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: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: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:The Geobacter species evolved respiratory versatility to utilize a wide range of terminal electron acceptors. To explore this adaptive mechanism, Fe(III) citrate, hydrous ferric oxide, and fumarate were selected as electron acceptors, and the methylome and metabolome of Geobacter sulfurreducens PCA grown on each electron acceptor were investigated via third-generation, single-molecule real-time DNA sequencing.Results showed that the patterns of 4-methylcytosine (m4C) and 6-methyladenine (m6A) modification were all varied in different electron acceptor cultures. Moreover, genes (e.g., GSU0466 and GSU1467) with low expression levels generally had high methylation levels. These findings suggest that m4C and m6A modifications play a role in the adaption of G. sulfurreducens to diverse electron acceptors, and DNA methylation may be involved in the adaption mainly via gene expression regulation.
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:Gene expression in a Geobacter sulfurreducens strain adapted for faster Fe(III) oxide reduction grown with ferric citrate as an electron acceptor
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: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. Three biological replicates were hybridized in duplicate. Experimental (V1) was labeled with cy5, control (wild type PCA) was labeled with cy3.
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:G. sulfurreducens (ATCC #51573) was obtained from the laboratory culture collection of Dr. Derek Lovley. Cells were grown under strict anaerobic conditions at 30 °C in chemostats, (see Esteve-Núñez, A., M. M. Rothermich, M. Sharma, and D. R. Lovley. 2004. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ. Microbiol.:in press. for more information), with acetate (5 mM) as the electron donor and Fe(III) citrate (55 mM) or fumarate (27.5 mM) as the electron acceptor. Under these conditions acetate is the substrate limiting growth. Cultures were maintained at a dilution rate of 0.05 h-1 for 5 culture vessel volumes to ensure that cells were at steady-state prior to harvesting. Cells were harvested by centrifugation at 4 °C and the cell pellet was flash-frozen in liquid nitrogen and then stored at 80 °C prior to RNA extraction. Three cultures of acetate limited growth with fumarate as the electron acceptor and three cultures of acetate limited growth with Fe(III) as the electron acceptor were grown. Each culture vessel was harvested and extracted for total RNA separately to produce three biological replicates for this experiment.