The Functional Characterization of the Quorum Sensing E. Coli Regulators B and C in EHEC
Clarke, Marcie B.
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Enterohemorrhagic E. coli (EHEC) is the causative agent of hemorrhagic colitis. During an infection, EHEC can sense and respond to environmental cues, including the cell density of the intestinal normal flora (through the floral-derived AI-3 signal) and the epinephrine/norepinephrine produced naturally by the host. This cell-to-cell signaling may aid in colonization and disease by allowing EHEC to up-regulate its flagella and motility genes to swim closer to the intestinal epithelium. Previously, Sperandio et al. (2002) have shown that the quorum sensing E. coli regulators B and C (QseB&C), a two-component system in EHEC, are responsible for the regulation of the master regulator of flagella and motility genes, flhDC, in response to cell-to-cell signaling . Here, we show that QseC, the membrane-bound sensor kinase, can autophosphorylate itself in response to AI-3 or epinephrine/norepinephrine and then transfer this phosphate to the response regulator, QseB. The autophosphorylation of QseC is not affected by the addition of autoinducer-2 or intestinal hormones, including gastrin, galanin, and secretin. Additionally, autophosphorylation can be antagonized upon the addition of phentolamine, an a-adrenergic receptor antagonist. Given that enterocytes harbor a-adrenergic receptors, it would be consistent for a microbial adrenergic sensor (QseC) to mostly resemble (in an orthologous and not a homologous fashion) an a- and not a ᭡drenergic receptor. Taken together, these results suggest that QseC may be a microbial adrenergic receptor conserved amongst different bacterial and fungal species. After QseC has autophosphorylated and transferred its phosphate to QseB, QseB acts as a transcription factor to activate the expression of flhDC, the master regulator of flagella and motility genes. Nested deletion analyses of the flhDC promoter suggest that QseB may bind to three promoter regions, to either repress or activate transcription. Further transcriptional studies suggest that phosphorylated QseB autoregulates its own transcription in a similar manner. These analyses have identified a QseB consensus binding sequence, which was utilized in an in silico search to identify novel potential targets of QseB. Through the use of both biochemistry and genetics, a comprehensive model of the QseB&C signaling cascade was generated.