Brint J M, Ohman D E
Department of Medicine, University of Tennessee, Memphis, USA.
J Bacteriol. 1995 Dec;177(24):7155-63. doi: 10.1128/jb.177.24.7155-7163.1995.
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.
在化学诱变的存活菌株中检测到了铜绿假单胞菌PAO1的突变体，这些突变体缺乏产生能降解酪蛋白的蛋白酶的能力。其中一个这样的突变体（PDO31）表现出弹性蛋白酶活性、β-溶血活性和绿脓菌素的产量降低。从PAO1的基因文库中发现了一个4.3 kb的EcoRI片段，它能互补PDO31的缺陷。利用转座子诱变和该克隆的缺失衍生物，结合互补试验来确定目标基因的物理位置。核苷酸序列分析揭示了一个开放阅读框（rhlR），它编码一种假定的27.6 kDa蛋白（RhlR），与群体感应系统的自诱导物响应调节因子具有同源性，如费氏弧菌的LuxR和铜绿假单胞菌的LasR。对rhlR下游的进一步序列分析揭示了一个独立转录的基因（rhlI），它编码一种假定的22.2 kDa蛋白，与自诱导物合成酶家族的成员具有同源性，如费氏弧菌的LuxI和铜绿假单胞菌的LasI。其他人（U.A. Ochsner和J. Reiser，《美国国家科学院院刊》92: 6424 - 6428，1995）最近也报道了rhlRI序列是铜绿假单胞菌PG201中鼠李糖脂生物表面活性剂合成的一种自诱导物介导的调节机制。通过基因置换，利用经Tn501插入修饰的克隆，在PAO1中构建了rhlR或rhlI缺陷的突变体。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均表现出缺陷。用lasB - cat融合体测量弹性蛋白酶基因的转录，结果表明PAO1中弹性蛋白酶的产生受细胞密度依赖性基因激活的调控。然而，在rhlI突变体中，lasB - cat的转录显示出低基础活性，并且失去了lasB所有的细胞密度依赖性转录，该突变体已失去了假定的自诱导物合成酶（预计可激活RhlR）。因此，RhlR - RhlI代表了在铜绿假单胞菌中发现的第二种自诱导物响应调节机制，它控制多种毒力因子外毒素产物的表达，包括弹性蛋白酶。
