Construction of reaction model for class 1 integron capturing antibiotic resistance gene cassettes on chromosome of bacteria

Abstract:

[Background] Integron plays important roles in acquisition and spread of antibiotic resistance among bacteria. The research on the improvement of detection methods and reaction mechanisms of integron integration reaction can deepen the understanding of the integron’s contribution in antibiotic resistance acquisition and spread, and provide new ways in suppression of the emergence and spread of the resistant strains. [Objective] To construct class 1 integron reaction model on bacterial chromosome to evaluate integrase-mediated gene cassette site-specific recombination. [Methods] The CM fragment containing chloramphenicol resistance gene cat, the LacA5 fragment containing gene cassette aadA5, the PcS fragment containing integron recombination site attI1 and strong promoter of variable region, the homologous arms on both sides of the knock-in site were amplified by polymerase chain reaction separately. The above five fragments were linked by overlap extension polymerase chain reaction to prepare the knock-in fragment of integron reaction model, and the constructed fragment was knocked into the chromosome of Escherichia coli JM109 by homologous recombination. After transferred with the class 1 integron integrase high expression plasmid pHSint, the integrated strains were screened on streptomycin plate and identified by polymerase chain reaction and sequencing. [Results] The sequencing results of the constructed fragment of integron reaction model were identity with that of expected. The constructed fragment was successfully knocked into the chromosome of E. coli JM109. After transferred with the integrase high expression plasmid pHSint, the strains with gene cassette aadA5 integrated into attI1 were successfully screened on streptomycin plate. The results of polymerase chain reaction and sequencing were identity with that of expected. [Conclusion] Reaction model of class 1 integron integrase-mediated gene cassette site-specific recombination was successfully constructed on E. coli chromosome. It will lay the foundation for further revealing the reaction mechanism of integron capture antibiotic resistance gene cassettes.

Key words:Integron, Integrase, Gene cassettes, Antibiotic resistance, Homologous recombination