A Bioelectrochemical Approach to Characterize Extracellular Electron Transfer by Synechocystis sp. PCC6803

Biophotovoltaic devices employ photosynthetic organisms at the anode of a microbial fuel cell to generate electrical power. Although a range of cyanobacteria and algae have been shown to generate photocurrent in devices of a multitude of architectures, mechanistic understanding of extracellular electron transfer by phototrophs remains minimal. Here we describe a mediatorless bioelectrochemical device to measure the electrogenic output of a planktonically grown cyanobacterium, Synechocystis sp. PCC6803. Light dependent production of current is measured, and its magnitude is shown to scale with microbial cell concentration and light intensity. Bioelectrochemical characterization of a Synechocystis mutant lacking Photosystem II demonstrates conclusively that production of the majority of photocurrent requires a functional water splitting aparatus and electrons are likely ultimately derived from water. This shows the potential of the device to rapidly and quantitatively characterize photocurrent production by genetically modified strains, an approach that can be used in future studies to delineate the mechanisms of cyanobacterial extracellular electron transport

Genome-scale constraint-based modeling of Geobacter metallireducens

Background: Geobacter metallireducens was the first organism that can be grown in pure culture to completely oxidize organic compounds with Fe(III) oxide serving as electron acceptor. Geobacter species, including G. sulfurreducens and G. metallireducens, are used for bioremediation and electricity generation from waste organic matter and renewable biomass. The constraint-based modeling approach enables the development of genome-scale in silico models that can predict the behavior of complex biological systems and their responses to the environments. Such a modeling approach was applied to provide physiological and ecological insights on the metabolism of G. metallireducens. Results: The genome-scale metabolic model of G. metallireducens was constructed to include 747 genes and 697 reactions. Compared to the G. sulfurreducens model, the G. metallireducens metabolic model contains 118 unique reactions that reflect many of G. metallireducens\u27 specific metabolic capabilities. Detailed examination of the G. metallireducens model suggests that its central metabolism contains several energy-inefficient reactions that are not present in the G. sulfurreducens model. Experimental biomass yield of G. metallireducens growing on pyruvate was lower than the predicted optimal biomass yield. Microarray data of G. metallireducens growing with benzoate and acetate indicated that genes encoding these energy-inefficient reactions were up-regulated by benzoate. These results suggested that the energy-inefficient reactions were likely turned off during G. metallireducens growth with acetate for optimal biomass yield, but were up-regulated during growth with complex electron donors such as benzoate for rapid energy generation. Furthermore, several computational modeling approaches were applied to accelerate G. metallireducens research. For example, growth of G. metallireducens with different electron donors and electron acceptors were studied using the genome-scale metabolic model, which provided a fast and cost-effective way to understand the metabolism of G. metallireducens. Conclusion: We have developed a genome-scale metabolic model for G. metallireducens that features both metabolic similarities and differences to the published model for its close relative, G. sulfurreducens. Together these metabolic models provide an important resource for improving strategies on bioremediation and bioenergy generation

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

Secondary mineral formation associated with respiration of nontronite, NAu-1 by iron reducing bacteria

Experimental batch and miscible-flow cultures were studied in order to determine the mechanistic pathways of microbial Fe(III) respiration in ferruginous smectite clay, NAu-1. The primary purpose was to resolve if alteration of smectite and release of Fe precedes microbial respiration. Alteration of NAu-1, represented by the morphological and mineralogical changes, occurred regardless of the extent of microbial Fe(III) reduction in all of our experimental systems, including those that contained heat-killed bacteria and those in which O(2), rather than Fe(III), was the primary terminal electron acceptor. The solid alteration products observed under transmission electron microscopy included poorly crystalline smectite with diffuse electron diffraction signals, discrete grains of Fe-free amorphous aluminosilicate with increased Al/Si ratio, Fe-rich grains, and amorphous Si globules in the immediate vicinity of bacterial cells and extracellular polymeric substances. In reducing systems, Fe was also found as siderite. The small amount of Fe partitioned to the aqueous phase was primarily in the form of dissolved Fe(III) species even in the systems in which Fe(III) was the primary terminal electron acceptor for microbial respiration. From these observations, we conclude that microbial respiration of Fe(III) in our laboratory systems proceeded through the following: (1) alteration of NAu-1 and concurrent release of Fe(III) from the octahedral sheets of NAu-1; and (2) subsequent microbial respiration of Fe(III)

