Studies on homoserine lactone and its antagonists which regulate the expression of virulence genes in Pseudomonas aeruginosa

1)铜绿假单胞菌自身诱导物生物测定在铜绿假单胞菌中，Las R蛋白是激活几种毒力基因所必需的。铜绿假单胞菌（P.aeruginosa）产生的一种可扩散的信号分子--自诱导物（派）是lasR活性所必需的，它由细菌细胞产生并释放到培养基中。pCRlasI通过将PCR制备的lasI的启动子区（666 bp）插入pCRII的EcoR 1位点，提供了含有氨苄青霉素抗性标记的lacZ片段。将pMC 1871的（3.1Kb，Sal消化）插入pCRlasI（Sal 1位点），形成pCRlasI/lacZ（7.5Kb），其中插入了来自pKK 232-8的rrnBT（终止子，EcoR 1180 bp片段）。由此获得pCRTlasI/lacZ（7.7Kb）用XbaI消化并钝化，其中lasR插入来自pGEX-3X 1asR 3455的（NarI + TthuIII 1 2.4Kb片段）以形成最终质粒，pCRTlasI/lacZ/GSTlasR（10.2 Kb）.2）高丝氨酸内酯类似物的合成合成以下高丝氨酸内酯类似物用于在PAI测定系统中测定其激动剂或拮抗剂活性上述; N-癸酰基-DL-高丝氨酸内酯、N-癸酰基-DL-高半胱氨酸硫代内酯、N-十二酰基-环己胺、N-（2-酮丁酰基）-DL-高丝氨酸内酯、N-十二酰基-DL-高丝氨酸内酯、4-氧代-5-氮杂十一酰基-DL-高丝氨酸内酯、3-氧代-4-氮杂十二酰基-DL-高丝氨酸内酯。

1) Pseudomonas aeruginosa autoinducer bioassayIn 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 analoguesThe 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.