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

Lysine-91 of the tetraheme c-type cytochrome CymA is essential for quinone interaction and arsenate respiration in Shewanella sp. strain ANA-3

The tetraheme c-type cytochrome, CymA, is essential for arsenate respiratory reduction in Shewanella sp. ANA-3, a model arsenate reducer. CymA is predicted to mediate electron transfer from quinols to the arsenate respiratory reductase (ArrAB). Here, we present biochemical and physiological evidence that CymA interacts with menaquinol (MQH2) substrates. Fluorescence quench titration with the MQH2 analog, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), was used to demonstrate quinol binding of E. coli cytoplasmic membranes enriched with various forms of CymA. Wild-type CymA bound HOQNO with a Kd of 0.1–1 μM. It was also shown that the redox active MQH2 analog, 2,3-dimethoxy-1,4-naphthoquinone (DMNH2), could reduce CymA in cytoplasmic membrane preparations. Based on a CymA homology model made from the NrfH tetraheme cytochrome structure, it was predicted that Lys91 would be involved in CymA-quinol interactions. CymA with a K91Q substitution showed little interaction with HOQNO. In addition, DMNH2-dependent reduction of CymA-K91Q was diminished by 45% compared to wild-type CymA. A ΔcymA ANA-3 strain containing a plasmid copy of cymA-K91Q failed to grow with arsenate as an electron acceptor. These results suggest that Lys91 is physiologically important for arsenate respiration and support the hypothesis that CymA interacts with menaquinol resulting in the reduction of the cytochrome

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

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

Towards Electrosynthesis in Shewanella: Energetics of Reversing the Mtr Pathway for Reductive Metabolism

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

Long-term population dynamics of dreissenid mussels (Dreissena polymorpha and D. rostriformis): a cross-system analysis

Dreissenid mussels (including the zebra mussel Dreissena polymorpha and the quagga mussel D. rostriformis) are among the world's most notorious invasive species, with large and widespread ecological and economic effects. However, their long-term population dynamics are poorly known, even though these dynamics are critical to determining impacts and effective management. We gathered and analyzed 67 long-term (>10 yr) data sets on dreissenid populations from lakes and rivers across Europe and North America. We addressed five questions: (1) How do Dreissena populations change through time? (2) Specifically, do Dreissena populations decline substantially after an initial outbreak phase? (3) Do different measures of population performance (biomass or density of settled animals, veliger density, recruitment of young) follow the same patterns through time? (4) How do the numbers or biomass of zebra mussels or of both species combined change after the quagga mussel arrives? (5) How does body size change over time? We also considered whether current data on long-term dynamics of Dreissena populations are adequate for science and management. Individual Dreissena populations showed a wide range of temporal dynamics, but we could detect only two general patterns that applied across many populations: (1) Populations of both species increased rapidly in the first 1–2 yr after appearance, and (2) quagga mussels appeared later than zebra mussels and usually quickly caused large declines in zebra mussel populations. We found little evidence that combined Dreissena populations declined over the long term. Different measures of population performance were not congruent; the temporal dynamics of one life stage or population attribute cannot generally be accurately inferred from the dynamics of another. We found no consistent patterns in the long-term dynamics of body size. The long-term dynamics of Dreissena populations probably are driven by the ecological characteristics (e.g., predation, nutrient inputs, water temperature) and their temporal changes at individual sites rather than following a generalized time course that applies across many sites. Existing long-term data sets on dreissenid populations, although clearly valuable, are inadequate to meet research and management needs. Data sets could be improved by standardizing sampling designs and methods, routinely collecting more variables, and increasing support.Deutsche Forschungsgemeinschaft Cary Institute of Ecosystem Studies (DLS) NSF‐LTREB. Grant Number: DEB‐1556246 NSF‐OPUS. Grant Numbers: DEB‐1456532, JE 288/9‐1, JE 288/9‐2, TÁMOP‐4.2.2.A‐11/1/KONV‐2012‐0038, GINOP‐2.3.2‐15‐2016‐00019, LG‐T; USGS G14AC000263, US EPA GL00E01184 Cornell Agricultural Experiment Station. Grant Number: NYC‐0226747 New York State Department of Environmental Conservation. Grant Number: 1517823 Belarusian Republican Foundation for Fundamental Researc