Mechanisms of Bacterial Extracellular Electron Exchange.

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 ~ 6 nm 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 Acidothiobacillus ferrioxydans, 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 extentions and soluble flavin shuttles, all of which require the outer membrane cytochromes MtrC and OmcA in addition to secreted flavin

A combined EPR and MD simulation study of a nitroxyl spin label with restricted internal mobility sensitive to protein dynamics

EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions

Exploring the biochemistry at the extracellular redox frontier of bacterial mineral Fe(III) respiration

Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural features of two of these outer-membrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily

Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners

Many subsurface microorganisms couple their metabolism to the reduction or oxidation of extracellular substrates. For example, anaerobic mineral-respiring bacteria can use external metal oxides as terminal electron acceptors during respiration. Porin–cytochrome complexes facilitate the movement of electrons generated through intracellular catabolic processes across the bacterial outer membrane to these terminal electron acceptors. In the mineral-reducing model bacterium Shewanella oneidensis MR-1, this complex is composed of two decaheme cytochromes (MtrA and MtrC) and an outer-membrane β-barrel (MtrB). However, the structures and mechanisms by which porin–cytochrome complexes transfer electrons are unknown. Here, we used small-angle neutron scattering (SANS) to study the molecular structure of the transmembrane complexes MtrAB and MtrCAB. Ab initio modeling of the scattering data yielded a molecular envelope with dimensions of ∼105 × 60 × 35 Å for MtrAB and ∼170 × 60 × 45 Å for MtrCAB. The shapes of these molecular envelopes suggested that MtrC interacts with the surface of MtrAB, extending ∼70 Å from the membrane surface and allowing the terminal hemes to interact with both MtrAB and an extracellular acceptor. The data also reveal that MtrA fully extends through the length of MtrB, with ∼30 Å being exposed into the periplasm. Proteoliposome models containing membrane-associated MtrCAB and internalized small tetraheme cytochrome (STC) indicate that MtrCAB could reduce Fe(III) citrate with STC as an electron donor, disclosing a direct interaction between MtrCAB and STC. Taken together, both structural and proteoliposome experiments support porin–cytochrome–mediated electron transfer via periplasmic cytochromes such as STC

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO2.

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In $\textit{Shewanella oneidensis}$ MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porin$\cdot$cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1. We show photoreduction of MtrC and OmcA adsorbed on Ru$^{\text{II}}$-dye-sensitized TiO$_2$ nanoparticles and that these protein-coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer-membrane-associated cytochromes of MR-1 for solar-driven microbial synthesis in natural and engineered bacteria.This work was supported by the UK Biotechnology and Biological Sciences Research Council (grants BB/K009753/1, BB/K010220/1, BB/K009885/1, and BB/K00929X/1), the Engineering and Physical Sciences Research Council (EP/M001989/1, PhD studentship 1307196 to E.V.A.), a Royal Society Leverhulme Trust Senior Research Fellowship to J.N.B., the Christian Doppler Research Association, and OMV group

Effects of soluble flavin on heterogeneous electron transfer between surface exposed bacterial cytochromes and iron oxides

Dissimilatory iron-reducing bacteria can utilize insoluble Fe(Mn)-oxides as a terminal electron acceptor under anaerobic conditions. For Shewanella species specifically, multiple evidences suggest that iron reduction is associated with the secretion of flavin mononucleotide (FMN) and riboflavin. However, the exact mechanism of flavin involvement is unclear; while some indicate that flavins mediate electron transfer (Marsili et al., 2008), others point to flavin serving as cofactors to outer membrane proteins (Okamoto et al., 2013). In this work, we used methyl viologen (MV•+)-encapsulated, porin-cytochrome complex (MtrCAB) embedded liposomes (MELs) as a synthetic model of the Shewanella outer membrane to investigate the proposed mediating behavior of microbially produced flavins. The reduction kinetics of goethite, hematite nand lepidocrocite (200 μM) by MELs ([MV•+] ~ 42 μM and MtrABC ≤ 1 nM) were determined in the presence FMN at pH 7.0 in N2 atmosphere by monitoring the concentrations of MV•+ and FMN through their characteristic UV-visible absorption spectra. Experiments were performed where i) FMN and Fe(III)-oxide were mixed and then reacted with the reduced MELs and ii) FMN was reacted with the reduced MELs followed by addition of Fe(III)-oxide. The redox reactions proceeded in two steps: a fast step that was completed in a few seconds, and a slower one lasting over 400 seconds. For all three Fe(III)-oxides, the initial reaction rate in the presence of a low concentration of FMN (≤ 1 μM) was at least a factor of five faster than those with MELs alone, and orders of magnitude faster than those by FMNH2, suggesting that FMN may serve as a co-factor that enhances electron transfer from outer-membrane c-cytochromes to nFe(III)-oxides. The rate and extent of the initial reaction followed the order of lepidocrocite > hematite > goethite, the same as their reduction potentials, implying thermodynamic control on reaction rate. For LEP, with the highest reduction potential among the three Fe(III)-oxides, its reduction by FMNH2 completed in less than 10 minutes, suggesting that FMN is capable of mediating electron transfer to LEP. At higher FMN concentrations (> 1 μM), the reaction rates for both steps decreased and varied inversely with FMN concentration, indicating that FMN inhibited the MEL to Fe(III)-oxide electron transfer reaction under these conditions. The implications of the observed kinetic behaviors to flavin-mediated Fe(III) oxide reduction in natural environments are discussed

