|Budget Amount *help
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
The alternative sigma factor sigB is the master regulator of a lot of general stress and energy depletion in Staphylococcus aureus. We examined the role of rsbU and rsbV in the sigB locus (rsbU-rsbV-rsbW-sigB). To obtain rsbV knockout mutant, we used homologous recombination method. We constructed knockout plasmid, pEMVKO, by inserting rsbV, which was disrupted by CAT gene, into pSPT181 with TC-resistant gene in frame and then transformed by electroporation. After incubation at 42 C, we selected Cm-resistant and Tc-sensitive strains, which should be rsbV-knockout mutant, on replica plates containing Cm5 or Tc5. However, we failed to obtain the strain. The failure may attribute to the heat-dependent recombination. Next, we complemented intact sigB locus (rsbU-sigB) into RN6390, an rsbU mutant (11bp-depletion in rsbU), and EM1001, a sigB KO mutant, and named RN1002 and EM1002, respectively. When we injected these strains to the hairless mice, RN6390, EM1001, and EM1002 made subcutaneous abscess, which were similar to the ulcer. On the other hand, RN1002 showed the small nodular abscess. The abscess size of RN6390 tended to be larger than EM1001 (440mm^2 versus 370mm^2), but there is no statistical difference. EM1002 showed smaller than ant other strain (50mm^2). When exposed to heat after incubation inM9 minimal solution, RN1002 grew slower than any other strain. Our studies indicate that sigB locus including rsbU and rsbV contributes to the stress response and virulence of Staphylococcus aureus.