, 2001). Template plasmids and oligonucleotides used for genetic constructions are listed in Tables 1 and 2, respectively. The sequence of STY1365 was amplified by PCR and the product was purified using the Nucleotide Removal Kit (Qiagen). Selleck EPZ6438 The purified DNA was digested by PstI/EcoRI (Invitrogen) and cloned in the PstI/EcoRI-digested mid-copy-number vector pSU19
(Bartolome et al., 1991) to yield pRP005 plasmid. To generate pRP010, a PCR product of STY1365 was directly cloned in the pCC1™ vector according to the manufacturer’s instructions (CopyControl™ PCR Cloning Kit, Epicentre). The plasmids were confirmed by PCR, restriction endonuclease assays and sequencing (Macrogen Corp., Rockville, MD). Finally, these plasmids were introduced into the corresponding mutant strain by electroporation. Primers for cloning as well as sequencing are described in Table 2. Salmonella Typhi Akt assay strains carrying lacZY fusions were grown routinely in LB broth and OD600 nm was monitored. β-Galactosidase activity was measured as described previously (Bucarey et al., 2005). β-Galactosidase activity was calculated as follows:
103× (A420 nm−1.75 × A550 nm) mL−1 min−1/A600 nm, and expressed in Miller Units where A is the absorbance units. Each assay was made in duplicate and repeated at least three times. Isolation of total RNA was performed as described P-type ATPase previously (Rodas et al., 2010). RT-PCR amplification was performed
with 5 μg of DNAse I-treated RNA using Superscript II RT (Invitrogen). Amplification included 35 cycles (94 °C for 30 s, 58 °C for 45 s and 72 °C for 90 s) followed by a 5-min extension at 72 °C to ensure full extension of amplified fragments. Primers used to amplify STY1365 are described in Table 2. Reverse transcription of 16S rRNA was used as a positive control (Bucarey et al., 2005). DNAse-treated RNA that had not been transcribed was used as negative control. Thirty-microliter aliquots were resolved in 1.5% agarose gels, stained with ethidium bromide and visualized under UV source. The STY1365-3xFLAG fusion protein was detected by Western blotting using an anti-FLAG M2 monoclonal antibody (Sigma). Overnight cultures of S. Typhi strain carrying the FLAG epitope was subcultured in 25 mL of LB broth and grown to an OD600 nm of 0.2 at 37 °C with shaking. Cells were collected by centrifugation, and subcellular fractionation of inner- and outer-membrane proteins was performed (Santiviago et al., 2001; Bucarey et al., 2006). Cytoplasmic fraction was obtained according to the protocol described by Ludwig et al. (1995). Protein fractions were concentrated by precipitation with ice-cold trichloroacetic acid (final concentration 10%) and washed with acetone. Proteins were quantified using the Pierce® BCA Protein Assay Kit (Thermo Scientific).