Evolution of malaria mortality and morbidity after the emergence of chloroquine resistance in Niakhar, Senegal

Background: Recently, it has been assumed that resistance of Plasmodium to chloroquine increased malaria mortality. The study aimed to assess the impact of chemoresistance on mortality attributable to malaria in a rural area of Senegal, since the emergence of resistance in 1992, whilst chloroquine was used as first-line treatment of malaria, until the change in national anti-malarial policy in 2003. Methods: The retrospective study took place in the demographic surveillance site (DSS) of Niakhar. Data about malaria morbidity were obtained from health records of three health care facilities, where diagnosis of malaria was based on clinical signs. Source of data concerning malaria mortality were verbal autopsies performed by trained fieldworkers and examined by physicians who identified the probable cause of death. Results: From 1992 to 2004, clinical malaria morbidity represented 39% of total morbidity in health centres. Mean malaria mortality was 2.4 parts per thousand and 10.4 parts per thousand among total population and children younger than five years, respectively, and was highest in the 1992-1995 period. It tended to decline from 1992 to 2003 (Trend test, total population p = 0.03, children 0-4 years p = 0.12 - children 1-4 years p = 0.04 - children 5-9 years p = 0.01). Conclusion: Contrary to what has been observed until 1995, mortality attributable to malaria did not continue to increase dramatically in spite of the growing resistance to chloroquine and its use as first-line treatment until 2003. Malaria morbidity and mortality followed parallel trends and rather fluctuated accordingly to rainfall

The effect of dose on the antimalarial efficacy of artemether-lumefantrine: a systematic review and pooled analysis of individual patient data

Background: Artemether-lumefantrine is the most widely used artemisinin-based combination therapy for malaria, although treatment failures occur in some regions. We investigated the effect of dosing strategy on efficacy in a pooled analysis from trials done in a wide range of malaria-endemic settings. Methods: We searched PubMed for clinical trials that enrolled and treated patients with artemether-lumefantrine and were published from 1960 to December, 2012. We merged individual patient data from these trials by use of standardised methods. The primary endpoint was the PCR-adjusted risk of Plasmodium falciparum recrudescence by day 28. Secondary endpoints consisted of the PCR-adjusted risk of P falciparum recurrence by day 42, PCR-unadjusted risk of P falciparum recurrence by day 42, early parasite clearance, and gametocyte carriage. Risk factors for PCR-adjusted recrudescence were identified using Cox's regression model with frailty shared across the study sites. Findings: We included 61 studies done between January, 1998, and December, 2012, and included 14 327 patients in our analyses. The PCR-adjusted therapeutic efficacy was 97·6% (95% CI 97·4-97·9) at day 28 and 96·0% (95·6-96·5) at day 42. After controlling for age and parasitaemia, patients prescribed a higher dose of artemether had a lower risk of having parasitaemia on day 1 (adjusted odds ratio [OR] 0·92, 95% CI 0·86-0·99 for every 1 mg/kg increase in daily artemether dose; p=0·024), but not on day 2 (p=0·69) or day 3 (0·087). In Asia, children weighing 10-15 kg who received a total lumefantrine dose less than 60 mg/kg had the lowest PCR-adjusted efficacy (91·7%, 95% CI 86·5-96·9). In Africa, the risk of treatment failure was greatest in malnourished children aged 1-3 years (PCR-adjusted efficacy 94·3%, 95% CI 92·3-96·3). A higher artemether dose was associated with a lower gametocyte presence within 14 days of treatment (adjusted OR 0·92, 95% CI 0·85-0·99; p=0·037 for every 1 mg/kg increase in total artemether dose). Interpretation: The recommended dose of artemether-lumefantrine provides reliable efficacy in most patients with uncomplicated malaria. However, therapeutic efficacy was lowest in young children from Asia and young underweight children from Africa; a higher dose regimen should be assessed in these groups. Funding: Bill and Melinda Gates Foundation