Abstract
Horizontal gene transfer through natural competence is an important driving force of bacterial evolution and antibiotic resistance development. In several Gram-negative pathogens natural competence is regulated by the concerted action of cAMP receptor protein (CRP) and the transcriptional co-regulator Sxy through a subset of CRP-binding sites (CRP-S sites) at genes encoding competence factors. Despite the wealth of knowledge on CRP’s structure and function it is not known how CRP and Sxy act together to activate transcription. In order to get an insight into the regulatory mechanism by which these two proteins activate gene expression, we performed a series of mutational analyses on CRP and Sxy. We found that CRP contains a previously uncharacterized region necessary for Sxy dependent induction of CRP-S sites, here named “Sxy Interacting Region” (SIR) encompassing residues Q194 and L196. Lost promoter induction in SIR mutants could be restored in the presence of specific complementary Sxy mutants, presenting evidence for a direct interaction of CRP and Sxy proteins in transcriptional activation. Moreover, we identified constitutive mutants of Sxy causing higher levels of CRP-S site promoter activation than wild-type Sxy. Both suppressor and constitutive mutations are located within the same area of Sxy.
Highlights
The cyclic adenosine monophosphate (cAMP) receptor protein (CRP; known as the catabolite activator protein, CAP) is a versatile transcriptional regulator, regulating more than a hundred genes in Escherichia coli
It has been shown that most of the competence genes found in E. coli are preceded by DNA motifs homologous to the CRPS sites found in H. influenzae, and that induction of these are dependent on cAMP-cAMP receptor protein (CRP) and Sxy as well (Sinha et al, 2009)
Previous studies hypothesized that Sxy binds cAMP-CRP directly to facilitate its binding to the otherwise low affinity CRP-S sites (Redfield et al, 2005; Cameron and Redfield, 2006; Sinha et al, 2009)
Summary
The cAMP receptor protein (CRP; known as the catabolite activator protein, CAP) is a versatile transcriptional regulator, regulating more than a hundred genes in Escherichia coli. CRP Interacts With Sxy region 1 (AR1) of the DNA-bound cAMP-CRP protein and the C-terminal domain of the RNAP α subunit (αCTD) facilitates RNAP binding to the promoter and thereby stimulates transcription (Kolb et al, 1993; Busby and Ebright, 1994; Benoff et al, 2002; Lawson et al, 2004). It has been shown that most of the competence genes found in E. coli are preceded by DNA motifs homologous to the CRPS sites found in H. influenzae, and that induction of these are dependent on cAMP-CRP and Sxy as well (Sinha et al, 2009). Based on our results we suggest a model where cAMP-CRP and Sxy interact directly, while cAMP-CRP contacts RNAP in a Class I like manner
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
