Typhimuriuma rec deletion Intrachromosomal recombination Plasmid integration   Strain Frequency (10 -5 ) Strain Frequency (10 -6 ) None χ9931 6.02 ± 0.38 χ9935 5.59 ± 0.94 ΔrecF126 χ9932 7.05 ± 1.40 χ9936 2.13 ± 0.60** ΔrecJ1315 χ9933 9.18 ± 2.18 χ9937 4.89 ± 0.41 ΔrecA62 χ9934 1.29 ± 0.51** χ9938b <0.00071** see more a Mean ± STD from 3-5 assays were shown in the table. b Upon introduction of pKD46 (30°C, 0.2% arabinose), the frequency was 6.41 ± 0.85 × 10-6 (P = 0.425). ** P < 0.01, relative to the parental rec + strain. To examine plasmid integration, the 5'tet gene was introduced into the S. Typhimurium chromosome at cysG. The resulting strains were transformed with plasmid pYA4464 (3'tet) (Figure

1B). The 789 bp of overlapping sequence between 5′tet on the chromosome and the 3′tet on the plasmid could result click here in

plasmid integration into the chromosome, generating an intact tetA gene (Figure 2B). Deletion of recA had a profound effect, reducing the integration frequency to less than 7 × 10-10, which was below the limits of detection in this assay (P < 0.01), indicating a strict requirement for RecA in this process. Introduction of plasmid pKD46, which encodes the λ Red recombinase, into χ9938 (ΔrecA) carrying pYA4464 restored the integration frequency to the level of the Rec+ strain χ9935. Deletion of recF reduced the frequency of integration less than 3-fold (P < 0.01; Table 4) and the ΔrecJ deletion had no effect. Effect of rec deletions on the virulence of S. Typhimurium BALB/c mice were orally inoculated with the highly virulent S. Typhimurium strain χ3761 and its rec mutant derivatives. The LD50s of χ3761, χ9070 (ΔrecF) Cytoskeletal Signaling inhibitor and χ9072 (ΔrecJ) were similar, 3.2 × 104, 6.8 × 104 and 1.5 × 105 CFU, respectively (Table 5). The LD50 of the ΔrecF ΔrecJ double mutant was approximately 100-fold higher than χ3761, at 2.2 × 106 CFU. All mice inoculated with 1.3 × 109 CFU of the ΔrecA mutant survived, indicating that the LD50 was > 1.3 × 109 CFU. Two months following the initial inoculation with the Δ recA mutant strain, surviving mice were challenged with either 1.5 × 108 or 1.5 × 109 CFU of wild-type strain χ3761. All mice survived

the challenge, indicating that Δ recA mutant strain χ9833 was both attenuated and immunogenic. Table 5 Virulence of S. Typhimurium rec mutants in BALB/c mice (oral inoculation) Strain rec deletion Dose (CFU) Survivor/total LD50 (CFU) χ3761 None 1.5 × 106 0/4 3.2 × 104     1.5 × 105 1/4       1.5 × 104 3/4       1.5 × 103 4/4   χ9070 ΔrecF126 1.0 × 107 0/4 6.8 × 104     1.0 × 106 1/4       1.0 × 105 1/4       1.0 × 104 4/4   χ9072 ΔrecJ1315 1.0 × 107 0/4 1.5 × 105     1.0 × 106 0/4       1.0 × 105 3/4       1.0 × 104 3/4   χ9081 ΔrecJ1315 Δ recF126 1.0 × 107 1/4 2.2 × 106     1.0 × 106 3/4       1.0 × 105 4/4       1.0 × 104 3/4   χ9833 ΔrecA62 1.3 × 109 10/10 >1.3 × 109 Discussion We began our studies using information gathered in E.