Cyclic AMP Receptor Protein Regulates cspD , a Bacterial Toxin Gene, in Escherichia coli

Abstract

ABSTRACT cspD , a member of cspA family of cold shock genes in Escherichia coli , is not induced during cold shock. Its expression is induced during stationary phase. CspD inhibits DNA replication, and a high level of the protein is toxic to cells. Recently, CspD was proposed to be associated with persister cell formation in E. coli . Here, we show that cyclic AMP receptor protein (CRP) upregulates cspD transcription. Sequence analysis of the cspD upstream region revealed two tandem CRP target sites, CRP site-I (the proximal site centered at −83.5 with respect to the transcription start) and CRP site-II (the distal site centered at −112.5). The results from electrophoretic mobility shift assays showed that CRP indeed binds to these two target sites in P cspD . The promoter-proximal CRP target site was found to play a major role in P cspD activation by CRP, as studied by transcriptional fusions carrying mutations in the target sites. The results from in vitro transcription assays demonstrated that CRP activates P cspD transcription in the absence of additional factors other than RNA polymerase. The requirement for activating region 1 of CRP in P cspD activation, along with the involvement of the 287, 265, and 261 determinants of the α-CTD, suggest that CRP activates by a class I-type mechanism. However, only moderate activation in vitro was observed compared to high activation in vivo , suggesting there might be additional activators of P cspD . Overall, our findings show that CRP, a global metabolic regulator in E. coli , activates a gene potentially related to persistence.