Benzoate induced transcriptomic response in Bacillus subtilis

Bacillus subtilis strain AG174 was grown in the presence and absence of benzoate (30 mM). Benzoate was used in order to equalize the external and internal pH. The cultures were grown at external pH 7.0, and the addition of benzoate did not change the external pH. Microarray analysis was performed on cDNA synthesized from bacterial RNA. Real-time PCR of highly up-regulated genes confirmed the results of the microarray. These data were compared to previous B.subtilis microarray results, where genes regulated by external pH were identified. Overnight cultures were diluted 1:500 in potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM MOPS at pH 7.0. Bacteria were cultured in baffled flasks (less than 10% volume) with rotation at 220 rpm, incubated at 37 °C to an optical density at 600 nm of 0.2. Four cultures were grown in the presence of 30 mM benzoate and four cultures were grown without benzoate. One sample was taken from each of four replicate cultures of 0 mM benzoate and 30 mM benzoate. RNA was isolated using 10% saturated phenol-ethanol solution and the RNeasy Kit. Gene expression profiles were obtained with standard Affymetrix procedures.