A kanamycin resistance cassette from pACYC177 was amplified using primers kana1 and kana2

(Table 1) and then cloned into the ApaI-XbaI site of the pYG1 to generate pYG2. The sacB gene of pYLTAC7 was removed by EcoRI-restriction, generating a 1.7-kb check details fragment. Then, the sacB-containing fragment was cloned into the EcoRI site of the pYG2 resulting in pYG3. Finally, the vector pYG3 was digested by ApalI to remove the ampicillin resistance and was self-ligated to create the final plasmid pYG4. As described by Link et al. (1997), the 2067-bp in-frame deletion of the yncD gene was constructed by cross-over PCR with primers k1, k2, k3 and k4 (Table 1). The product was ligated directly to the pMD18-T vector (Takara Co., Dalian, China)

and confirmed by sequencing. The recombinant plasmid was digested by NdeI and the fragment containing the deletion copy of the yncD gene was ligated to pYG4. The resulting vector was introduced into E. coli S17-1/λpir by electroporation. The hybrid plasmid was transferred into YGC101 (wild type) by electroporation to perform mutagenesis. Selleckchem FK866 Integrons were selected from the LB plates containing kanamycin and were confirmed through PCR analysis. Overnight cultures of the identified integron grown in the absence of antibiotics were streaked onto LB agar containing 5% sucrose. Selected colonies with normal colony phenotypes were patched onto LB agar with and without kanamycin. The colonies that were sensitive to kanamycin were analyzed for the deletion by PCR with the primers O1 and O2, as well as I1 and I2 (Table 1). The strain carrying the desired deletion was selected

and designated as YGC102. The gene yncD was PCR-amplified from the wild-type strain using the primers C1 and C2 (Table 1), which were designed based on sequences external to the yncD coding region. After amplification, the DNA fragment was digested by EcoRI and HindIII and ligated to the pBR322 to obtain PYN plasmid. The resulting vector was introduced into the mutant strain YGC102 by electroporation to produce the strain YGC103. To determine the involvement of yncD in virulence, 3-mercaptopyruvate sulfurtransferase the median lethal dose (LD50) of YGC101, YGC102 and YGC103 was determined as described by Wang et al. (2001) with minor modifications. Female BALB/c mice aged 6–8 weeks (three mice per group, three groups per strain) were injected intraperitoneally with various dilutions of the different strains mixed with 7% (w/v) mucin from porcine stomach (Sigma) at a final volume of 0.5 mL in phosphate-buffered saline (PBS). The number of deaths that occurred within 72 h after inoculation was counted. The LD50 was calculated as described by Reed & Muench (1938). To evaluate the effect of yncD gene deletion on the survival capability in vivo, we performed bacterial competition experiments in the mouse model.