Project description:Geobacter sulfurreducens is a dissimilatory metal-reducing bacterium capable of forming thick electron-conducting biofilms on solid electrodes in the absence of alternative electron acceptors. The remarkable ability of such biofilms to transfer electrons, liberated from soluble organic electron donors, over long distances has attracted scientific interest as to the mechanism for this process, and technological interest for application to microbial fuel and electrolysis cells and sensors. Here, we employ comparative proteomics to identify key metabolic pathways involved in G. sulfurreducens respiration by planktonic cells versus electron-conducting biofilms, in an effort to elucidate long-range electron transfer mechanisms.
Project description:The conductive pili of Geobacter sulfurreducens are essential for optimal extracellular electron transfer to Fe(III) and long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene for PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, slow rates of Fe(III) reduction were detected after extended (> 30 days) incubation in the presence of Fe(III) oxide. After seven consecutive transfers the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, proteomic, and gene deletion studies indicated that this adaptation was associated with greater production of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every three days, the wild-type strain out-competed the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron-shuttling producing Fe(III) reducers in most soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current; consistent with the concept that long-range electron transport through G. sulfurreducens biofilms cannot be achieved without PilA-pili.
Project description:There is a wide diversity of potential applications for direct electron transfer from electrodes to microorganisms, which might be better optimized if the mechanisms for this novel electrode-biofilm interaction were further understood. Geobacter sulfurreducens is one of the few microorganisms available in pure culture that is known to be capable of directly accepting electrons from a negatively poised electrode. Gene transcript abundance in cells of G. sulfurreducens using electrons delivered from a graphite electrode as the sole electron donor for fumarate reduction was compared with transcript abundance in cells growing on the same graphite material, but without an electrical connection and acetate as the electron donor.
Project description:The conductive pili of Geobacter sulfurreducens are essential for optimal extracellular electron transfer to Fe(III) and long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene for PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, slow rates of Fe(III) reduction were detected after extended (> 30 days) incubation in the presence of Fe(III) oxide. After seven consecutive transfers the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, proteomic, and gene deletion studies indicated that this adaptation was associated with greater production of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every three days, the wild-type strain out-competed the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron-shuttling producing Fe(III) reducers in most soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current; consistent with the concept that long-range electron transport through G. sulfurreducens biofilms cannot be achieved without PilA-pili. An eight-chip study using total RNA recovered from four separate cultures of Geobacter sulfurreducens JS-1 (experimental condition) or Geobacter sulfurreducens KN400 (control condition) grown with acetate (10mM)-Fe(III) oxide (100 mmol l-1) exponential growth. Each chip measures the expression level of 3,328 genes from Geobacter sulfurreducens KN400 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Coculture Geobacter sulfurreducens and Enterococcus faecium were grown in open circuit (control) and closed circuit (current collecting) to observe the difference in extracellular electron transfer (EET) mechanisms. mRNA was extracted, amplified and used on Combimatrix custom arrays to obtain an expression profile. Analysis was performed using R.
Project description:Geobacter sulfurreducens has a complex metabolism that adapts to use electron acceptors at a wide range of redox potentials. In this study, we used RNA-seq to identify genes associated with electron transfer pathways at different redox potentials. By correlating the RNA-seq data with cyclic voltammetry, we associated several multiheme cytochromes with specific electron transfer pathways.
