Project description:Anaerobic ammonium-oxidising (anammox) bacteria, members of the ‘Candidatus Brocadiaceae’ family, play an important role in the nitrogen cycle and are estimated to be responsible for about half of the oceanic nitrogen loss to the atmosphere. Anammox bacteria combine ammonium with nitrite and produce dinitrogen gas via the intermediates nitric oxide and hydrazine (anammox reaction) while nitrate is formed as a by-product. These reactions take place in a specialized, membrane-bound compartment called the anammoxosome. Therefore, the substrates ammonium, nitrite and product nitrate have to cross the outer-, cytoplasmic- and anammoxosome membranes to enter or exit the anammoxosome. The genomes of all anammox species harbour multiple copies of ammonium-, nitrite- and nitrate transporter genes. Here we investigated how the distinct genes for ammonium-, nitrite- and nitrate- transport were expressed during substrate limitation in membrane bioreactors. Transcriptome analysis of Kuenenia stuttgartiensis planktonic cells under ammonium-limitation showed that three of the seven ammonium transporter genes and one of the six nitrite transporter genes were significantly upregulated, while another ammonium and nitrite transporter gene were downregulated in nitrite limited growth conditions. The two nitrate transporters were expressed to similar levels in both conditions. In addition, genes encoding enzymes involved in the anammox reaction were differentially expressed, with those using nitrite as a substrate being upregulated under nitrite limited growth and those using ammonium as a substrate being upregulated during ammonium limitation. Taken together, these results give a first insight in the potential role of the multiple nutrient transporters in regulating transport of substrates and products in and out of the compartmentalized anammox cell.
Project description:This SuperSeries is composed of the following subset Series: GSE21312: Gene expression in a Geobacter sulfurreducens strain adapted for faster Fe(III) oxide reduction grown with ferric citrate as an electron acceptor GSE21313: Gene expression in a Geobacter sulfurreducens strain adapted for faster Fe(III) oxide reduction grown with fumarate as an electron acceptor Refer to individual Series
Project description:Nitric oxide (NO) has several important functions in biology and atmospheric chemistry as a toxin, signaling molecule, ozone depleting agent and the precursor of the greenhouse gas nitrous oxide (N2O). Even though NO is a potent oxidant, and was available on earth earlier than oxygen, its direct use by microorganisms for growth was not demonstrated before. Using physiological experiments, metatranscriptomics and metaproteomics, here we show that anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis grow by coupling ammonium oxidation to NO reduction, and produce only N2. Such a metabolism could have existed on early earth, and has implications in controlling N2O and NO emissions both from natural and manmade ecosystems, where anammox bacteria contribute significantly to N2 release to the atmosphere.
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. One of these five mutations inactivates dcuB, a fumarate/succinate antiporter necessary for growth with fumarate as an electron acceptor. V1 dcuB+ is a V1 strain containing a wild type copy of dcuB. Whole genome DNA microarray analysis was performed in order to determine which genes are up- or down-regulated in V1 dcuB+ compared to PCA, both grown with fumarate 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 M-NM-<g) 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 manufacturerM-bM-^@M-^Ys instructions. RNA samples from three biological replicates were hybridized in duplicate on 12K Combimatrix antisense-detecting arrays. The experimental condition (DL1 grown with Mn(IV) oxide as acceptor) was labeled with cy5, the control condition (DL1 grown with Fe(III) citrate as acceptor) was labeled with cy3
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 M-NM-<g) 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 manufacturerM-bM-^@M-^Ys instructions. RNA samples from three biological replicates were hybridized in duplicate on 12K Combimatrix antisense-detecting arrays. The experimental condition (DL1 grown with Fe(III) oxide as acceptor) was labeled with cy5, the control condition (DL1 grown with Fe(III) citrate as acceptor) was labeled with cy3
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. One of these five mutations inactivates dcuB, a fumarate/succinate antiporter necessary for growth with fumarate as an electron acceptor. V1 dcuB+ is a V1 strain containing a wild type copy of dcuB. Whole genome DNA microarray analysis was performed in order to determine which genes are up- or down-regulated in V1 dcuB+ compared to PCA, both grown with fumarate as an electron acceptor. Three biological replicates were hybridized in duplicate. Experimental (V1) was labeled with cy5, control (wild type PCA) was labeled with cy3.
Project description:Genome-based Metagenomic Analysis of Candidatus Scalindua with Anammox consortia in Long-term Cultivated Marine Anammox Bacteria Community