Construction And Biological Analysis Of Serratia Marcescens Cpx Two-component Mutants

Serratia marcescens is one kind of Gram-negative bacterium,which is widely distributed in nature environment,belonging to Enterobacteriaceae.S.marcescens is an opportunistic pathogen of some vertebrates and invertebrates,such as silkworms,dairy cows,and Chinese soft-shelled turtles.It has gradually become a serious threat to hospitals,which can cause respiratory infections,wound infections,septicemia and so on.In the sericulture industry,S.marcescens is one of the main pathogenic bacteria.It can cause septicemia of silkworms and the diseased silkworms die in a short period of time,which has brought huge economic losses.Many studies have shown that the Cpx AR two-component system is widely distributed in multiple pathogenic bacteria and plays an important regulatory role in the pathogenicity,the formation of biofilms,and antibiotic resistance and other aspects of bacteria.However,it is rarely reported in S.marcescens,and the function is still unclear.In this article,we constructed the cpx A,cpx R,cpx AR gene deletion strains of S.marcescens SCQ1 and the complement strains.By detecting and analysising the biological characteristics of the knockout strains,we speculated the function of Cpx AR two-component system in SCQ1 to help us further understand the pathogenic mechanism of S.marcescens.The main results are as follows:1.The gene knockout and complement strains were constructed by homologous replacement and screening by PCR and sequencing,the mutants?cpx A,?cpx R,?cpx AR and their complement strains C?cpx A,C?cpx R,C?cpx AR were successfully obtained.2.According to the related biological characteristics test results,compared with the wild-type strain SCQ1,there were no significant changes in colony morphology,growth,protease and lecithinase hydrolysis activity,motility,stress tolerance,antibiotic tolerance,hemolytic activity of single knockout strain?cpx A.The biofilm formation ability of the?cpx A was no significantly different from SCQ1 before 72 hours,and it was significantly decreased at 96 hours.The activities of lecithin,protease,and hemolysis and stresses tolerance of single knockout strain?cpx R were no significantly different from SCQ1.The colony phenotype of?cpx R was pink when cultured for 12 h,and the colony was red when cultured for 36 h,which was consistent with SCQ1.The growth rate of?cpx R was slightly lower than SCQ1,but not the final cell density.The area of the swimming and the swarming circle of SCQ1 were 2.04-and 1.375-folds of?cpx R,respectively.The motility and the biofilm formation ability of?cpx R were significantly weaker than that of SCQ1.The results of antibiotic resistance experiments showed that the MIC values of?cpx R of cephalosporin,ceftazidime,gentamicin,and amikacin were increased by 16-,8-,4-,and 8-folds compared with SCQ1.The resistance of other antibiotics was similar to SCQ1.3.In our study,we found that the growth rate of the double knockout strain?cpx AR was significantly inhibited compared with SCQ1.With the extension of the culture time,the colony of?cpx AR gradually changed from white to light pink plate on LB medium.Moreover,?cpx AR loses the motility and hemolytic ability.The protease hydrolysis ability,the enzymatic hydrolysis ability of lecithin,and the ability of biofilm formation of?cpx AR were significantly weakened.The stress tolerance of?cpx AR to 5%KCl and 0.05%SDS did not change significantly,but to 1%H₂O₂ was weakened.Antibiotic tolerance tests of?cpx AR showed,when compared with SCQ1,the MIC values of cephalosporin,ceftazidime,gentamicin,amikacin,norfloxacin,and ofloxacin were increased by 8-,4-,8-,16-,2-and 2-folds,and the MIC value of azithromycin and chloramphenicol decreased 16-and 4-folds.4.The virulence tests showed that the pathogenicity of?cpx A and?cpx R to silkworms were similar to SCQ1,while the virulence of?cpx AR was significantly attenuated.The LD₅₀ of?cpx AR was 8.92-folds that of SCQ1.The gene expression profile of 12 virulence related genes were detected and the q RT-PCR assays with SCQ1as control.The results showed that the shl A,Pho P,and Pho Q genes were significantly up-regulated in?cpx A and?cpx R and the expression of hfq in?cpx A was significantly down-regulated.The expression of other genes in?cpx A and?cpx R were no significant changes.In?cpx A,?cpx R,and?cpx AR,shl B was nearly not expressed.In?cpx AR,shl A was up-regulated,and Flh C,Flh D,hfq,Pho P,Pho Q,and wec A were decreased significantly.Preliminary results showed that the transcription level of some virulence-related genes in the mutants had changed.Whether these changes were regulated by the Cpx two-component system remains to be further studied.In conclusion,the Cpx AR two-component system was involved in the growth,extracellular enzyme production,motility,stress tolerance,biofilm formation,antibiotic resistance and pathogenicity of S.marcescens SCQ1.The effect of cpx AR double genes knockout on the biological characteristics of the strain was more significant than cpx R or cpx A single gene knockout.