Two recent papers have addressed the issue of how MexT regulates the expression of the mexEF-oprN operon. First, nod boxes, the binding site of the NodD protein required in the nodulation of Rhizobia, were found upstream of the mexEF-oprN
operon, and MexT might regulate the operon upon binding at those boxes. Secondly, an ATCA(N5)GTCGAT(N4)ACYAT consensus sequence was found upstream of the mexEF-oprN operon and this sequence was proposed to be the MexT-binding site (Goethals et al., 1992; Köhler et al., 1997, 1999; Tian et al., 2009). However, the precise mechanism by which they act on the regulatory element(s) of mexEF-oprN mTOR inhibitor remained to be elucidated. We report here the molecular interaction between MexT and the mexT-mexE intergenic DNA, and the positive and negative regulation of mexEF-oprN gene expression. The bacterial strains and plasmids used are listed in Table 1. The P. aeruginosa cells were cultured at 37 °C in Luria–Bertani (LB) broth Selleck SB431542 supplemented with 150 μg mL−1 of gentamicin, or an appropriate amount of isopropyl β-d-thiogalactopyranoside (IPTG) as needed. Escherichia coli DH5α was the host for DNA manipulation. DNA was manipulated by standard methods (Sambrook et al., 1989). The plasmid DNA was extracted from E.
coli using a GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol. Transformation-competent P. aeruginosa cells were prepared according to the instruction manual of GenePulser II (Bio-Rad, Hercules, CA). Pseudomonas aeruginosa cells harboring appropriate reporter plasmid were grown in LB broth containing 2 mM IPTG at 37 °C. The cells were harvested at the desired time and β-galactosidase activity in the cell-free extracts was determined according to the method of Miller (Sambrook et al., 1989). The mexT-mexE intergenic DNA was amplified using the primers nmexE1-Eco and nmexE2-Hin (Table 2). The PCR products were digested with EcoRI and HindIII, and ligated into pME4510 carrying the promoter-less lacZ,
yielding pME4510-Ep. Pseudomonas aeruginosa PAO1S and PAO1SC, producing nonfunctional and intact MexT, respectively, were transformed with pME4510-Ep. The role of MexT in the expression of mexE (for Chlormezanone the mexEF-oprN expression) was assessed using the mexE∷lacZ reporter gene. The plasmid carrying the 3′- or 5′-end deletion was constructed in pME4510-Ep by inverted PCR of the desired length of mexT-mexE intergenic DNA. EcoRI-tagged primers (Ep31, Ep51, Ep71, and Ep91, respectively) paired with 4510-1Eco were used for the construction of pME4510-Ep31, pME4510-Ep51, pME4510-Ep71, and pME4510-Ep91 (Table 2). The resulting fragments were digested with EcoRI and self-ligated. HindIII-tagged primers (Ep42, Ep62, Ep82, and Ep42) paired with 4510-4Hin were used for the construction of pME4510-Ep42, pME4510-Ep62, pME4510-Ep82, and pME4510-Ep54 (Table 2). The resulting fragments were digested with HindIII and self-ligated.