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Anaerobic growth in M9 + glucose + fumarate,genomic DNA reference

ABSTRACT: Responses of Escherichia coli MG1655 as they grow anaerobically in M9 + glucose + fumarate Keywords: time course

ORGANISM(S): Escherichia coli

PROVIDER: GSE4374 | GEO | 2006/03/08

SECONDARY ACCESSION(S): PRJNA94665

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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Anaerobic growth in M9 + glucose + fumarate,genomic DNA reference

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