Project description:The formation of electroactive biofilms is a crucial process for the generation of bioelectricity and bioremediation. G. sulfurreducens is a dissimilatory metal-reducing microorganism that can couple oxidation of organic matter with extracellular electron transfer to different insoluble electron acceptors. It has the capability to form biofilms in insoluble metal oxides and electroconductive biofilms in electrodes in bioelectrochemical systems. The formation of electroactive biofilms in this microorganism is a process that has been studied from a physiological, genetic, physical, and electrochemical approach. In G. sulfurreducens, we found that the transcriptional regulator GSU1771 participates in the gene expression of essential genes involved in electron transfer and biofilm formation. Strains deficient in GSU1771 increases Fe(III) reduction, produces more c-type cytochromes and exopolysaccharides. Furthermore, the biofilms produced are thicker and more electroactive than wild-type. In this work, we investigate the global gene expression profile performing RNA-seq comparing Δgsu1771 mutant biofilm grown in non-conductive support (glass) and respiring-graphite electrode. RNA-seq analysis of Δgsu1771 biofilm grown in glass support revealed a total of 467 (167 upregulated and 300 downregulated) differentially expressed genes versus the wild-type biofilm. Meanwhile, in Δgsu1771 biofilm developed in respiring-electrode graphite, we detect 119 (79 upregulated and 40 downregulated) differentially expressed genes with respect to wild-type biofilm. Moreover, transcriptional changes of 67 (56 with the same regulation and in 11 counterregulation) genes were shared in Δgsu1771 biofilm developed in glass and graphite electrodes. We locate upregulated in Δgsu1771 biofilms potential target genes, involved in exopolysaccharide synthesis (gsu1961-63, gsu1959, gsu1972-73, gsu1976-77). We confirmed the upregulation of gsu1979, gsu0972, gsu0783, pgcA, omcM, aroG, panC gnfK, gsu2507, and the downregulation of asnA, ato-1, gsu0810, pilA, csrA, ppcD, and gsu3356 genes by RT-qPCR. DNA-protein binding assay shows direct binding of the GSU1771 regulator to the promoter region of pgcA, pulF, relA, and gsu3356. Also, heme-staining and western blotting revealed an increase of c-type cytochromes in Δgsu1771 biofilms such as OmcS and OmcZ. In general, our data shows that GSU1771 is a global regulator involved in controlling the extracellular electron transfer and exopolysaccharide synthesis, processes required for electroconductive biofilm development.
Project description:Mahadevan2006 - Genome-scale metabolic
network of Geobacter sulfurreducens (iRM588)
This model is described in the article:
Characterization of
metabolism in the Fe(III)-reducing organism Geobacter
sulfurreducens by constraint-based modeling.
Mahadevan R, Bond DR, Butler JE,
Esteve-Nuñez A, Coppi MV, Palsson BO, Schilling CH, Lovley
DR.
Appl. Environ. Microbiol. 2006 Feb;
72(2): 1558-1568
Abstract:
Geobacter sulfurreducens is a well-studied representative of
the Geobacteraceae, which play a critical role in organic
matter oxidation coupled to Fe(III) reduction, bioremediation
of groundwater contaminated with organics or metals, and
electricity production from waste organic matter. In order to
investigate G. sulfurreducens central metabolism and electron
transport, a metabolic model which integrated genome-based
predictions with available genetic and physiological data was
developed via the constraint-based modeling approach.
Evaluation of the rates of proton production and consumption in
the extracellular and cytoplasmic compartments revealed that
energy conservation with extracellular electron acceptors, such
as Fe(III), was limited relative to that associated with
intracellular acceptors. This limitation was attributed to lack
of cytoplasmic proton consumption during reduction of
extracellular electron acceptors. Model-based analysis of the
metabolic cost of producing an extracellular electron shuttle
to promote electron transfer to insoluble Fe(III) oxides
demonstrated why Geobacter species, which do not produce
shuttles, have an energetic advantage over shuttle-producing
Fe(III) reducers in subsurface environments. In silico analysis
also revealed that the metabolic network of G. sulfurreducens
could synthesize amino acids more efficiently than that of
Escherichia coli due to the presence of a pyruvate-ferredoxin
oxidoreductase, which catalyzes synthesis of pyruvate from
acetate and carbon dioxide in a single step. In silico
phenotypic analysis of deletion mutants demonstrated the
capability of the model to explore the flexibility of G.
sulfurreducens central metabolism and correctly predict mutant
phenotypes. These results demonstrate that iterative modeling
coupled with experimentation can accelerate the understanding
of the physiology of poorly studied but environmentally
relevant organisms and may help optimize their practical
applications.
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