Project description:Previous studies demonstrated that an outer membrane c-type cytochrome, OmcB (GSU_2737), was involved in Fe(III) reduction in Geobacter sulfurreducens. An OmcB-deficient mutant was greatly impaired in its ability to reduce both soluble and insoluble Fe(III). Reintroducing omcB restored the capacity for Fe(III) reduction at a level proportional to the level of OmcB production. Here, we report that the OmcB-deficient mutant gradually adapted to grow on soluble Fe(III) but not insoluble Fe(III). The adapted OmcB-deficient mutant reduced soluble Fe(III) at a rate comparable to that of the wild type, but the cell yield of the mutant was only ca. 60% of that of the wild type under steady-state culturing conditions. Analysis of proteins and transcript levels demonstrated that expression of several membrane-associated cytochromes was higher in the adapted mutant than in the wild type. Further comparison of transcript levels during steady-state growth on Fe(III) citrate with a whole-genome DNA microarray revealed a significant shift in gene expression in an apparent attempt to adapt metabolism to the impaired electron transport to Fe(III). These results demonstrate that, although there are many other membrane-bound c-type cytochromes in G. sulfurreducens, increased expression of these cytochromes cannot completely compensate for the loss of OmcB. The concept that outer membrane cytochromes are promiscuous reductases that are interchangeable in function appears to be incorrect. Furthermore, the results indicate that there may be different mechanisms for electron transfer to soluble Fe(III) and insoluble Fe(III) oxides in G. sulfurreducens, which emphasizes the importance of studying electron transport to the environmentally relevant Fe(III) oxides. (From Leang C, Adams LA, Chin KJ, Nevin KP, Methé BA, Webster J, Sharma ML, Lovley DR. Adaptation to disruption of the electron transfer pathway for Fe(III) reduction in Geobacter sulfurreducens. J Bacteriol. 2005 Sep;187(17):5918-26.) Keywords: Geobacter, gene expression, genetic modification
Project description:The diazotroph Trichodesmium is an important contributor to marine dinitrogen (N2) fixation, supplying so-called new N to phytoplankton in typically N-limited ocean regions. Identifying how iron (Fe) and phosphorus (P) influence Trichodesmium activity and biogeography is an ongoing area of study, where predicting patterns of resource stress is complicated in part by the uncertain bioavailability of organically complexed Fe and P. Here, a comparison of 26 metaproteomes from picked Trichodesmium colonies identified significantly different patterns between three ocean regions: the western tropical South Pacific, the western North Atlantic, and the North Pacific Subtropical Gyre. Trichodesmium metaproteomes across these regions significantly differed in KEGG submodule signals, and vector fitting showed that dissolved Fe, phosphate, and temperature significantly correlated with regional proteome patterns. Populations in the western tropical South Pacific appeared to modulate their proteomes in response to both Fe and P stress, including a comparatively low relative abundance of the N2 fixation marker protein, NifH. Significant increases in the relative abundance of both Fe and P stress marker proteins previously validated in culture studies suggested that Trichodesmium populations in the western North Atlantic and North Pacific were P-stressed and Fe-stressed, respectively. These patterns recapitulate established regional serial and co-limitation patterns of resource stress on phytoplankton communities. Evaluating community stress patterns may therefore predict resource controls on diazotroph biogeography. These data highlight how Trichodesmium modulates its metabolism in the field and provide an opportunity to more accurately constrain controls on Trichodesmium biogeography and N2 fixation.
Project description:Four-day-old seedlings germinated in wet paper were transferred to solution culture with 0.1 mM Fe(III)-EDTA for five days. Then seedlings were transferred to solution culture without Fe or with 0.1 mM Fe(III)-EDTA for five days. Roots and shoots were separated and used for RNA extraction.
