Abstract
TfoX (Sxy) and CRP are two important competence activators. The link between tfoX and CRP has been shown in H. influenza but lacking evidence of direct interaction. Recently a Sxy-dependent CRP (CRP-S) site autoregulating Sxy was reported in E. coli. Here, we show that the cAMP-CRP complex transcriptionally regulates tfoX expression through multiple canonical CRP (CRP-N) sites in Vibrios. This conclusion is supported by an analysis of the tfoX mRNA levels and tfoX transcriptional reporter fusions. The reduced expression of tfoXVC was restored by trans-complementation of crp in ∆crp and by exogenous cAMP in ∆cya. A promoter deletion analysis and the site-directed mutagenesis of the putative CRP-N sites revealed the presence of two functional CRP-N sites. The direct binding of cAMP-CRP to the tfoXVCpromoter was demonstrated by EMSA assays. Additionally, the transcriptional start site (TSS) of tfoXVF in V. fluvialis was determined, and −10/−35 regions were predicted. Further comparison of the tfoX promoter in Vibrios revealed the existence of similar −10 motifs and putative CRP-N sites, indicating the conserved mechanism of CRP regulation on tfoX. Our study demonstrates the direct binding of the cAMP-CRP complex to tfoX promoter, and broadens the understanding of the molecular mechanism regulating tfoX in Vibrios.
Highlights
Many bacterial species can take up environmental DNA under natural conditions, most of which is degraded in the cytoplasm for reuse
Yamamoto’s works greatly improved the knowledge of the regulation of tfoXVC in V. cholerae by identifying chitin disaccharide (GlcNAc)[2] as the minimum inducer of chitin-dependent competence, by mapping the transcriptional start site (TSS), and the transcriptional and translational cis-acting elements, and by distinguishing the sRNA tfoR regulation[21,22]. Using this knowledge, combined with the prior clues provided by microarray data[27], we reexamined the promoter region of tfoXVC and found a potential cAMP receptor protein (CRP)-N binding site centered at − 84.5 relative to the TSS, which contained a perfect TGTGA half-site and a 4/5 matched TCTCA half-site for the DNA binding domains of the active CRP dimer
To determine whether CRP was involved in the expression of tfoXVC, we examined the tfoXVC mRNA level in wild type and isogenic mutant crp via Quantitative reverse transcription PCR (qRT-PCR) (Fig. 1A)
Summary
Many bacterial species can take up environmental DNA under natural conditions, most of which is degraded in the cytoplasm for reuse. The DNA that escapes degradation might be incorporated into the host chromosomes by RecA-dependent homologous recombination[1,2,3,4,5,6] This is a very complicated phenomenon with regard to its biological function, machinery components and the regulated and regulatory genes involved. That study first demonstrated that chitin, a polymer of β -1,4-linked N-acetylglucosamine (GlcNAc), induces natural competence in V. cholerae[5]. In V. cholerae, competence development is triggered by a chitin-induced transcription regulator, TfoxVC, an ortholog of Sxy in H. influenzae, and a quorum-sensing regulator, HapR5. Sxy (TfoX) and CRP are two major shared activators that control the development of competence in the families Pasteurellaceae, Enterobacteriaceae, and Vibrionaceae[12]. A small regulator RNA, tfoR, activates the translation of tfoX in response to the presence of (GlcNAc)[2] or chitin[22]
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