Effect Of Phosphate-depletion On Pseudomonas Aeruginosa Quorum Sensing System And Study Of Relatedtranscriptional Regulator

Pseudomonas aeruginosa,a Gram- negative bacteria, is widely distributed in nature.It can be found in the water?air?soil, and even on the animal and human's skin?digestive tract?respiratory? urinary and intestinal tract.It can cause the incidence of infection in animals and humans throughtout the year. Pseudomonas aeruginosa has become a serious concern treateninghuman health and aquaculture industry. Quorum sensing(QS) system is a cell-cell communication mechanism. Bacterial cells produce, releaseand respond to small diffusible signal mo lecules to coordinate gene expression and groupbehaviors.When the accumulated number of the signal molecules reach a certain threshold, sgnals bind to a receptor protein to induce the expression of related genes.In Pseudomonas aeruginosa, the QS network contains four systems:Las, Rhl, PQS and IQS.Among them,IQS can be activatedby phosphate-depletion stress, and is controlled by Las and Pho B systems.Phosphate depletion is a common stress encountered by the patients undergone surgical operations, and the stress is frequently associated with severe microbial infections, including those caused by P. aeruginosa.This thesis aims to study the influence of phosphate stress on the IQS quorum sensing system of Pseudomonas aeruginosa. The IQS signal was detected by HPLC analysis.Then, the related regulatory genes, unveiled by transcriptome analysis,were knocked out to determine their roles in bacterialphysiology.The findings may provide clues for futher understanding the regulatorymechanisms and signaling networks of IQS quorum sensing system.HPLC analysis showed that IQS was affected by phosphate concentration.Transcriptome analysis identified two regulatory genes, i.e PA3045, PA2665. PA3045 encodes a response regulator of a two-component system, and its corresponding sensorykinase gene is PA3044. PA2665, located at the upstream of the gene for flavinhemoglobin(fhp), encodes an anaerobic nitric oxide reductase transcriptional regulator.It can mediate the NO-sensing regulation of fhppromoter, therefor regulating the expression of fhp gene.To investigate the specific functions of PA3044, PA3045 and PA2665 inregulation of bacterial pathogenesis, we constructed the mutants by in- frame deletion andperformed phenotype analysis. The results of physiology and biochemistry bioassays showed thatknocking out PA3044 did not cause noticible phenotype changes. Whereas, the deletion mutants of PA3045 and PA2665 showed enhance biofilm formation, and increased production of AHL signal, elastase and pyocyanin, compared with the wild type strain PAO1. In contrast, the motility of ?3045, ?2665, ?pho B, ?las R were less than that of the wild-type strain PAO1.It has seemed that PA3045, PA2665 are negative regulatory factors, affecting biofilm formation and production of a range of virulence related factors, including elastase, pyocyanin and AHL signals.This thesis unveiled the regulatoryroles of PA3045 and PA2665 in bacterial physiology and virulence.The findings laid a solid foundation for further study oftheir roles in bacterial thequorum-sensing networks andtheir regulatory mechanisms in modulation of bacterial pathogenesis.