The Research On The Regulation Of Class 1 Integrase And Gene Cassettes In The Resistant Bacteria From Aquatic Environments

The use and abuse of antibiotics has led to the rapid dissemination of drug-resistant bacteria around the world. Horizontal gene transfer contributes to the spread of resistant bacteria. As the common mobile genetic element in bacteria, integron plays an important role on the transfer of resistance gene. Class 1 integron is the most common among all the integrons, it allows bacteria to capture or, exchange as well as express gene cassettes. The integron platform is composed of a gene, called intl, that encodes a site-specific recombinase IntI, a recombination site attl, a functional promoter Pc with divergent to the integrase gene and a Pint promoter which regulates the transcription of integrase. The Pc promoter can occasionally be combined with a second promoter designated P2. Although several Pc variants with different strengths have been described, their relative distribution is not known in aquatic environments.The Pc promoter in class 1 integrons is located within the intll coding sequence, so the structure and transcription of it may have an effect on the integrase. Studies have shown that a conserved LexA-binding motif overlapping the promoter regions. As LexA is the key protein of the SOS response in bacteria, research about the SOS regulation on the integrase and gene cassettes had been carrying on. Studies have shown that the expression of integrase and gene cassette is co-regulated in V. cholerae. Some antibiotics would induce the SOS response in bacteria, thereby enhancing their expression of integrase activity. Both of Pc and Pint can be up-regulated in the environmental press of nutrient-rich medium, high temperature, or high salt concentration. However, the researches on the regulation of integrase and gene cassettes expression are rarely reported in E. coli.In this study, the prevalence of Pc variants was analyzed the antibiotic bacteria from five different sampling sites in Jinan. By PCR, we found four kinds of Pc promoter variants. They were PcW(54.29%)、PcWTGN-10(34.26%), PcH1(8.57%) and PcW-P2(2.88%) respectively. Our results indicated that weak promoter variants were more frequently in the water aquatic environments.On the basis of previous studies, we constructed three bidirectional promoter vectors with the three Pc variants(PcW, PcWTGN-10, PcH1) and Pint, observing the changes of Pc and Pint when they were induced by different conditions and analyzing their regulatory factors. Results showed that without any stress pressure, the strength of Pc was as follows, according to their strength:PcWTGN-10≈PcH1>PcW, while the strength of Pint was on the contrary When there were low concentrations of antibiotics in the environments, the strength of Pint could be stronger and so was Pc. What’s more, the regulation on Pint by antibiotics belongs to SOS response. When there were low concentrations of chemicals in the environments, it could have an influence on the activity of bidirectional promoter vector Pc-Pint, PTA and BHT could up-regulate the expression of integrase and this might very helpful for bacteria adapt to different stresses. When the water temperature was not the same, high temperature could activate Pc and Pint, but this had little relationship with SOS response.Studies have shown that the antibiotics and chemical residues in domestic aquatic environments can regulate the recombination and expression of resistance gene through SOS response, and promote the generation and dissemination of drug-resistant microbes. Therefore, hazardous substances in environment can not only accumulate the drug-resistant microbes (the expression of resistance gene),but also promote the evolution of resistance genes,This study provide more theoretical basis on controlling the spread of drug-resistant microbes, regulating and controlling the use of antibiotics, and optimizing processes of various wastewater and strengthen the detection of wastewater discharge.