The resulting PCR product AZD8186 datasheet was digested with isocaudarner SpeI and XbaI and ligated into XbaI-digested pRE112 to yield plasmid pYA4680. In addition, undigested, agarose-gel purified PCR product was electroporated into the cat-sacB Salmonella strains carrying plasmid pKD46 and spread onto LB plates containing 5% sucrose to select for

deletion of the cat-sacB cassette. Chloramphenicol-sensitive isolates were verified as ΔrecA62 by PCR using primers P15 and P16 (ΔrecA62: 1360 bp; wt: 2412 bp). S. Typhimurium strains χ9833 and χ9939 were constructed by this method (Table 2). For construction of a ΔrecA62 GANT61 order mutant of S. Typhi, wild-type strain Ty2 was mated with E. coli strain χ7213(pYA4680). Transconjugants were selected on LB plates containing chloramphenicol, followed by counterselection on sucrose plates as described above. The resulting ΔrecA62 strain was designated χ11159. The S. Paratyphi A strain χ11243 was generated from wild-type strain χ8387 using the same strategy. The ΔrecF deletion strains were constructed using suicide vectors pYA3886 and pYA4783. From the S. Typhimurium

chromosome, a 397-bp sequence upstream Selleckchem Bucladesine of the recF gene was amplified with primers P17 and P18, which were engineered with XbaI and KpnI sites, respectively. The downstream 296-bp sequence (including 78 bp from the 3′ ORF of recF) was amplified with primers P19 and P20 containing KpnI and SphI sites, respectively. The two fragments were digested and inserted into XbaI-SphI digested pRE112, resulting in plasmid pYA3886. The corresponding deletion was designated ΔrecF126. Strains χ9070, χ9081 and χ11244 were generated by conjugation using E. coli strain χ7213(pYA3886). Phage P22HTint mediated transduction was used to construct Typhi strain χ11053 [56]. The ΔrecF126 deleted 996 bp from the 5′end of recF in serovars Typhimurium and Paratyphi. The upstream flanking sequence of S. Typhi is different with the other serotypes. To construct a serovar Typhi-specific Casein kinase 1 ΔrecF mutation, we constructed a new suicide vector. The recF upstream flanking sequence in plasmid

pYA3886 was replaced with the corresponding DNA sequence (447 bp) from S. Typhi Ty2. Primers P21 and P22 were used for this modification. The resulting plasmid was designated as pYA4783. The Typhi-specific ΔrecF1074 mutation was introduced into S. Typhi strains ISP1820 and Ty2 by conjugation with E. coli strain χ7213(pYA4783) to yield strains χ11133 and χ11134, respectively. Primers P23 and P24 were used to verify the recF126 and recF1074 deletions. Similar strategies were used to construct the Δ recJ1315 deletion with suicide vector pYA3887. From the S. Typhimurium chromosome, 330 bp upstream of the recJ gene was amplified with primers P25 and P26, which were engineered with XbaI and KpnI sites, respectively. The 299-bp downstream sequence was amplified with primers P27 and P28, engineered with KpnI and SphI sites, respectively.