A library from strain TT1704-OS was constructed in cosmid pLA2917 (see Materials and methods for details). Analysis of the flanking sequences to MudJ revealed a large ORF. We searched for homologies against the S. Typhimurim LT2 genome annotation, and it matched to the yfeR gene, reported as a putative LysR transcriptional regulator (McClelland et al., 2001). Its gene product, the YfeR protein, shows features that are shared by members of the LTTRs. It exhibits high similarity to other described LTTRs, contains the consensus helix–turn–helix DNA-binding domain (amino
acids 5–64, pfam 00126), and shows the anomalous Lys/Arg ratio (0.19). Strain TT1704-OS was grown in LB medium containing variable concentrations of NaCl, and its β-galactosidase www.selleckchem.com/products/PLX-4720.html activity was evaluated. In all conditions tested, the growth rate was similar to that of the parental strain (data not shown). When compared with high osmolarity conditions, growth under low osmolarity conditions resulted in a fourfold increase in the β-galactosidase activity (Fig. 1a). Growth in LB medium rendered intermediate β-galactosidase values (data not shown). An osmotic challenge was also used to provide further evidence of yfeR osmoregulation. Selleckchem CHIR 99021 Strain TT1704-OS was grown in LB medium at low and high osmolarity conditions to the mid-exponential growth phase, and then a shift of LB medium was done: β-galactosidase activity was evaluated before
and after the medium shift (Fig. 1b). As expected, cultures switched to high and low osmolarity conditions decreased and increased its
β-galactosidase activity, respectively. Lastly, to confirm osmoregulation of the yfeR gene, we detected yfeR mRNA by RNase-ONE protection assay. As predicted (Fig. 1c), yfeR-specific mRNA increases when cells grow under low osmolarity conditions. Many members of the LTTR family autorepress their transcription. To test this, we cloned the yfeR sequence in the low copy number plasmid pLG338-30. The resulting Dichloromethane dehalogenase plasmid (pLGYFER) was introduced into strain TT1704-OS. β-Galactosidase values obtained (Fig. 2) confirmed that the YfeR protein represses its expression both at low and at high osmolarity. A common property of members of the LysR family is that they regulate the adjacent gene, located in inverted orientation. An ORF (yfeH) is located upstream of yfeR and in inverted orientation (Fig. 3a). The yfeH gene is predicted to encode a putative Na+-dependent transporter. To map the 5′ end of transcription of both genes a 5′RACE experiment was carried out. The nucleotide sequence of the 5′RACE products showed that transcription of yfeR and yfeH genes started at the adenosines located, respectively, 26 and 20 bp upstream of yfeR and yfeH genes translation start points (Fig. 3a). The −35 and −10 boxes for each promoter were bioinformatically determined. The 89-bp yfeR-yfeH intergenic region (Fig.