Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstraße 3, 48149 Münster, Germany.
Appl Microbiol Biotechnol. 2013 Jan;97(2):751-60. doi: 10.1007/s00253-012-4236-4. Epub 2012 Jun 28.
The quorum sensing signalling molecules 2-heptyl-3-hydroxy-4(1H)-quinolone, termed the "Pseudomonas quinolone signal" (PQS), and 2-heptyl-4(1H)-quinolone (HHQ) play an important role in the control of virulence gene expression in Pseudomonas aeruginosa. To construct a bioreporter for the specific and sensitive detection of these compounds, a plasmid with the pqsR gene encoding the PQS- and HHQ-responsive transcriptional regulator PqsR, and with the PqsR-controlled pqsA promoter fused to the lacZ gene, was established in Pseudomonas putida KT2440. The bioreporter responds to HHQ and PQS at concentrations in the range of 0.1-10 and 0.01-5 μM, respectively, with EC(50) values of 1.50 ± 0.25 μM for HHQ and 0.15 ± 0.02 μM for PQS. 2,4-Dihydroxyquinoline, a metabolite produced abundantly by P. aeruginosa, did not elicit an increase in reporter enzyme activity. To test whether the bioreporter can be used for the detection of enzymes active on AQ signalling molecules, the hodC gene coding for 2-methyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase was expressed in the reporter strain. This dioxygenase catalyses the cleavage of PQS, albeit with very low activity. The response of the bioreporter to PQS was significantly quenched by co-expression of the hodC gene, and HPLC analysis of culture extracts verified that the PQS levels decreased during cultivation. The bioreporter can be applied to screen for AQ-converting enzymes, which will be useful tools to interfere with quorum sensing and thus virulence in P. aeruginosa.
群体感应信号分子 2-庚基-3-羟基-4(1H)-喹啉，称为“铜绿假单胞菌喹诺酮信号”（PQS）和 2-庚基-4(1H)-喹啉（HHQ）在铜绿假单胞菌毒力基因表达的调控中起着重要作用。为了构建一种用于这些化合物的特异性和灵敏检测的生物传感器，构建了一个质粒，其中包含编码 PQS 和 HHQ 反应性转录调节因子 PqsR 的 pqsR 基因，以及与 pqsA 启动子融合的 lacZ 基因，该启动子受 PqsR 控制，位于恶臭假单胞菌 KT2440 中。生物传感器对 HHQ 和 PQS 的响应浓度分别在 0.1-10 和 0.01-5 μM 范围内，HHQ 和 PQS 的 EC(50)值分别为 1.50±0.25 μM 和 0.15±0.02 μM。2,4-二羟基喹啉是铜绿假单胞菌大量产生的一种代谢产物，不会引起报告酶活性的增加。为了测试生物传感器是否可用于检测对 AQ 信号分子有活性的酶，在报告菌株中表达了编码 2-甲基-3-羟基-4(1H)-喹啉 2,4-二加氧酶的 hodC 基因。该双加氧酶虽然活性很低，但能催化 PQS 的裂解。hodC 基因的共表达显著抑制了生物传感器对 PQS 的响应，并且培养物提取物的 HPLC 分析证实了在培养过程中 PQS 水平下降。该生物传感器可用于筛选 AQ 转化酶，这将是干扰群体感应和因此铜绿假单胞菌毒力的有用工具。
