Each culture was divided in halves, one half was used as a control and the other half was transferred to a new flask containing cAMP (5-mM). modulator. Keywords:Vibrio cholerae, signal transduction, quorum sensing, cAMP, cAMP receptor protein, carbon catabolite repression == 1. Introduction == Vibrio choleraeis a Gram-negative highly motile bacterium that colonizes the human small intestine and produce cholera toxin (CT) which causes the profuse watery diarrhea typical of cholera [1,2]. The expression of CT is regulated by quorum sensing. Quorum sensing is a process by which bacterial cells communicate with one another by secreting extracellular signaling molecules termed autoinducers. Two autoinducer systems function inV. cholerae. The cholera autoinducer 1 (CAI-1) synthase CqsA is responsible for the biosynthesis of CAI-1 [(S)-3-hydroxytridecan-4-one] which at high cell density binds to its receptor CqsS [3]. Autoinducer 2 (AI-2) is a furanosyl borate diester [(2S, 4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate] synthesized by the activity of LuxS [4] and recognized by receptor LuxPQ. Accumulation of CAI-1 and AI-2 activates the expression of the master quorum sensing regulator HapR [5]. HapR plays a pivotal role in regulating virulence gene expression, protease production and biofilm formation [6-8] by modulating intracellular cyclic diguanylate (c-di-GMP) levels, as well as by acting Lubiprostone at specific promoters that control virulence gene expression and exopolysaccharide biosynthesis [9,10]. The cAMP receptor protein (CRP) is a global regulator FASN well known for its role in carbon catabolite repression: the inhibition of gene expression by the presence of a rapidly metabolizable carbon source (i.e. glucose) in the growth medium [11-13]. In carbon catabolite repression, the presence of glucose indirectly inhibits adenylate cyclase through a component of the posphoenolpyruvate-dependent phosphotransferase system (PTS) leading to low intracellular cAMP levels [14]. Conversely, when glucose is limited (or replaced by a poor carbon source), the activity of adenylate cyclase increases leading to high intracellular levels of cAMP and formation of the cAMP-CRP complex [12]. Lubiprostone The cAMP-CRP complex binds as a dimer to the consensus sequence TGTGA-(N6)-TCACA which can be found within, adjacent or upstream of responsive promoters [11-13]. In a previous study we demonstrated that CRP is required for expression of the CAI-1 synthase CqsA [15]. Consequently, deletion ofcrpwas shown to impact the expression of HapR, multiple HapR-regulated genes and phenotypes such as production of hemagglutinin (HA)/protease, CT, exopolysaccharide biosynthesis and biofilm formation [15,16]. In this study we show that cAMP levels post-transcriptionally activate the expression of CqsA by enhancing the stability of thecqsAmRNA. Thus, cAMP acts upstream of quorum sensing to regulate CAI-1 biosynthesis and modulate the cell density required to activate HapR. Finally, this is the first report of the cAMP-CRP complex modulating gene expression at the level of mRNA stability to integrate carbon catabolite repression and quorum sensing. == 2. Materials and methods == == 2.1. Strains and media == V. choleraemutants used in this study were constructed from the El Tor biotype strain C7258 (Per isolate, 1991). Construction of strains AJB61 (C7258cqsA), WL7258 (C7258crp), C7258lacZand WL7258lacZhave been described previously [15,17].E. coliTOP10 (Invitrogen) and S171pir[18] were used for cloning purposes.V. choleraestrains were grown in Bacto tryptic soy broth (TSB) (Becton, Dickinson & Co.) or LB at 37C with agitation (250 rpm). When necessary, culture media were supplemented with ampicillin (Amp, 100-g/ml), tetracycline (Tet, 5-g/ml), kanamycin (Km, 25-g/ml), X-gal (20-g/ml) or polymyxin B (100-units/ml). Plasmid DNA was introduced inV. choleraeby electroporation [19]. == 2.2 Construction of mutants == Strain WL7259 (C7258cya) containing a deletion ofcyaencoding adenylate cyclase was constructed by transferring the suicide vector pCVDcya-km [16] to C7258 followed by sucrose selection as described previously [16]. To construct the crpluxOdouble mutant SZS013, chromosomal DNA fragments flanking theluxOopen reading frame (ORF) were amplified from C7258 genomic DNA with the Advantage Lubiprostone 2 PCR kit (Clontech) using the primer sets 5-GCGCGAGCTCGTGATTTACGATTAGGCG/5-GCGCGGATCCAAACATCACACATCTAGAC and 5-GCGCGGATCCGTGAACTCAATGATTAC/5-GCGCGTCGACAGATAACCTTTCGGTGC and the amplicons were sequentially cloned in pCVD442 [20]. The resulting suicide vector containing the chromosomalluxOdeletion was transferred to strain WL7258lacZby conjugation and the luxOmutant was obtained by sucrose selection as described previously [15,16]. == 2.3. Construction of plasmids and chromosomally integratedlacZfusions == A 1.6-kb DNA fragment encompassing thecqsAORF flanked by 5 and 3 non-coding (intergenic) sequences was amplified using primers 5-GGGGATCCTGACCGTGATGTATTGCTA and 5-GAACTGCAGCGCTCAGTAAACTCCTAA and cloned as aBamHI-SphI fragment in pBR322 to yield pBRCqsA2383. To construct acqsA-lacZtranscriptional fusion we sequentially cloned therrnBT1T2transcription terminator [21] and a 214-bp fragment containing thecqsApromoter region amplified with primers 5-GGCCAAGCTTTCGCAATATATCCTAGTT and 5-GAACTGCAGCGCTCAGTAAACTCCTAA in pUC19 to generate pTTcqsA. A 1-kb Lubiprostone internal fragment of the C7258lacZgene (lacZVC) was amplified using primers 5-CGAAGGTACCAATCCCCGATTCA and 5-GCCTCTAGATCGCCACCGTTTTACACTG and cloned as aKpnI-XbaI fragment in pTTcqsA upstream of therrnBT1T2transcription terminator to generate pZTC. Next, a 1.6-kbKpnI-HindIII fragment containing thelacZVCfragment,.