1In strains JE22070 and JE6692 the genotype preceding
the forward slash is: metE205 ∆araB9.
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 (metE205∆araB9 / 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).