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:Fe(II) can be oxidized by mixotrophic nitrate-reducing Fe(II)-oxidizing bacteria to Fe(III), leading to mineral precipitation. However, the effects of this process on the metabolism and reproduction ability of the Fe(II)-oxidizing bacterial cells have not yet been quantified. In this study, we investigated the effects of Fe(II) on the mixotrophic nitrate-reducing Fe(II)-oxidizing bacteria Acidovorax sp. BoFeN1 by performing cell plate counts, chemical analyses, and scanning electron microscopy (SEM) imaging. The result showed that nitrate-reducing Fe(II)-oxidizing bacteria Acidovorax sp. BoFeN1 undergoes a striking physiological state in which respiration persists while cell reproduction collapses after their oxidation of Fe(II). Across 0.1–10 mM Fe(II), colony-forming units dropped by up to 103-fold, yet acetate consumption remained largely unaffected and nitrate reduction was even slightly promoted. SEM-EDS reveals extensive Fe(III) encrustation on cells. Adding an Fe(III) chelator, citrate, restored reproductive capacity without notably changes in Fe(II) oxidation or nitrate reduction, indicating that it was the products of Fe(II) oxidation, the secondary Fe(III) minerals, that impaired cell reproduction. These findings uncover a mineral encrustation-driven uncoupling between respiration and reproduction, suggest how natural organic ligands may buffer NRFeOx bacterial communities in Fe-rich anoxic environments, and point to cellular-level levers for mitigating denitrification failures and N2O risks in engineered and natural systems.
Project description:Geobacter sulfurreducens PCA was put under selective pressure for rapid Fe(III) oxide reduction. The resultant strain, V1, contained five confirmed mutations and reduced Fe(III) oxide 17 times faster. Whole genome DNA microarray analysis was performed in order to determine which genes are up- or down-regulated in V1 compared to PCA, both grown with ferric citrate as an electron acceptor.
Project description:Whole-genome DNA microarray analysis of Geobacter sulfurreducens cells grown on Fe(III)-oxide or Mn(IV)-oxide versus cells grown on soluble Fe(III) citrate indicated that there were significant differences in transcription patterns during growth on the insoluble metal oxides compared to growth on soluble Fe(III). Many of the genes that appeared to be up-regulated during growth on the metal hydroxides were involved in electron transport. The most highly up-regulated genes for both conditions were omcS and omcT, which encode co-transcribed c-type cytochromes exposed on the outer surface of the cell that are known to be required for Fe(III) and Mn(IV)-oxide reduction. Other electron transport genes that were up-regulated on both insoluble metals included the gene coding for the outer membrane c-type cytochrome, OmcG, genes for the outer membrane proteins, OmpB and OmpC, and the gene that codes for the structural protein of electrically conductive pili, PilA. Genes that were up-regulated in cells grown on Fe(III)-oxide but not Mn(IV)-oxide, included outer membrane c-type cytochromes including OmcE, a putative DMSO reductase protein, and proteins from the cytochrome bc1 complex. Electron transport genes that were only up-regulated in Mn(IV)-oxide grown cells included the genes that code for the outer membrane c-type cytochromes, OmcZ and OmcB, the periplasmic c-type cytochrome, MacA, and fumarate reductase. Genetic studies indicated that the c-type cytochrome proteins, PpcH, OmcJ, OmcM, OmcV, MacA, OmcF, OmcI, and OmcQ, and the iron sulfur subunit of the cytochrome b/b6 complex, QcrA, are important for reduction of insoluble Fe(III)-oxides but do not appear to be important for Mn(IV) reduction. These results demonstrate that the physiology of Fe(III) reducing bacteria differ significantly during growth on insoluble electron and soluble electron acceptors and emphasizes the importance of c-type cytochromes in extracellular electron transfer in G. sulfurreducens. Geobacter sulfurreducens cells were grown with acetate (5 mM) provided as the electron donor and either Fe(III) oxide or Fe(III) citrate provided as the electron acceptor. Cells were harvested at mid-log and total RNA was extracted. Total RNA (0.5 μg) was amplified using the MessageAmp II-Bacteria Kit (Ambion, Foster City, CA) according to the manufacturers instructions. Ten micrograms of amplified RNA (aRNA) was chemically labeled with Cy3 (for the control or soluble electron acceptor condition) or Cy5 (for the experimental or insoluble electron acceptor condition) dye using the MicroMax ASAP RNA Labeling Kit (Perkin Elmer, Wellesley, MA) according to the manufacturer’s instructions.
