SYNTHESIS OF MULTIPLE EXOPRODUCTS IN PSEUDOMONAS-AERUGINOSA IS UNDER THE CONTROL OF RHLR-RHLI, ANOTHER SET OF REGULATORS IN STRAIN PAO1 WITH HOMOLOGY TO THE AUTOINDUCER-RESPONSIVE LUXR-LUXI FAMILY
SYNTHESIS OF MULTIPLE EXOPRODUCTS IN PSEUDOMONAS-AERUGINOSA IS UNDER THE CONTROL OF RHLR-RHLI, ANOTHER SET OF REGULATORS IN STRAIN PAO1 WITH HOMOLOGY TO THE AUTOINDUCER-RESPONSIVE LUXR-LUXI FAMILY
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DOI：
10.1128/jb.177.24.7155-7163.1995
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发表时间：
1995-12-01
影响因子：
3.2
通讯作者：
OHMAN, DE
中科院分区：
文献类型：
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作者：
BRINT, JM;OHMAN, DE
在化学诱变的幸存者中检测到铜绿假单胞菌PAO 1的突变体，其缺乏产生降解酪蛋白的蛋白酶的能力。一个这样的突变体（PDO 31）显示出弹性蛋白溶解活性、β-溶血活性和绿脓菌素的产生减少。从PAO 1的基因库中发现了一个4.3-kb的EcoRI片段，其补充了PDO 31中的缺陷，将克隆的转座子诱变和缺失衍生物与互补试验结合使用以确定目的基因的物理位置，核苷酸序列分析揭示了一个开放阅读框架（rhlR），其编码一个推定的27.6-kDa蛋白（RhlR），与自身诱导物-群体感应系统的响应调节因子，如费氏弧菌的LuxR和铜绿假单胞菌的LasR。rhlR下游的进一步序列分析揭示了一个独立转录的基因（rhlI），其编码一种推定的22.2 kDa蛋白质，与自诱导合成酶家族的成员具有同源性，如V的LuxI、Fischeri和铜绿假单胞菌的LasI。rhlRI序列最近也被其他人报道（U，A，Ochsner和J，雷泽，美国国家科学院学报，92：6424-6428，1995）作为铜绿假单胞菌PG 201中鼠李糖脂生物表面活性剂合成的自诱导物介导的调节机制。通过基因置换在PAO 1中构建具有rhlR或rhlI缺陷的突变体，使用经Tn 501插入修饰的克隆，与野生型相比，rhlR和rhlI突变体在弹性蛋白酶、LasA蛋白酶、鼠李糖脂和绿脓菌素的产生方面都表现出缺陷。用lasB-cat融合测量的弹性蛋白酶基因的转录表明，弹性蛋白酶的产生受到细胞密度的影响。PAO 1中的基因激活，然而，rhlI突变体中lasB-cat的转录，其已经失去了假定的自诱导物合成酶（预测激活Rh 1 R），显示出低的基础活性，并且已经失去了lasB的所有细胞密度依赖性转录。因此，Rh 1 R-Rh 1 I代表了在铜绿假单胞菌中发现的第二种自诱导物应答调节机制，其控制多种毒力因子外产物（包括弹性蛋白酶）的表达。
Mutants of Pseudomonas aeruginosa PAO1 that were deficient in the ability to produce proteases that degrade casein were detected among the survivors of chemical mutagenesis, One such mutant (PDO31) showed reduced production of elastolytic activity, beta-hemolytic activity, and pyocyanin, A 4.3-kb EcoRI fragment from a gene bank of PAO1 that complemented defects in PDO31 was found, Transposon mutagenesis and deletion derivatives of the clone were used in conjunction with complementation tests to determine the physical location of the gene of interest, Nucleotide sequence analysis revealed an open reading frame (rhlR) encoding a putative 27.6-kDa protein (RhlR) with homology to autoinducer-responsive regulators of quorum sensing systems such as LuxR of Vibrio fischeri and LasR of P. aeruginosa. Further sequence analysis downstream of rhlR revealed an independently transcribed gene (rhlI) that encodes a putative 22.2-kDa protein with homology to members of the family of autoinducer synthetases, such as LuxI of V, fischeri and LasI of P. aeruginosa. The rhlRI sequences were also recently reported by others (U, A, Ochsner and J, Reiser, Proc, Natl, Acad, Sci, USA 92: 6424-6428, 1995) as an autoinducer-mediated regulation mechanism for rhamnolipid biosurfactant synthesis in P, aeruginosa PG201, Mutants with defects in rhlR or rhlI were constructed in PAO1 by gene replacement, using clones modified by Tn501 insertion, Compared with the wild type, the rhlR and rhlI mutants both showed defects in the production of elastase, LasA protease, rhamnolipid, and pyocyanin, Transcription from the gene for elastase, as measured with a lasB-cat fusion, demonstrated that production of elastase was subject to cell density-dependent gene activation in PAO1, However, transcription of lasB-cat in the rhlI mutant, which had lost the presumptive autoinducer synthetase (predicted to activate RhlR), showed low basal activity and had lost all cell density-dependent transcription of lasB, Thus, RhlR-RhlI represent the second autoinducer-responsive regulatory mechanism found in P, aeruginosa that controls expression of multiple virulence factor exoproducts, including elastase.