The Research Of Regulation Mechanism For Integron Capturing Gene Cassettes

At present antibiotic resistance of bacteria become serious,especially multidrug resistant are having a significant impact on clinical treatment of infectious disease. Single-drug-resistance phenotype could be anticipated,it may be attributed to the result of gene mutation.Conversely,it is difficult to interpret the phenomenon of multidrug resistance exsisting the same host merely by gene mutation.It is clear that the horizontal dissemination of exogenous genes among bacteria is the most efficient means by which bacteria acquire new resistance.The prevalence of integrons in E.coli isolates from healthy sujects who lived in communities and not took antibiotic for at least 1 month showed 15%.More than 70 different antibiotic genes covering most classes of antimicrobials presently in use are structured as gene cassette.These data indicates that integrons play a pivotal role in the spread of antibiotic resistance determinants and appearance of multiple resistances.Under some condition integrons can capture profitable gene cassette to get more possibilities to survie,but also can acquire deleterious cassette,or obtain excessive gene cassettes to result in genetic burden.So there must be a ingenious mechanism for which the host regulates the integration of gene cassette.This mechanism still remains elusive so far.The investigation of that will contribute to promote understanding the effect of integron on the horizontal dissesimation of resistance gene and provide help to block or delay the spread of resistance among bacteria.Meanwhile it will lay some foundation to develop a powerful site- and orientation-specific recombination tool by use of characteristic of integron system.In this study we will focus on some factors that influence integation efficiency including host factors,the length of gene cassette, environmental condition and expression level of integrase.PartⅠ.Development of a novel method based on real-time PCR to determine integration frequency mediated by integrase intI1.The widely used quantifiable assay to determine recombination frequency, mediated by integrase,was performed by a conjugation test with phenotypic screening. The results were severely influenced by the orientation in which the fragment carrying the attC site was cloned into the plasmid pACYC184.This method proved to be time consuming,and the precision of the data obtained in this manner was relatively low. So we developed a novel method based on quantitively florescent real-time PCR. There are two plasmids in the E.coli host strain,BL21(DE3).One is pUCINT which overexpresses integrase,catalyzing cassette recombination between the attI site and an attC site.The other is the plasmid pACINAD,which contains the integron and aadA2 gene cassette at different sites of plasmid pACYC 184.The copy numbers of cointgrates and total integrons were determined,and the integration frequency is the result of the former divided by the latter.The results indicated the integration frequency in host BL21(DE3) was 1.87×10^-4,while the background integration frequency was less than 5.23×10^-8.This novel method laid a foundation for further research of mechanism for integration.PartⅡ.Effect of host factor on the integration frequencyLateral gene transfer has been proposed as a fundamental process underlying bacterial diversity and evolution.Recent discoveries about integron system indicate it has the potential to play a role in this process.However,regulation mechanism for integration process and factors in the host bacteria which influence integration efficiency is poorly known.In this study we used transposon(Tn) mutagenesis to identify E.coli genes involved in regulation of recombination frequency mediated by integrase.A total of more than 700 mutants were determined for integration frequency by real-time PCR on a LightCycler instrument.Consequently a mutant with dramatically increasing integration frequency compared with wild type was selected. Expression difference between this mutant and the wild type was screened by use of E.coli gene chip.Compared with the wild-type strain,37 genes were up-regulated and 19 genes were down-regulated.Among those 8 genes including ybcT,peaD',ybcW, exoD',ybcX,nohB,renD',ybcV were clustered in the prophage DLP 12 and they were unanimously up-regulated.This is extremely striking,because many bacteria's genome published to date reside prophages and just as integrons they are also considered as powerful tools for gene evolution.These expression differences were confirmed by real-time PCR.The result was consistent with observation obtained by the microarry assay.Our further investigation showed that ybcV,ybcW and nohB genes could enhance the integration reaction. PartⅢ.Analysis of the impact of length of gene cassette on the integration frequencyMost classes of antimicrobials presently in use have their own resistance cassettes, however these cassettes seldom exceed l kb.We constructed a variety of plasmids containing different length of cassettes with identical attC site,then the deletion, integration and capture frequencies of those cassettes mediated by integron integrase 1 were determined by real-time fluorescent quantitative PCR.The data indicated that during the integron capturing gene cassette the integration process other than the excision of gene cassette may be the rate limiting step.Meanwhile the results showed that recombination frequency of 699 bp(approximately equal to the length of one cassette) cassette was 32-fold higher than that of 1908 bp(approximately equal to the length of two cassettes) cassette,142-fold higher than that of 2805 bp(approximately equal to the length of three cassettes) cassette.The implication of this feature of integrase was discussed.PartⅣ.Analysis of relationship between recombination frequency catalyzed by integrase intⅠ1 and antibiotic concentration.To date,there is no report on the relationship between antibiotic concentration and recombination efficiency mediated by integron integrase 1.We used above newly developed method to determine the recombination frequency at the attⅠsite of aadA2 gene cassette by overexpressing the integrase at different streptomycin concentration levels.The resulting frequencies were 1.97×10^-3,3.23×10^-3,3.27×10^-3,0.45 and 1.32, with respective streptomycin concentrations of 0,20,30,40 and 50μg/ml.The background frequency of recombination without integrase overexpression was less than 1.75×10^-7.These findings indicate that antibiotic concentration significantly influences recombination frequency of gene cassette,catalyzed by integron integrase1.Western blotting analysis demonstrated that the expression level of the aadA2 gene cassette at the attⅠsite was about ten times higher than it at its native site.Together, this provides evidence that under the selective pressure of antibiotic,the expression level of the resistance gene significantly increased by gene rearrangement within the integron.PartⅤ.Effect of expression level of integrase intI1 on the integration frequency.We measured integration frequencies mediated by integrase from plasmid pUCINT induced by various concentrations of IPTG.The results showed that changes of integration frequencies under different expression level were not obvious.In order to further prove this phenomenon the integration frequencies were determined when integrase gene was cloned into plasmid pET28a induced by different levels of IPTG. The data revealed that these integration frequencies remained unchanged when indued by IPTG.SDS-PAGE and Western blotting assay showed that expression level of integrase drastically increased after induced by IPTG.These results suggest that the expression level of integrase is not the exclusive factor affecting integration efficiency, other aspects such as stability of integrase complex are also involved.