Optimization Of Haemophilus Parasuis Natural Transformation And Primary Studies Of ComA

Haemophilus parasuis is the agent of Glasser's disease,which is characterized by fibrinous polyserositis,arthritis,and meningitis in clinically.Natural transformation is that bacteria take up and integrate foreign DNA into their own chromosomes under natural conditions.The experiment of Natural transformation is an operating system to research the function of virulence-associated genes.To improve the natural transformation efficiency of gene deleted mutant and screen the natural competence between field isolation,this study optimized the operation of original natural transformation.Bacterial series gene of com are concerned with the ability of transformation,playing different roles during the transformation.The research considered the HPS's ComA as the object,studying its function in different biological specificities by constructing and comparing the inactive mutation and complemented strain.1.Optimization and evaluation of natural transformation conditionA recombinant plasmid and a field-isolate strain M-3 had been selected as the natural competence in this study.We optimized the different aspects of HPS natural transformation control variable method,which including bacterial growth phase,the time of hatching,the concentration of bacteria and exogenous cAMP in the transformation system,the amount of exogenous plasmid and the time of plate hatching after transformation.The natural transformation frequency corresponding to the different conditions were calculate,to evaluate the influence of each variable on the frequency of natural transformation.Then,we use the optimum condition to improve the probability of positive transformants.2.Screening the natural compentence15 standard reference strains and 75 isolates were screened by using the optimized natural transformation method.The 15 standard reference strains did not detect the occurrence of natural transformation.The 11 strains of isolates were identified as natural compentence,and three strains have high natural transformation ability.3.Construction of recombinant suicide plasmid and complemented plasmidIn order to clone the homologous fragment and Kan resistance gene,the specific primers were designed according to the whole genome sequence of SC 1401.The resistance cassette was ligated downstream of the gene by overlap PCR and the fragment was ligated into the pK18mobsacB plasmid vector to obtain the recombinant plasmid RLcomA-Kan-pK18.The primers were designed according to the whole genome sequence to clone the whole sequence of the comA gene containing the promoter.The recombinant plasmid pMC-comA was obtained by ligating the whole sequence into the pMC-express shuttle expression plasmid vector.4.Construction of a comA inactive mutation and complemented strainThe recombinant plasmid RLcomA-Kan-pK18 was transformed into SC 1401 strain to obtain the inactivated strain comA::Kan-SC1401.The recombinant plasmid pMC-comA was transformed into Escherichia coli S17(X)to obtain pMC-comA?S17(X).The complemented strain CcomA-SC1401 was constructed by using pMC-comA?S17(X)and comA::Kan?SC1401 through conjugation.The strains were identified by PCR,RT-PCR and Western Blotting.5.Characterization of the function of ComABy measuring the growth curve of SC 1401,comA inactive mutation and complemented strain,it was found that the inactivation of comA had a definite effect on growth.Observation of bacterial morphology under the optical microscope showed that the comA gene had a significant effect on the morphology of HPS,and the SC1401 possessed polymorphism from long to short rod,but the inactive mutation was short or even spherical.The complemented strain recovered the polymorphism based on the short rod.It can be observed some low electronic density area in the cytoplasm of comA inactive mutation using transmission electron microscopy.When cultured in different conditions,the comA::Kan?SC1401 was more sensitive to acidic condition than the parental strain(p>0.05),but no obvious difference was observed between the two strains to the high temperature and alkaline condition.In the serum bactericidal test,the parental strain was more sensitive to the serum bactericidal activity compared to the comA inactive mutation(p<0.05).The autoagglutination activity of the comA::Kan-SC1401 was significantly higher than SC 1401 in the autoagglutination experiment.In the biofilm formation experiment,the comA inactive mutation formed less biofilm than the parental strain(p<0.05).The result indicated that ComA plays an important role in the process of HPS natural transformation by comparing the ability of SC1401?comA::Kan?SC1401 and CcomA?SC1401(p<0.05).In summary,this study analyzes transformation frequency influenced by different conditions.Using the optimal conditions can improve the frequency signally and increase the probability of selection of positive transformants.We obtained 11 natural competent strains by screening 75 wild strains isolated from the laboratory.By contributing comA inactive mutation and complemented strain and analysing their biological characteristics,verify preliminarily that ComA plays a certain role in HPS bacterial morphology,self-agglutination ability,biofilm formation and natural transformation,which provides a reference for further exploring the ComA's role of HPS.