The Mechanism Of Bacterial Quorum Sensing System Involved In The Defense Of Phage Infection

Bacterial Quorum Sensing(QS)system allowed bacteria can detect the change of signal molecules concentration in external environment and activate or inhibit corresponding genes to monitor the cell density,modify the biophysical function and defense environmental stress.In nature,phages are "predators" of bacteria they play an important role in control of bacterial population and community structure,but we still know less about the role of QS in defense of phage infection.In this paper,both gram positive and negative bacteria and their phages have been used to illuminate the QS system involved in defense of phage infections.At the first,Escherichia coli and T4 phage have been investigated.Based on Red recombination technology,the receptor and their downstream genes including sdiA,lsrB and ydiV,lsrR corresponding to QS system of AI-1、AI-2 have selected to construct deficient and complementary bacterial strains.All recombination constructions have been finished,except the construction of △lsrR failed,due to essential role of lsrR in growth of E.coli.The result of T4 infection rate showed that significant changes can be observed in strains of △ydiV and △lsrB,which increased or decreased nearly 20%accordingly.And all complementary strains presented same phenotypes with wild type strain.No significant change can be observed in △sdiA strain,because of deficient AI-1 in E.coli,in which there is no signal molecule synthesis genes but receptor gene of SdiA.All these results indicated that QS system of AI-1 and AI-2 were involved in defense of T4 infection in E.coli.Because of the fail in construction of △lsrR and effect from endogenous molecules in AI-2,we focused on analysis of AI-1 signal pathway in this section.The applications of signal molecules have been optimized,the best signal molecule is C4-HSL and optimal concentration is 5μM.After addition of C4-HSL,the infection rate decreased 15%in wild type,but no significant change can be observed in △sdiA,complementary strain of pCA24N-sdiA showed same results with wild type,which demonstrate that QS AI-1 system involved in defense of T4 infection.Base on knockout and RT-PCR technology,several genes involved in AI-1 pathway have been investigated.After treated with C4-HSL,transcriptional level of ydiV was up regulated and leading to up-regulation of ompC、waaA、waaQ.Neither △ydiV nor △sdiA can be observed same result.This result also can be validated in T4 adsorption experiments.Although the results showed that the addition of signal molecules lead to phage adsorption rate increased by 13%,but the final infection rate is reduced by 15%.What is the reason involved in it?We found that ydiV is a global regular gene in E.coli,and its activation can also activate the important anti-phage system of CRISPR,but is that really the case?The functions of leuO,hns,bglJ rcsB and casE genes related to CRISPR were investigated.The results showed that the addition of signal molecules led to the up regulation of the transcriptional levels of bglJ and rcsB,and finally increased the transcriptional level of leuO greatly.Although the gene hns which is a LeuO negative regulator was also partially up-regulated,but due to leuO/hns ratio significantly increased,the CRISPR system was activated greatly.The transcription level of casE was 2.4 times increased.In order to confirm this result,a △leuO strain was constructed.The results showed that the deletion of leuO resulted in a 13%increase in the infection rate of T4,which completely proved our previous hypothesis.In order to reveal the mechanism of QS involved in defense of phage infection,a Gram-positive bacteria Bacillus cereus MYB41-22 and its phage VMY22,both are isolated by our research group,were be used as model in this study.The genome sequencing of MYB41-22 and VMY22 were completed by us.The results showed that the genome size of phage VMY22 was 18,609 bp and the GC content was 36.4%.There were 25 ORFs in the whole genome.The genome size of MYB41-22 was 4,428,864 bp and the GC content was 36.06%.There were 5,481 predicted ORFs,and potential QS systems of PlcR-PapR and LuxS were found in it,and the function of PlcR-PapR and LuxS were characterized briefly.Several signal molecular peptides for PlcR-PapR system also have been synthesized.All in all,we can draw conclusions below:The QS system of E.coli is absolutely involved in defense of phage,in which the activation of AI-1 system not only requires the presence of signal molecules,but also sdiA,ydiV and leuO genes.Although the addition of signal molecules causes the incensement of membrane proteins,including phage receptor proteins,in response to external stimuli;but it also stimulates the CRISPR system to resist phage infection.The ultimate result based on these two roles,and QS system played a anti-phage function in E.coli.In this paper,we also tried to illuminate QS system involved in Gram-positive Bacillus cereus MYB41-22 and its phage VMY22.Both genomes were sequenced,and the QS system composition were detected and characterized briefly.Due to my limited working time and energy constraints,the relevant research can only be left to the other members in lab to complete.