Project description:Whole-genome DNA microarray analysis of Geobacter sulfurreducens cells grown on Fe(III)-oxide or Mn(IV)-oxide versus cells grown on soluble Fe(III) citrate indicated that there were significant differences in transcription patterns during growth on the insoluble metal oxides compared to growth on soluble Fe(III). Many of the genes that appeared to be up-regulated during growth on the metal hydroxides were involved in electron transport. The most highly up-regulated genes for both conditions were omcS and omcT, which encode co-transcribed c-type cytochromes exposed on the outer surface of the cell that are known to be required for Fe(III) and Mn(IV)-oxide reduction. Other electron transport genes that were up-regulated on both insoluble metals included the gene coding for the outer membrane c-type cytochrome, OmcG, genes for the outer membrane proteins, OmpB and OmpC, and the gene that codes for the structural protein of electrically conductive pili, PilA. Genes that were up-regulated in cells grown on Fe(III)-oxide but not Mn(IV)-oxide, included outer membrane c-type cytochromes including OmcE, a putative DMSO reductase protein, and proteins from the cytochrome bc1 complex. Electron transport genes that were only up-regulated in Mn(IV)-oxide grown cells included the genes that code for the outer membrane c-type cytochromes, OmcZ and OmcB, the periplasmic c-type cytochrome, MacA, and fumarate reductase. Genetic studies indicated that the c-type cytochrome proteins, PpcH, OmcJ, OmcM, OmcV, MacA, OmcF, OmcI, and OmcQ, and the iron sulfur subunit of the cytochrome b/b6 complex, QcrA, are important for reduction of insoluble Fe(III)-oxides but do not appear to be important for Mn(IV) reduction. These results demonstrate that the physiology of Fe(III) reducing bacteria differ significantly during growth on insoluble electron and soluble electron acceptors and emphasizes the importance of c-type cytochromes in extracellular electron transfer in G. sulfurreducens. Geobacter sulfurreducens cells were grown with acetate (5 mM) provided as the electron donor and either Fe(III) oxide or Fe(III) citrate provided as the electron acceptor. Cells were harvested at mid-log and total RNA was extracted. Total RNA (0.5 μg) was amplified using the MessageAmp II-Bacteria Kit (Ambion, Foster City, CA) according to the manufacturers instructions. Ten micrograms of amplified RNA (aRNA) was chemically labeled with Cy3 (for the control or soluble electron acceptor condition) or Cy5 (for the experimental or insoluble electron acceptor condition) dye using the MicroMax ASAP RNA Labeling Kit (Perkin Elmer, Wellesley, MA) according to the manufacturer’s instructions.
Project description:All diazotrophic bacteria and archaea isolated so far utilise a nitrogenase enzyme containing molybdenum in the active site co-factor to fix atmospheric dinitrogen to ammonia. However, in addition to the Mo-dependent nitrogenase, some nitrogen-fixing prokaryotes also express genetically distinct alternative nitrogenase isoenzymes, namely the V-dependent and Fe-only nitrogenases respectively. Nitrogenase isoenzymes are expressed hierarchically according to metal availability and catalytic efficiency. In proteobacteria, this hierarchy is maintained via stringent transcriptional regulation of gene clusters by dedicated bacterial enhancer binding proteins (bEBPs). The model diazotroph Azotobacter vinelandii contains two paralogs of the vanadium nitrogenase activator VnfA (henceforth, VnfA1), designated VnfA2 and VnfA3, with unknown functions. Here we demonstrate that the VnfA1 and VnfA3 bEBPs bind to the same target promoters in the Azotobacter vinelandii genome and co-activate a subset of genes in the absence of V, including the structural genes for the Fe only nitrogenase. Co-activation is inhibited by the presence of V and is dependent on an accessory protein VnfZ that is co-expressed with VnfA3. Our studies uncover a plethora of interactions between bEBPs required for nitrogen fixation, revealing unprecedented potential for fine tuning expression of alternative nitrogenases in response to metal availability.