1In strains JE22070 and JE6692 the genotype preceding the forward slash is: metE205araB9.
2Unless otherwise indicated, all strains used in this study were generated during the work.
3S. Typhimurium = Salmonella entericasubsp. enterica sv. Typhimurium strain LT2.
Deletion strain construction. Primers used in this study were synthesized by Integrated DNA Technologies, Inc. (IDT, Coralville, IA), and are listed in Table 2. ThepagR1 ::kan + andtktDE1 ::cat + markers were engineered as follows: PFU Ultra II Fusion DNA polymerase (Stratagene) was used to amplify flanking regions of plasmids pKD3 (cat +marker) or pKD4 (kan + marker) using primers designed with 36 bp of overlapping region to the beginning or the end ofpagR or tktD-tktE genes. Polymerase chain reaction (PCR) amplicons were visualized by post-staining with ethidium bromide (0.5 mg /mL) for 10 min. Products were cleaned with the Wizard SV gel and PCR clean up kit (Promega), and ~200 ng of PCR product was electroporated into S. Typhimurium strain JE6692 (metE205araB9 / pKD46 bla +) using a 0.2-cm electroporation cuvette (MidiSci) and a microPulser electroporator (Bio-Rad Laboratories) on Ec2 setting. Cells were recovered by incubation at 37 ºC with shaking and plated on lysogeny broth (LB, Difco) agar supplemented with either 12.5 µg/mL of chloramphenicol for the cat + marker or 25 µg/mL of kanamycin for the kan + marker. Correct antibiotic insertions were PCR verified then moved by P22-mediated transduction into strain JE6583 as described elsewhere (Davis et al., 1980). Colonies were streaked to isolation and the genomes of isolated colonies were sequenced using Sanger sequencing technology to verify correct insertion location and sequence using primers flanking genes of interest.
Construction of pagR3::lacZY chromosomal fusion strain.The kanamycin resistance cassette was resolved from strain JE21107 (pagR1 ::kan +, yielding ΔpagR2 ) using pCP20 and a method described elsewhere (Datsenko and Wanner, 2000). The lacZY+ fusion was created by FLP mediated integration of pKG136 (Ellermeier et al., 2002). Briefly, pCP20 was transformed into the resolved ΔpagR strain (JE24887). Plasmid pKG136 was transformed into the resulting strain and cells were recovered overnight in LB at 37 ºC. The next morning cells were plated on LB agar with 50 µg/mL kanamycin to select for integration. CorrectlacZY+ kan+ insertions were PCR verified then moved by P22-mediated transduction into strain JE6583 using a described protocol (Davis et al., 1980).