Staphylococcus aureus strains were aerobically cultured in tryptic soy broth at 37 °C, and 12.5 μg mL−1 chloramphenicol, 50 μg mL−1 kanamycin or 1 μg mL−1 tetracycline was added to maintain the chromosomally integrated plasmids. Bacterial strains and plasmids www.selleckchem.com/products/poziotinib-hm781-36b.html used in this study are listed in Table 1. Transformation of S. aureus with plasmids was performed by electroporation (Schenk & Laddaga, 1992). Phage transduction was performed using phage 80α (Novick, 1991). Transformation of E. coli, extraction of plasmid DNA, PCR and Southern blot hybridization were performed according to Sambrook & Russell (2001). Genomic DNA from S. aureus cells was extracted using a QIAamp DNA Blood Kit

(Qiagen) after digestion of cell-wall components with lysostaphin. The S. aureus gene was disrupted by the integration of a suicide vector into a chromosome by single cross-over homologous recombination (Kaito et al., 2005). The internal region of the target Linsitinib research buy ORF near the translation initiation site was amplified by PCR using primer pairs (Table 2) and NCTC8325-4 genomic DNA as template. The amplified DNA fragment was cloned into the multi-cloning site of pCK20 or pSF151, resulting in targeting vectors. Staphylococcus aureus

RN4220 was transformed with the targeting vector, resulting in the gene-disrupted mutant of RN4220. The gene disruption was transferred to strain NCTC8325-4 using phage 80α, resulting in the gene-disrupted

mutant of NCTC8325-4. The gene disruption was confirmed by Southern blot hybridization analyses (Supporting Information, Fig. S1). To delete the psmα and psmβ operons, the deletions in strain RN4220 (Kaito et al., 2011b) were transferred to NCTC8325-4 using phage 80α, resulting in M0406-7 and M1056-7. Silkworms were raised from fertilized eggs at 27 °C in an air incubator (MIR-554; Sanyo Electric Co., Tokyo, Japan) (Kaito Florfenicol et al., 2002, 2005). The fertilized eggs were purchased from Ehime Sansyu Co. (Ehime, Japan). Hatched larvae were fed an artificial diet (Silkmate 2S; Nosan Corp., Kanagawa, Japan). Fifth instar larvae were fed an antibiotic-free artificial diet (Katakura Industries Co., Ltd, Tokyo, Japan) for 1 day and injected with serial dilutions of S. aureus overnight cultures using a 1-mL syringe equipped with a 27G needle and maintained at 27 °C in a safety cabinet (Airtech Japan). Silkworms that did not move when picked up with a platinum loop at 24 h after the injection were confirmed dead. We injected silkworms with twofold serial dilutions of overnight culture of NCTC8325-4 and monitored the survival of silkworms at 24 h after the injection. The lethal dose (50%; LD50) of NCTC8325-4 was 1 × 107 CFU (Table 4), identical to that of strain RN4220 (Kaito et al., 2005), indicating no difference between these two strains in their silkworm killing abilities.