The ΔacfB fragment was digested with BamHI and EcoRI and the ΔtcpI∷Cm fragment was digested with KpnI, and then each was ligated into pKAS32 (Skorupski & Taylor, 1996), which was digested appropriately to generate pKEK870 and pKEK1117, respectively. The expression plasmid containing acfB was created by PCR amplification using
the oligonucleotides acfBMet and acfBXbaI. The PCR fragment was digested with XbaI and ligated into pBAD24 (Guzman et al., 1995) that had been digested with NcoI, treated with Klenow fragment to fill in the 5′ overhand, and then digested with XbaI, to form pKEK149. The expression plasmid containing tcpI was created by PCR amplification with oligonucleotides tcpI F BamHI and tcpI R EcoRI, followed by digestion with BamHI and EcoRI, and ligation into pWSK30 (Wang & Kushner, 1991) digested similarly to form pKEK1306. Table 1 contains a list of the bacterial strains MG-132 cost used in this study. Escherichia coli strain DH5α (Hanahan, 1983) was used for all cloning experiments, while the E. coli strain WM3046 (a gift from William Metcalf, University of Illinois) was used to transfer plasmids to V. cholerae by conjugation. The ΔacfB, ΔtcpI∷Cm, and ΔcheY-3 V. cholerae strains KKV2089, KKV2060, and KKV2090 were constructed as described previously (Skorupski & Taylor, 1996) by mating pKEK870, pKEK1117, and pSB27, respectively, into
V. cholerae strain O395. The ΔacfB, ΔtcpI∷Cm strain KKV2061 was constructed by CPT1ts transduction (Hava & Camilli, 2001) of ΔtcpI∷Cm
into strain KKV2089. The correct construction Selleck Proteasome inhibitor of all strains was verified by PCR and sequencing. CT in the culture supernatants was measured using a GM1-enzyme-linked immunosorbent assay with rabbit polyclonal antiserum against the purified B subunit of CT (Svennerholm & Holmgren, 1978). TCP was measured by CTXφ-Kan phage transduction (Waldor & Mekalanos, 1996). The in Tolmetin vivo colonization assays were performed as described by Gardel & Mekalanos (1996) using 5–6-day-old CD-1 suckling mice. The inocula consisted of ∼105 CFU for both wild-type and mutant strains, and intestines were harvested 22 h postinoculation. For strains carrying pKEK149, inocula also contained 0.1% arabinose. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the University of Texas, San Antonio. Within the VPI lie the acfB (VC0825) and tcpI (VC0840) genes, which are predicted to encode putative MCPs (Everiss et al., 1994; Harkey et al., 1994) (Fig. 1). The tcpI gene is in a single gene operon divergently transcribed from the regulatory genes tcpPH, while the acfB gene lies within an operon downstream of toxT and tcpJ. Both AcfB and TcpI have been demonstrated to be positively regulated by ToxT (Peterson & Mekalanos, 1988; DiRita et al.