Pearson J P, Pesci E C, Iglewski B H
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
J Bacteriol. 1997 Sep;179(18):5756-67. doi: 10.1128/jb.179.18.5756-5767.1997.
Two quorum-sensing systems (las and rhl) regulate virulence gene expression in Pseudomonas aeruginosa. The las system consists of a transcriptional activator, LasR, and LasI, which directs the synthesis of the autoinducer N-(3-oxododecanoyl) homoserine lactone (PAI-1). Induction of lasB (encoding elastase) and other virulence genes requires LasR and PAI-1. The rhl system consists of a putative transcriptional activator, RhlR, and RhlI, which directs the synthesis of N-butyryl homoserine lactone (PAI-2). Rhamnolipid production in P. aeruginosa has been reported to require both the rhl system and rhlAB (encoding a rhamnosyltransferase). Here we report the generation of a delta lasI mutant and both delta lasI delta rhlI and delta lasR rhlR::Tn501 double mutants of strain PAO1. Rhamnolipid production and elastolysis were reduced in the delta lasI single mutant and abolished in the double-mutant strains. rhlAB mRNA was not detected in these strains at mid-logarithmic phase but was abundant in the parental strain. Further RNA analysis of the wild-type strain revealed that rhlAB is organized as an operon. The rhlAB transcriptional start was mapped, and putative sigma 54 and sigma 70 promoters were identified upstream. To define components required for rhlAB expression, we developed a bioassay in Escherichia coli and demonstrated that PAI-2 and RhlR are required and sufficient for expression of rhlA. To characterize the putative interaction between PAI-2 and RhlR, we demonstrated that [3H]PAI-2 binds to E. coli cells expressing RhlR and not to those expressing LasR. Finally, the specificity of the las and rhl systems was examined in E. coli bioassays. The las system was capable of mildly activating rhlA, and similarly, the rhl system partly activated lasB. However; these effects were much less than the activation of rhlA by the rhl system and lasB by the las system. The results presented here further characterize the roles of the rhl and las quorum-sensing systems in virulence gene expression.
两种群体感应系统（las和rhl）调控铜绿假单胞菌中毒力基因的表达。las系统由转录激活因子LasR和LasI组成，LasI指导自诱导物N-（3-氧代十二烷酰）高丝氨酸内酯（PAI-1）的合成。lasB（编码弹性蛋白酶）和其他毒力基因的诱导需要LasR和PAI-1。rhl系统由一种假定的转录激活因子RhlR和RhlI组成，RhlI指导N-丁酰高丝氨酸内酯（PAI-2）的合成。据报道，铜绿假单胞菌中鼠李糖脂的产生需要rhl系统和rhlAB（编码一种鼠李糖基转移酶）。在此，我们报道了PAO1菌株的lasI缺失突变体以及lasI rhlI双缺失突变体和lasR rhlR::Tn501双突变体的构建。在lasI单突变体中鼠李糖脂的产生减少，而在双突变体菌株中则完全消失。在对数中期这些菌株中未检测到rhlAB mRNA，但在亲本菌株中其含量丰富。对野生型菌株的进一步RNA分析表明，rhlAB是作为一个操纵子组织的。确定了rhlAB的转录起始位点，并在其上游鉴定出假定的σ54和σ70启动子。为了确定rhlAB表达所需的成分，我们在大肠杆菌中开发了一种生物测定法，并证明PAI-2和RhlR是rhlA表达所必需且足够的。为了表征PAI-2与RhlR之间假定的相互作用，我们证明[3H]PAI-2与表达RhlR的大肠杆菌细胞结合，而不与表达LasR的细胞结合。最后，在大肠杆菌生物测定中检测了las和rhl系统的特异性。las系统能够轻度激活rhlA，同样，rhl系统能部分激活lasB。然而，这些效应远小于rhl系统对rhlA的激活以及las系统对lasB的激活。本文给出的结果进一步表征了rhl和las群体感应系统在毒力基因表达中的作用。
