| Pseudomonas aeruginosa is an opportunistic gram negative bacillus that grows ubiquitously,can cause serious infections in patients with compromised immunity,cystic fibrosis sufferers.The pathogen is also the third most common pathogen causing hospital-aquired infections,and it can infect both plants and animals.Additionally, treatment of such infections is rather difficult due to strong innate antibiotic and acquired antibiotic resistance. Therefore,investigation of the regulatory mechanisms that govern the pathogenicity and antimicrobial resistance for designing and developing new strategies to prevent and treat of infections caused by Pseudomonas aeruginosa has become extremely important.Discovery of quorum sensing(QS) provides a new way to study the regulation mechanisms of pathogenic micro-organisms. QS is a cell to cell communication mechanism widely conserved in bacteria,and with which bacteria coordinate community activities and virulence. It has become obvious that QS holds a key for the control and prevention of bacterial infections. Pseudomonas aeruginosa employs a complex QS network consisting of las,rhl,pqs and newly discovered IQS systems to coordinate their virulence gene expression. IQS system connects the central las sysyem and phosphate-stress response mechanism to the down stream pqs and rhl regulatory systems. IQS system is strictly controlled by las system under phosphate rich conditions,but also activated by phosphate deletion condition,which is a common stress signal during bacterial infection.Disruption of IQS biosynthesis paralyses the pqs and rhl QS systems and attenuates bacterial virulence.Production of IQS signal is tightly controlled by las under normal culture conditions but also activated by phosphate limitation. Although IQS signal has been successfully purified and structurally characterized,but the signaling mechanisms of IQS systemhas not yet been characterized. Further investigation of the IQS regulatory mechanisms is important to identify new targets for treatment of the infections by P. aeruginosa. This study focuses on the IQS regulatory mechanisms,the main work includes the following two parts:First,construction of a Tn5 insertion mutant library of P. aeruginosa strain PAO1. Under low phosphate condition,the mutant library was screened by using pyocyanin as selection marker,which led to identify a mutant,named as Pch996,showing decreased pyocyanin production out from 37,189 transposon mutants. Analysis of the transposon disrupted sequence of the mutant Pch996 by using hi TAIL-PCR and DNA sequencing led to identify an ORF PA5295,which is 1677 bp and encodes a peptide with 558 amino acids. Domain analysis showed that the PA5295 protein comprises two functional GGDEF and EAL domain. Compared to wild type PAO1,the mutant pyocyanin yield was decreased by about 35%,which was restored by complementation with the wild type PA5295 gene.Sequence alignment showed that the gene shares about 79% identies with a diguanylate cyclase of Pseudomonas nitroreducens and 77% identities with a diguanylate cyclase of Pseudomonas mendocina. The Tn5 disruption not only influenced the pyocyanin yield,but also affected biofilm formation,ability to kill nematodes,and swarming motility.Second,based on previous results and bioinformatics prediction,we further characterized three potentially IQS regulated genes,i.e., PA3192,PA3271,and PA4596. PA3192 encodes a response regulator of two-component system,PA3271 encodes a sensor of two-component system,PA4596 encodes a transcriptional regulator. The corresponding deletion mutants were obtained by gene knock-out. Phenotypic analysis showed that knocking out PA3192 and PA3271 influenced pyocyanin production, biofilm formation, swimming and swarming motility,and the capability of nematode fast killing,but did not influence the elastase production;whereas knocking out PA4596 influenced pyocyanin production,capability of nematode fast killing,but did not influence biofilm formation and the elastase production. RT-q PCR analysis showed that the transcriptional expression of las,rhl,pqs and IQS systems related genes were also affected by deletion of the three genes described above.In summary,through transposon mutagenesis and targeted gene disruption,this study identified four regulatory genes potentially associated with the IQS signaling pathway. The findings from this study provide new data for understanding the P. aeruginosa QS signaling pathway,which would be usful clues and framework for further characterization of the regulatory mechanisms and signaling pathways that control P. aeruginosa pathogenicity and virulence. |
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