Electron shuttle-mediated microbial Fe(III) reduction under alkaline conditions

Purpose: Extracellular Fe(III) reduction plays an important role in a variety of biogeochemical processes. Several mechanisms for microbial Fe(III) reduction in pH-neutral environments have been proposed, but pathways of microbial Fe(III) reduction within alkaline conditions have not been clearly identified. Alkaline soils are vastly distributed; thus, a better understanding of microbial Fe(III) reduction under alkaline conditions is of significance. The purpose of this study is to explore the dominant mechanism of bacterial iron reduction in alkaline environments. Materials and methods: We used antraquinone-2,6-disulfonate (AQDS) as a representative of quinone moities of humic substances and elemental sulfur and sulfate as sulfur species to investigate the potential role of humic substances and sulfur species in mediating microbial Fe(III) reduction in alkaline environments. We carried out thermodynamic calculations to predict the ability of bacteria to reduce Fe(III) (oxyhydr)oxides under alkaline conditions and the ability of AQDS and sulfur species to serve as electron acceptors for microbial anaerobic respiration in an assumed alkaline soil environments. A series of incubation experiments with two model dissimilatory metal reducing bacteria, Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA as well as mixed bacteria enriched from a soil were performed to confirm the contribution of AQDS and sulfur species to Fe(III) reduction under alkaline conditions. Results and discussion: Based on thermodynamic calculations, we predicted that, under alkaline conditions, the enzymatic reduction of Fe(III) (oxyhydr)oxides would be thermodynamically feasible but very weak. In our incubation experiments, the reduction of ferrihydrite by anaerobic cultures of Shewanella oneidensis MR-1, Geobacter sulfurreducens PCA or microbes enriched from a soil was significantly increased in the presence of S0 or AQDS. Notably, AQDS contributed more to promoting Fe(III) reduction as a soluble electron shuttle than S0 did under the alkaline conditions probably because of different mechanisms of microbial utilization of AQDS and S0. Conclusions: These results suggest that microbial reduction of Fe(III) (oxyhydr)oxides under alkaline conditions may proceed via a pathway mediated by electron shuttles such as AQDS and S0. Considering the high ability of electron shuttling and vast distribution of humic substances, we suggest that humic substance-mediated Fe(III) reduction may potentially be the dominant mechanism for Fe(III) reduction in alkaline environments

Influence of Uranium on Bacterial Communities: A Comparison of Natural Uranium-Rich Soils with Controls

This study investigated the influence of uranium on the indigenous bacterial community structure in natural soils with high uranium content. Radioactive soil samples exhibiting 0.26% - 25.5% U in mass were analyzed and compared with nearby control soils containing trace uranium. EXAFS and XRD analyses of soils revealed the presence of U(VI) and uranium-phosphate mineral phases, identified as sabugalite and meta-autunite. A comparative analysis of bacterial community fingerprints using denaturing gradient gel electrophoresis (DGGE) revealed the presence of a complex population in both control and uranium-rich samples. However, bacterial communities inhabiting uraniferous soils exhibited specific fingerprints that were remarkably stable over time, in contrast to populations from nearby control samples. Representatives of Acidobacteria, Proteobacteria, and seven others phyla were detected in DGGE bands specific to uraniferous samples. In particular, sequences related to iron-reducing bacteria such as Geobacter and Geothrix were identified concomitantly with iron-oxidizing species such as Gallionella and Sideroxydans. All together, our results demonstrate that uranium exerts a permanent high pressure on soil bacterial communities and suggest the existence of a uranium redox cycle mediated by bacteria in the soil

MIP/Aquaporin 0 Represents a Direct Transcriptional Target of PITX3 in the Developing Lens

The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations

Microbial sulfate reduction and metal attenuation in pH 4 acid mine water

Sediments recovered from the flooded mine workings of the Penn Mine, a Cu-Zn mine abandoned since the early 1960s, were cultured for anaerobic bacteria over a range of pH (4.0 to 7.5). The molecular biology of sediments and cultures was studied to determine whether sulfate-reducing bacteria (SRB) were active in moderately acidic conditions present in the underground mine workings. Here we document multiple, independent analyses and show evidence that sulfate reduction and associated metal attenuation are occurring in the pH-4 mine environment. Water-chemistry analyses of the mine water reveal: (1) preferential complexation and precipitation by H2S of Cu and Cd, relative to Zn; (2) stable isotope ratios of 34S/32S and 18O/16O in dissolved SO4 that are 2–3 ‰ heavier in the mine water, relative to those in surface waters; (3) reduction/oxidation conditions and dissolved gas concentrations consistent with conditions to support anaerobic processes such as sulfate reduction. Scanning electron microscope (SEM) analyses of sediment show 1.5-micrometer, spherical ZnS precipitates. Phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analyses of Penn Mine sediment show a high biomass level with a moderately diverse community structure composed primarily of iron- and sulfate-reducing bacteria. Cultures of sediment from the mine produced dissolved sulfide at pH values near 7 and near 4, forming precipitates of either iron sulfide or elemental sulfur. DGGE coupled with sequence and phylogenetic analysis of 16S rDNA gene segments showed populations of Desulfosporosinus and Desulfitobacterium in Penn Mine sediment and laboratory cultures