|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
1) Pseudomonas aeruginosa autoinducer bioassay
In Pseudomonas aeruginosa the Las R protein is required for activation of several virulence genes. A diffusible signal molecule, the P.aeruginosa autoinducer (PAI), produced by the bacteiial cell and released into the growth medium, is required for activity of lasR.By cloning a lasB : lacZ fusion and lasR gene under control of the lac promoter in Escherichia coli, we have developed a quantitative bioassay for PAI.The plasmid used (pCRTlasI/lacZ/GSTlasR, 10.2 Kb) was constructed, as follows. The pCRlasI (4.4 Kb), containing an ampicillin-resistance marker, was provided by insertion of the promoter region of lasI (666 bp) prepared by PCR into EcoR1 site of pCRII The lacZ fragment (3.1 Kb, Sal digest) of pMC 1871 was inserted in the pCRlasI (Sal1 site) to form the pCRlasI/lacZ (7.5 Kb), in which rrnBT (terminator, EcoRl 180 bp fragment) from pKK 232-8 was inserted. Thus obtained pCRTlasI/lacZ (7.7 Kb) was digested with Xbal and blunted, in which lasR (NarI + TthuIII1 2.4 Kb fragment) from pGEX-3X1asR3455 was inserted to form the final plasmid, pCRTlasI/lacZ/GSTlasR (10.2 Kb).
2) Synthesis of homoserine lactone analogues
The following homoserine lactone analogues were synthesized for determination of their agonist or antagonist activities in the PAl assay system mentioned above ; N-Decanoyl-DL-homo-serine lactone, N-Decanoyl-DL-homocystein thiolactone, N-Dodecanoyl-cyclohexylamine, N-(2-ketobutanoyl)-DL-homoserine lactone, N-Dodecanoyl-DL-homoserine lactone, 4-Oxo-5-azaundecanoyl-DL-homoserine lactone, 3-Oxo-4-azadodecanoyl-DL-homoserine lactone.