摘要
荧光假单胞菌2P24的PcoI-PcoR群体感应(QS)系统信号合成基因pcoI的表达受多种因子的调控,其中GacS-GacA双因子调控系统在转录水平正调控信号合成基因pcoI的表达。为进一步研究QS系统调控因子,将2P24基因组文库转入gacA缺失的pcoI基因转录报告菌株PM203(pcoI-lacZ,gacA?),筛选可提高pcoI表达的基因。结果表明粘粒pP32-24可显著提高pcoI转录水平,亚克隆实验证明其中的功能基因为pcoI;外源添加标准信号分子3-氧-己酰高丝氨酸内酯(3-oxo-C8-HSL)同样可显著提高pcoI基因的表达,表明pcoI基因的表达对自身有正调控作用。同时构建了QS系统的另外一个组分pcoR基因的缺失突变体,pcoR基因缺失后pcoI的表达和N-乙酰高丝氨酸内酯信号分子(AHL)的产量明显低于野生菌株及其互补菌株,并显著降低该菌株的生物膜(Biofilm)形成能力。这些结果表明菌株2P24的PcoI-PcoRQS系统中,信号合成基因pcoI的表达受自体反馈调控,pcoR基因参与pcoI基因表达的调控以及生物膜的形成。
PcoI-PcoR is a quorum-sensing （QS） system influencing the biofilm formation and rhizosphere colonization in Pseudomonas fluorescens 2P24. The expression of the pcoI, a N-acyl-homoserne lactone （AHL） biosynthase gene, is under the regulation of a number of chromosomal factors, such as the GacS-GacA two-component system. In this paper, we investigated the upstream regulators that influence the transcription of pcoI gene using a chromosomal pcoI-lacZ fusion reporter strain PM203. Cosmids containing genomic DNA of the wild-type strain 2P24 were introduced into the reporter strain PM203 （gacA—, pcoI-lacZ） to screen positive transcriptional regulators of pcoI gene. One of them named pP32-24, which contained a 5-kb Pst I functional fragment was selected. Further analysis identified that the pcoI was the gene responsible for the increase of the pcoI-lacZ expression. The expression of pcoI-lacZ reporter was also improved in both PM101 （pcoI-lacZ） and its gacA- mutant PM203 after addition of exogenous AHL, indicating that the expression of pcoI is positively regulated by AHL （autoinduction） in strain 2P24. In addition, deletion mutagenesis and complementation experiments demonstrated that the transcriptional regulator PcoR positively controlled the expression of pcoI and the formation of biofilm. These results suggest that, in strain 2P24, the expression of PcoI-PcoR QS system is auto-inducted, and the transcriptional factor PcoR is involved in the regulation of pcoI transcription and the biofilm formation.
出处
《微生物学通报》
CAS
CSCD
北大核心
2009年第7期981-987,共7页
Microbiology China
基金
国家自然科学基金项目(No.30671403
30871666)
国家863计划项目(No.2006AA10A211)
中澳MOST-DEST合作项目(No.2007DFA31570)
