Growth phase-differential quorum sensing regulation of anthranilate metabolism in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas quinolone signal (PQS) plays a role in the regulation of virulence genes and it is intertwined in the las/rhl quorum sensing (QS) circuits of Pseudomonas aeruginosa. PQS is synthesized from anthranilate by pqsA-D and pqsH whose expression is influenced by the las/rhl systems. Since anthranilate can be degraded by functions of antABC and catBCA, PQS synthesis might be regulated by the balance between the expression of the pqsA-D/phnAB, pqsH, antABC, and catBCA gene loci. antA and catA are repressed by LasR during log phase and activated by RhlR in late stationary phase, whereas pqsA-E/phnAB is activated by LasR in log phase and repressed by RhlR. QscR represses both but each repression occurs in a different growth phase. This growth phase-differential regulation appears to be accomplished by the antagonistic interplay of LasR, RhlR, and QscR, mediated by two intermediate regulators, AntR and PqsR, and their cofactors, anthranilate and PQS, where the expressions of antR and pqsR and the production of anthranilate and PQS are growth phase-differentially regulated by QS systems. Especially, the anthranilate level increases in an RhlR-dependent manner at late stationary phase. From these results, we suggest that RhlR and LasR regulate the anthranilate metabolism in a mutually antagonistic and growth phase-differential manner by affecting both the expressions and activities of AntR and PqsR, and that QscR also phase-differentially represses both LasR and RhlR functions in this regulation.     

The YebC family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production

Bacterial pathogenicity is often manifested by the expression of various cell-associated and secreted virulence factors, such as exoenzymes, protease, and toxins. In Pseudomonas aeruginosa, the expression of virulence genes is coordinately controlled by the global regulatory quorum-sensing systems, which includes the las and rhl systems as well as the Pseudomonas quinolone signal (PQS) system. Phenazine compounds are among the virulence factors under the control of both the rhl and PQS systems. In this study, regulation of the phzA1B1C1D1E1 (phzA1) operon, which is involved in phenazine synthesis, was investigated. In an initial study of inducing conditions, we observed that phzA1 was induced by subinhibitory concentrations of tetracycline. Screening of 13,000 mutants revealed 32 genes that altered phzA1 expression in the presence of subinhibitory tetracycline concentrations. Among them, the gene PA0964, designated pmpR (pqsR-mediated PQS regulator), has been identified as a novel regulator of the PQS system. It belongs to a large group of widespread conserved hypothetical proteins with unknown function, the YebC protein family (Pfam family DUF28). It negatively regulates the quorum-sensing response regulator pqsR of the PQS system by binding at its promoter region. Alongside phzA1 expression and phenazine and pyocyanin production, a set of virulence factors genes controlled by both rhl and the PQS were shown to be modulated by PmpR. Swarming motility and biofilm formation were also significantly affected. The results added another layer of regulation in the rather complex quorum-sensing systems in P. aeruginosa and demonstrated a clear functional clue for the YebC family proteins.     

假单胞菌株M18中pqsA突变株的构建及其对plt的调控

【目的】根际铜绿假单胞菌M18能产生藤黄绿菌素(Plt)和吩嗪-1-羧酸(PCA)两种主要的抗生素。其PqsR/PQS群体感应系统由应答调控蛋白PqsR与信号分子PQS组成。前期研究已经表明pqsR负调控Plt生物合成及基因簇表达。本论文旨在研究PQS分子对Plt合成及基因表达的调控作用。【方法】从M18基因组中扩增PQS合成基因pqsA,通过同源重组技术构建假单胞菌M18的pqsA突变菌株M18pqsA。利用lacZ报告基因分析、信号分子添加实验等,研究PQS对Plt合成及基因表达的调控作用。【结果】在KMB培养基中,分别比较野生型菌株M18和突变菌株M18pqsA的Plt产量,突变菌株的Plt产量存在较小幅度的升高,约为野生型菌株的1.53倍。添加PQS对plt表达存在一定程度但不是很显著的负调控作用。【结论】PQS分子对Plt生物合成及基因表达存在部分负调控作用。     

Identification of a novel regulator of the quorum-sensing systems in Pseudomonas aeruginosa

Quorum sensing is a global gene-regulatory mechanism in bacteria that enables individual bacterial cells to communicate and coordinate their population behaviors. Quorum sensing is central to the pathogenesis of many bacterial pathogens including Pseudomonas aeruginosa and therefore has been exploited as a target for developing novel antipathogenic drugs. In P. aeruginosa , three intertwined quorum-sensing systems, las, rhl , and the 2-alkyl-4(1 H )-quinolone system, which includes the Pseudomonas quinolone signal (PQS), control virulence factor production, and pathogenesis processes. Previously, we obtained a mutant with diminished expression of the phzA1B1C1D1E1F1G1 operon that is involved in the production of virulence factor phenazine compounds. In this study, the mutant was further characterized, and evidence indicating that the disrupted gene PA1196 in the mutant is a potential regulator of the rhl and PQS systems is presented. PA1196 positively controls the expression of the rhl and PQS systems and affects bacterial motility and multiple virulence factor expression via the quorum-sensing systems. This adds an important new player in the complex quorum-sensing network in P. aeruginosa .     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

Interspecies communication between Burkholderia cepacia and Pseudomonas aeruginosa

Burkholderia cepacia and Pseudomonas aeruginosa are opportunistic pathogens that commonly cause pulmonary infections in cystic fibrosis patients and occasionally co-infect patients' lungs. Both organisms possess quorum-sensing systems dependent on N-acyl homoserine lactone (N-acyl-HSL). Cross-feeding assays demonstrated that P. aeruginosa and B. cepacia were able to utilize heterologous N-acyl-HSL signaling molecules. The ability of quorum-sensing genes from one species to complement the respective quorum-sensing mutations in the heterologous species was also examined. These studies suggest that B. cepacia CepR can use N-acyl-HSLs synthesized by RhlI and LasI and that P. aeruginosa LasR and RhlR can use N-acyl-HSLs synthesized by CepI. It is possible that a mixed bacterial population of B. cepacia and P. aeruginosa can coordinately regulate some of their virulence factors and influence the progression of lung disease due to infection with these organisms.