Phenotype microarray screening of carbon sources used byVibrio choleraeidentifies genes regulated by the cAMP receptor protein

Abstract

The cyclic AMP receptor protein (CRP) regulates genes involved in carbon source metabolism, iron uptake, and virulence in bacteria. Identifying the carbon sources utilized by bacteria that are regulated by CRP will help elucidate the CRP regulation cascade and associated responses to environmental stimuli. CRP-dependent regulation of carbon source metabolism in Vibrio cholerae is not thoroughly understood. To identify the candidate carbon sources utilized by V. cholerae that are affected by CRP, we used high-throughput screening to compare the metabolic differences between wild-type and CRP mutant strains of V. cholerae O1 El Tor. Phenotype microarray was used for primary screening of the wild-type and mutant strains, followed by minimal media growth assays and quantitative RT-PCR to validate the candidate carbon sources. In total, 24 carbon sources were subject to CRP regulation, 11 of which have not been previously reported in bacteria. The genes known to be involved in the metabolism of 4 of the carbon sources identified were verified by quantitative RT-PCR. In addition, gel shift experiments showed that CRP bound directly to VCA0053 and VC0391 promoters. Overall, this comprehensive analysis of CRP-mediated catabolite control in V. cholerae has identified new candidate carbon sources for in-depth experimental studies.