|Budget Amount *help
¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥2,100,000 (Direct Cost : ¥2,100,000)
To investigate the mechanisms of the production of protease and hemolytic substance, cepalycin, produced by Burkholderia cepacia, we analyzed Tn5-generated protease-negative mutant and cepalycine-negative mutant derived from strain KF1 and strain JN106.
1. A protease-negative and lipase-positive mutant of KF1 was complemented by a clone having an open reading frame coding a 212-amino acid polypeptide which showed homology to Escherichia coli periplasmic disulfide bond oxidoreductase, DsbA.
2. The DsbA mutant showed the following phenotypes. (1)defective in the production of extracellular protease and alkaline phosphatase, (2)repression of growth, (3)defective in motility, (4)increase in sensitivity to CdィイD12+ィエD1 and ZnィイD12+ィエD1, (5)increase in sensitivity to a variety of antibiotics including β-lactams and sodium dodecyl sulfite.
3. Several protease- and lipase-negative mutants of KF1 were analyzed. The region complementing protease production of these mutants was located in a 9.9kb fragment. Analysis of the nucleotide sequence revealed gspD, -E, -F, -C, -G, -H, -I, -J, -K and gspL which encode for components involved in a type II secretion system, a Sec-dependent general secretory pathway of gram-negative bacteria. The gsp genes were presumed to be organized in three operons consisting of gspD-E-F, gspC(in opposite direction) and gspG-H-I-J-K-L.
These results suggested that B. cepacia protease is secreted by a type II secretion system and requires the disulfide oxidoreduction system for production/secretion.
4. A cepalycin- and protease-deficient mutant of JN106 had a mutation in an open reading frame for a 327-amino acid polypeptide belonging to LysR family proteins. A typical helix-turn-helix motif was found in the N-terminal region. Protease activity and cepalycin activity were restored when the lysR family gene termed cviR(B. cepacia virulence regulator) on a plasmid, suggesting that the cviR gene is involved in the production of both protease and cepalycin.