a range of bioprocesses.     

Expression in periplasmic space of Shewanella oneidensis

A Shewanella expression system has been used for an overproduction of c-type multiheme proteins. The proteins were exported to the periplasmic space for the maturation. Since the periplasmic expression system is attractive, especially for protease-sensitive proteins, an expression vector containing a signal peptide was constructed for expressions in the periplasmic space of Shewanella oneidensis. To evaluate the system, two eukaryotic proteins which originally do not have signal sequences and are difficult to express in Escherichia coli, were selected. The first is human cytochrome c. Properties of the recombinant cytochrome c were identical to those previously reported, indicating the protein is intact. The other was potato calcium-dependent protein kinase. The protein was expressed in periplasmic space. These results indicated that the system is generally applicable for any protein expression including c-type cytochromes, protease-sensitive proteins and those with multi-disulfide bonds because of transportation to the periplasmic space.     

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

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.     

Deciphering the electron transport pathway for graphene oxide reduction by Shewanella oneidensis MR-1

We determined that graphene oxide reduction by Shewanella oneidensis MR-1 requires the Mtr respiratory pathway by analyzing a range of mutants lacking these proteins. Electron shuttling compounds increased the graphene oxide reduction rate 3- to 5-fold. These results may help facilitate the use of bacteria for large-scale graphene production.     

Validating annotations for uncharacterized proteins in Shewanella oneidensis

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.     

Posttranslational Modification of Flagellin FlaB in Shewanella oneidensis

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

Addressing Shewanella oneidensis "cytochromome": the first step towards high-throughput expression of cytochromes c

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.     

Respiration and growth of Shewanella oneidensis MR-1 using vanadate as the sole electron acceptor

Shewanella oneidensis MR-1 is a free-living gram-negative gamma-proteobacterium that is able to use a large number of oxidizing molecules, including fumarate, nitrate, dimethyl sulfoxide, trimethylamine N-oxide, nitrite, and insoluble iron and manganese oxides, to drive anaerobic respiration. Here we show that S. oneidensis MR-1 is able to grow on vanadate as the sole electron acceptor. Oxidant pulse experiments demonstrated that proton translocation across the cytoplasmic membrane occurs during vanadate reduction. Proton translocation is abolished in the presence of protonophores and the inhibitors 2-heptyl-4-hydroxyquinoline N-oxide and antimycin A. Redox difference spectra indicated the involvement of membrane-bound menaquinone and cytochromes c, which was confirmed by transposon mutagenesis and screening for a vanadate reduction-deficient phenotype. Two mutants which are deficient in menaquinone synthesis were isolated. Another mutant with disruption in the cytochrome c maturation gene ccmA was unable to produce any cytochrome c and to grow on vanadate. This phenotype could be restored by complementation with the pEC86 plasmid expressing ccm genes from Escherichia coli. To our knowledge, this is the first report of E. coli ccm genes being functional in another organism. Analysis of an mtrB-deficient mutant confirmed the results of a previous paper indicating that OmcB may function as a vanadate reductase or may be part of a vanadate reductase complex.     

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.