, 1985), different virulence factors (Mekalanos, BIBF 1120 1992; Bajaj et al., 1996) and several other genes (Weber et al., 2006; Gunasekera et al., 2008). Hitherto, most of the well-characterized osmoregulated genes correspond to genes that are upregulated following an osmotic upshift (Cairney et al., 1985; Han et al., 2005; Weber et al., 2006; Gunasekera et al., 2008). Nevertheless, adaptation

to low-osmolarity conditions must also result in regulation of genes that are specifically required to cope with these conditions. In this work we designed a genetic strategy focused on identifying genes that are optimally expressed at low osmolarity in Salmonella enterica serovar Typhimurium (S. Typhimurium). We report here the identification of a novel LysR-type transcriptional regulator (LTTR) that shows osmolarity-dependent expression. Bacterial strains, plasmids and phages used are listed in Table 1. Cells were routinely grown in Luria–Bertani (LB) medium. For some experiments, LB was modified by adding NaCl up to 0.5 M (LB 0.5 M NaCl) or by not including NaCl (LB 0 M NaCl). When required, X-Gal (40 μg mL−1) was added to the culture medium. Antibiotics were used at the following concentrations: kanamycin

(Km) 50 μg mL−1; ampicillin (Ap) 25 and 50 μg mL−1; tetracycline (Tc) 15 μg mL−1. The growth temperature was 37 °C unless noted otherwise. To obtain phage-free isolates, transductants were purified by streaking on EBU plates (LB agar PD0325901 molecular weight supplemented with 0.25% glucose, 0.25% KH2PO4, 12.5 mg L−1 Evans Blue and 25 mg L−1

fluorescein). Restriction digestion, ligation, transformation, agarose gel electrophoresis and DNA manipulations were performed using standard procedures. For plasmid DNA preparations, the Wizard® Plus SV Minipreps kit (Promega) was used. DNA was recovered Hydroxychloroquine clinical trial from agarose gels by electroelution or Qiaquick® gel extraction kit (Qiagen). The Wizard® Clean-Up System (Promega) was used for purification of DNA fragments. PCR experiments were performed in the Perkin Elmer GeneAmp PCR System 2400 according to standard protocols, using DynaZyme™ (Finnzyme). Oligonucleotides used are listed in Table 1. DNA sequencing reactions were carried out according to the instructions of the BigDye® Terminator v3.1 Cycle Sequencing Kit from Applied Biosystems. A lysate of P22HTint4 phage grown on S. Typhimurium strain TT10288 (hisD9953∷MudJ) was used to transduce strain TT1704, selecting Km resistance (Kmr). The recipient strain carried the nontransducible deletion his-9953, which avoids homologous recombination with MudJ from donor lysate. To identify the gene in which MudJ was inserted, Sau3A-partially digested TT1704-OS chromosomal DNA was ligated with BglII-digested cosmid pLA2917. The ligation was packed following instructions from Gigapack III (Stratagene) and used to infect E. coli HB101.