Project description:This experiment tests the gene expression of the RelA mutant (ORF03691/GSU2236) versus wild type G. sulfurreducens during stationary phase growth. Batch culture, Acetate electron donor, Fumarate electron acceptor.
Project description:This experiment tests the gene expression of the RelA mutant (ORF03691/GSU2236) versus wild type G. sulfurreducens during log phase growth. Chemostat culture, Acetate-limiting electron donor, Fumarate electron acceptor.
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.
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, as previously described (for more information 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.), with acetate (5 mM) as the electron donor and fumarate (27.5 mM) as the electron acceptor. For growth in the absence of fixed nitrogen, the ammonium chloride (4.7 mM) was omitted from the medium and fumarate served as the electron acceptor. Therefore, cultures with ammonium chloride were limited by acetate while cultures without ammonium chloride were limited by nitrogen. 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. Cells grown in the absence of ammonium had acetylene reduction rates of 0.012 nmol/hr compared to 0.003 nmol/hr in ammonium-grown cells, providing evidence that these cells were fixing nitrogen.. The steady-state concentration of cells in the nitrogen-fixing chemostats (0.178 mg/mL + 0.011; mean + standard deviation, n=3) was significantly lower than chemostats provided with ammonium (0.45 mg/mL + 0.007). Three cultures of acetate limited growth with fumarate as the electron acceptor and three cultures of nitrogen limited growth with fumarate 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.
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: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:The expression profile of adapted G. sulfurreducens strain T9-1 grown with 40 mM fumarate without any other substrate versus the expression profile of G. sulfurreducens PCA (wild type) grown with 10 mM acetate and 40 mM fumarate
Project description:Wild type G. sulfurreducens DL1 strain (see Caccavo, F., Jr., D. J. Lonergan, D. R. Lovley, M. Davis, J. F. Stolz, and M. J. McInerney. 1994. Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism. Appl Environ Microbiol 60:3752-9. see also Coppi, M. V., C. Leang, S. J. Sandler, and D. R. Lovley. 2001. Development of a genetic system for Geobacter sulfurreducens. Appl Environ Microbiol 67:3180-7.) and DLCN16 mutant (.rpoS::Km) (see Nuñez, C., L. Adams, S. Childers, and D. R. Lovley. 2004. The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens. J Bacteriol 186:5543-6.) were grown under anaerobic conditions at 30 °C in continuous culture with a 200 ml working volume as previously described (see Esteve-Nunez, A., M. Rothermich, M. Sharma, and D. Lovley. 2005. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ Microbiol 7:641-8.). Cells were cultured at a growth rate of 0.05 h-1, steady-state cell growth was obtained after 5 volume refills and was confirmed by a constant cell density and concentrations of fumarate and succinate. Acetate (5.5 mM) was the electron donor and the limiting substrate. The electron acceptor was fumarate (30mM). Three biological replicates of control and treatment cells were obtained to produce hybridizations for this experiment.
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:Microbially-mediated uranium bioremediation has been demonstrated in uranium contaminated aquifers when acetate was artificially supplied and growth of the natural population of Geobacteraceae was stimulated. In order to mimic the environmental response to acetate, steady-state cells of G. sulfureducens were cultured in chemostats under conditions of either 1) acetate as the sole electron donor and limiting factor and fumarate as the sole electron acceptor or 2) acetate was supplied in excess with fumarate as sole electron acceptor and limiting factor. In silico fluxome modeling and transcriptome analysis were used as tools for investigating the cell response to the acetate availability. For global gene expression profiling, a DNA microarray of the complete G. sulfurreducens genome was used. Statistically significant results were obtained from two-color, dye swap hybridizations produced from a total of three biological replicates. Eight technical replicates were tested from two of the biological replicates and six technical replicates were tested from the third biological replicate. Major findings from this study are given as follows. The in silico model successfully predicted a higher TCA-cycle flux (ca. 2-fold) under acetate-excess conditions, suggesting that catabolism of acetate is favored with respect to anabolism, and thus more electrons are available for metal reduction. Transcriptome analyses offered a comprehensive picture of the regulation points subjected to the acetate availability. Under acetate-excess conditions, acetate transporters in the G. sulfurreducens genome were down-regulated. In addition the oxidation-related acetyl-CoA transferase was up-regulated approximately three-fold and the assimilatory-related acetate kinase was down-regulated approximately two-fold, respectively, indicating that the transcriptional regulation of acetate activation may be the key point for coping with the excess of acetate and increasing the TCA flux. The level of transcription for 10 c-type cytochromes was significantly increased in cells cultured with an excess of acetate. OmcS, an outer-membrane cytochrome which actively participates in electron transfer to Fe(III)-oxides and graphite electrodes from fuel cells, showed one of the highest fold increases in transcription. The integration of in silico modeling and genome-wide analysis shows for first time how G. sulffureducens adapts its metabolic flux and transcriptional network for optimizing the use of acetate as an electron donor for exocellular respiration instead of for use as a carbon source for biomass production. Keywords: Geobacter, gene expression, acetate limitation, fumarate limitation