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G. sulfurreducens growth with acetate as limiting electron donor versus fumarate as limiting electron acceptor

ABSTRACT: 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

ORGANISM(S): Geobacter sulfurreducens PCA Geobacter sulfurreducens

PROVIDER: GSE9830 | GEO | 2008/12/09

SECONDARY ACCESSION(S): PRJNA103799

REPOSITORIES: GEO

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Project description:1. Summary: SfrAB, the soluble ferric reductase of Geobacter sulfurreducens, is a two subunit complex that can catalyze the NADPH-dependent reduction of chelated Fe(III) and is encoded in a two gene cluster in which the sfrB gene (GSU_0510) is 126 bp upstream of the sfrA gene (GSU_0509)[Kaufmann and Lovley (2001) J. Bacteriol. 183:4468-4476]. In order to gain insight into the physiological function of SfrAB, a knockout mutant was constructed by replacing a 3.6 kb stretch of sequence encompassing the C-terminal 72% of the SfrB and the N-terminal 80% of the SfrA coding sequences with a kanamycin resistance cassette via homologous recombination. The SfrAB knockout mutant (Delta sfrAB::kan) was found to be initially unable to grow in media in which acetate served as the sole electron donor. However, following prolonged incubation (2-3 weeks) in acetate:fumarate medium, an acetate-adapted SfrAB-null strain with the ability to grow on fumarate with acetate serving as the sole electron donor was isolated. This acetate-adapted, SfrAB-null strain and wild type G. sulfurreducens were cultured in parallel in chemostats at an intermediate growth rate (0.05 hr-1) in acetate-limited freshwater fumarate medium (5 mM acetate:27.5 mM fumarate). After the chemostats reached steady state, cells were harvested by centrifugation and RNA was extracted. In order to identify genes that were involved in growth on acetate in the absence of SfrAB, this RNA was used to perform microarray analysis comparing gene expression in the acetate-adapted, SfrAB-null and wild type strains. 2. Growth condtions: Cells were cultured in chemostats at a dilution rate of 0.05 hr-1 in acetate-limited freshwater fumarate medium (5 mM acetate:27.5 mM fumarate) and harvested when the chemostats were at steady state. 3. Mutant designation: acetate-adapted SfrAB-null strain, genotype= Delta sfrAB::kan. (Note: there is an unadapted Delta sfrAB::kan strain which cannot grow on acetate and an adapted one which can. The acetate-adapted strain was used for the microarray analysis).

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G. sulfurreducens growth with acetate as limiting electron donor versus fumarate as limiting electron acceptor

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