Matthew J. Marshall

Characterization of an electron conduit between bacteria and the extracellular environment

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning β-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.

c-Type cytochrome-dependent formation of U(IV) nanoparticles by Shewanella oneidensis.

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 UO 2 nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), 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 UO 2 nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO 2-EPS matrix exhibited glycocalyx-like properties and 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 UO 2 nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO 2 nanoparticles. In the environment, such association of UO 2 nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O 2 or transport in soils and sediments.

Structure of a bacterial cell surface decaheme electron conduit

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along “nanowire” appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

Extracellular Polymeric Substances from Shewanella sp. HRCR-1 Biofilms: Characterization by Infrared Spectroscopy and Proteomics

The composition of extracellular polymeric substances (EPS) from Shewanella sp. HRCR-1 biofilms was investigated using infrared spectroscopy and proteomics to provide insight into potential ecophysiological functions and redox activity of the EPS. Both bound and loosely associated EPS were extracted from Shewanella sp. HRCR-1 biofilms prepared using a hollow-fibre membrane biofilm reactor. Fourier transform infrared spectra revealed the presence of proteins, polysaccharides, nucleic acids, membrane lipids and fatty acids in the EPS fractions. Using a global proteomic approach, a total of 58 extracellular and outer membrane proteins were identified in the EPS. These included homologues of multiple Shewanella 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.

Imaging Hydrated Microbial Extracellular Polymers: Comparative Analysis by Electron Microscopy

ABSTRACT Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigation of microscale associations. Electron microscopy has been used extensively for geomicrobial investigations, and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions by conventional electron microscopy approaches with imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding the nature of interactions between microbial extracellular polymers and their environment.

Direct Involvement of Type II Secretion System in Extracellular Translocation of Shewanella oneidensis Outer Membrane Cytochromes MtrC and OmcA

MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. Deletions of type II secretion system (T2SS) genes reduced their extracellular release and their accessibility to the proteinase K treatment, demonstrating the direct involvement of T2SS in translocation of MtrC and OmcA to the bacterial cell surface.

Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals

The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decaheme cytochromes, MtrC and MtrA, brought together inside a transmembrane porin, MtrB, to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system containing a pool of internalized electron carriers was used to investigate how the topology of the MtrCAB complex relates to its ability to transport electrons across a lipid bilayer to externally located Fe(III) oxides. With MtrA facing the interior and MtrC exposed on the outer surface of the phospholipid bilayer, the established in vivo orientation, electron transfer from the interior electron carrier pool through MtrCAB to solid-phase Fe(III) oxides was demonstrated. The rates were 103 times higher than those reported for reduction of goethite, hematite, and lepidocrocite by S. oneidensis, and the order of the reaction rates was consistent with those observed in S. oneidensis cultures. In contrast, established rates for single turnover reactions between purified MtrC and Fe(III) oxides were 103 times lower. By providing a continuous flow of electrons, the proteoliposome experiments demonstrate that conduction through MtrCAB directly to Fe(III) oxides is sufficient to support in vivo, anaerobic, solid-phase iron respiration.

Smart Align—a new tool for robust non-rigid registration of scanning microscope data

Many microscopic investigations of materials may benefit from the recording of multiple successive images. This can include techniques common to several types of microscopy such as frame averaging to improve signal-to-noise ratios (SNR) or time series to study dynamic processes or more specific applications. In the scanning transmission electron microscope, this might include focal series for optical sectioning or aberration measurement, beam damage studies or camera-length series to study the effects of strain; whilst in the scanning tunnelling microscope, this might include bias-voltage series to probe local electronic structure. Whatever the application, such investigations must begin with the careful alignment of these data stacks, an operation that is not always trivial. In addition, the presence of low-frequency scanning distortions can introduce intra-image shifts to the data. Here, we describe an improved automated method of performing non-rigid registration customised for the challenges unique to scanned microscope data specifically addressing the issues of low-SNR data, images containing a large proportion of crystalline material and/or local features of interest such as dislocations or edges. Careful attention has been paid to artefact testing of the non-rigid registration method used, and the importance of this registration for the quantitative interpretation of feature intensities and positions is evaluated.

Contribution of extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms to U(VI) immobilization.

The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) to U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells with minimal EPS, we show that (i) bEPS from Shewanella sp. HRCR-1 biofilms contribute significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; (ii) bEPS can be considered a functional extension of the cells for U(VI) immobilization and they likely play more important roles at lower initial U(VI) concentrations; and (iii) the U(VI) reduction efficiency is dependent upon the initial U(VI) concentration and decreases at lower concentrations. To quantify the relative contributions of sorption and reduction to U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(VI). We found that, when reduced, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated the reactivity of laEPS, while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, possibly facilitated U(VI) reduction.

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

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.