What Genetics Offers Geobiology

For over 50 years, the Parker Brothers’ board game “Clue” has maintained its position as the classic family detective game. A murder has been committed in the mansion, but we don’t know where, by whom, or how. Was it Professor Plum in the study with a knife, or Miss Scarlett in the ballroom with a candlestick? Through rolls of the dice, fragments of information patiently accumulated piece-by-piece, and the application of logic, players construct a case to figure out “whodunit”. Because there are several potential solutions to the problem, the key challenge is to figure out what happened by understanding how it happened

Stable sub-complexes observed in situ suggest a modular assembly pathway of the bacterial flagellar motor

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio- temporal synchronization of gene expression, protein localization and association of a dozen or more unique components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with each subsequent component stabilizing the last. Here, using electron cryo-tomography of intact Legionella pneumophila, Pseudomonas aeruginosa and Shewanella oneidensis cells, we observe stable outer-membrane-embedded sub-complexes of the flagellar motor. These sub- complexes consist of the periplasmic embellished P- and L-rings, in the absence of other flagellar components, and bend the membrane inward dramatically. Additionally, we also observe independent inner-membrane sub- complexes consisting of the C- and MS-rings and export apparatus. These results suggest an alternate model for flagellar motor assembly in which outer- and inner-membrane-associated sub-complexes form independently and subsequently join, enabling later steps of flagellar production to proceed

The ‘porin-cytochrome’ model for microbe-to-mineral electron transfer

Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via ‘porin–cytochrome’ electron transport modules. The molecular structure of an outer-membrane extracellular-facing deca-haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer-membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as ‘nanowires’, or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram-negative bacteria

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in Shewanella oneidensis MR-1

BackgroundEtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood.ResultsThe expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down-regulated at least 2-fold in the mutant, which, showed lower or no reduction of these electron acceptors when compared to the wild type, suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down-regulation of genes implicated in aerobic metabolism.ConclusionIn contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and, in conjunction with other regulators, fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1. Transcriptomic and sequence analyses of the genes differentially expressed showed that those mostly affected by the mutation belonged to the "Energy metabolism" category, while stress-related genes were indirectly regulated in the mutant possibly as a result of a secondary perturbation (e.g. oxidative stress, starvation). We also conclude based on sequence, physiological and expression analyses that this regulator is more appropriately termed Fnr and recommend this descriptor be used in future publications

Mind the gap: diversity and reactivity relationships among multihaem cytochromes of the MtrA/DmsE family

Shewanella oneidensis MR-1 has the ability to use many external terminal electron acceptors during anaerobic respiration, such as DMSO. The pathway that facilitates this electron transfer includes the decahaem cytochrome DmsE, a paralogue of the MtrA family of decahaem cytochromes. Although both DmsE and MtrA are decahaem cytochromes implicated in the long-range electron transfer across a ~300 Å (1 Å=0.1 nm) wide periplasmic ‘gap’, MtrA has been shown to be only 105 Å in maximal length. In the present paper, DmsE is further characterized via protein film voltammetry, revealing that the electrochemistry of the DmsE haem cofactors display macroscopic potentials lower than those of MtrA by 100 mV. It is possible this tuning of the redox potential of DmsE is required to shuttle electrons to the outer-membrane proteins specific to DMSO reduction. Other decahaem cytochromes found in S. oneidensis, such as the outer-membrane proteins MtrC, MtrF and OmcA, have been shown to have electrochemical properties similar to those of MtrA, yet possess a different evolutionary relationship.National Science Foundation (U.S.) (Grant MCB 0546323)National Science Foundation (U.S.) (Grant CHE 0840418)Research Corporation for Science Advancement (Scialog Award)National Institutes of Health (U.S.) (Grant F32GM904862

Reconstruction of Extracellular Respiratory Pathways for Iron(III) Reduction in Shewanella Oneidensis Strain MR-1

Shewanella oneidensis strain MR-1 is a facultative anaerobic bacterium capable of respiring a multitude of electron acceptors, many of which require the Mtr respiratory pathway. The core Mtr respiratory pathway includes a periplasmic c-type cytochrome (MtrA), an integral outer-membrane β-barrel protein (MtrB), and an outer-membrane-anchored c-type cytochrome (MtrC). Together, these components facilitate transfer of electrons from the c-type cytochrome CymA in the cytoplasmic membrane to electron acceptors at and beyond the outer-membrane. The genes encoding these core proteins have paralogs in the S. oneidensis genome (mtrB and mtrA each have four while mtrC has three) and some of the paralogs of mtrC and mtrA are able to form functional Mtr complexes. We demonstrate that of the additional three mtrB paralogs found in the S. oneidensis genome, only MtrE can replace MtrB to form a functional respiratory pathway to soluble iron(III) citrate. We also evaluate which mtrC/mtrA paralog pairs (a total of 12 combinations) are able to form functional complexes with endogenous levels of mtrB paralog expression. Finally, we reconstruct all possible functional Mtr complexes and test them in a S. oneidensis mutant strain where all paralogs have been eliminated from the genome. We find that each combination tested with the exception of MtrA/MtrE/OmcA is able to reduce iron(III) citrate at a level significantly above background. The results presented here have implications toward the evolution of anaerobic extracellular respiration in Shewanella and for future studies looking to increase the rates of substrate reduction for water treatment, bioremediation, or electricity production

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 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⋅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 RuII -dye-sensitized TiO2 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

Tensor transformation technique for the transport equation

A step-wise tensor transformation technique is presented for the transformation of the single energy group transport equation to an arbitrary spatial coordinate system. Both gradient and divergence forms of the equation are given and the same method is applied to the derivation of the diffusion approximation. It is demonstrated that using an orthogonal representation of the propagation vector will simplify the divergence form of the equation. The application of this technique is in the representation of the transport equation in coordinate systems other than the usual rectangular, cylindrical and spherical ones. Its use is demonstrated by transforming the transport equation to a toroidal coordinate system consisting of nested circular toroids. (auth