The Research Of Regulation Mechanism For Integron Capturing And Expressing Antibiotic Resistance Gene Cassettes In Bacteria

Under the selective pressure of antibiotics, drug resistant strains emerged unceasingly since extensively utilization of antibiotics. Capture exogenous antibiotic resistance genes through horizontal transfer is one of the important way to facilitate the emerging and dissemination of the clinical drug resistant strains. By capturing exogenous antibiotic resistance gene cassettes and ensuring the expression of the genes within gene cassettes, integron play important roles in the horizontal dissemination of antibiotic resistance genes in bacteria. Meanwhile, integron can locate on plasmid or as a part of transposon and transfer along with them, all these facilitate the spread of antibiotic resistance genes among bacteria. Up to now, more than 80 different gene cassettes have been discovered in integron and most of them are antibiotic resistant gene cassettes, their coding products can confer resistance to nearly all classes of antibiotics. More than one antibiotic resistance gene cassettes can be contained in the variable region of a integron and lead to multidrug resistance. As it played important roles in horizontal dissemination of antibiotic resistance genes among bacteria, integron has been paid close attention by reseachers.It has been reported that under the disadvantage environment, bacteria can regulate the expression of the integron integrase thus the efficiency of capture gene cassettes through SOS response system, and up to now, the reaction system of the site specific recombination that catalyzed by integron integrase can not be successfully constructed in vitro, these indicated that there must be certain mechanism in regulating integron capturing and expressing exogenous antibiotic resistance genes. The expression of the antibiotic resistance genes in the variable region of integron is associated with the antibiotic resistance phenotype of bacteria, and the capture of the antibiotic resistance gene cassettes is associated with the emergence and dissemination of antibiotic resistance genes in bacteria, thus the investigation of the regulation mechanism for integron capture and expressing antibiotic resistance gene cassettes in bacteria will contribute to promote understanding on the integron as an evolution platform to adapt the change of the environment in bacteria. Meanwhile it will lay some foundation to develop certain drugs to down regulate or to prevent the expression of the antibiotic resistance genes in gene cassttes, and to lower the capture efficiency of exogenous antibiotic resistance genes by integron, so as to decrease the emerging and dissemination of antibiotic resistance genes in bacteria.In this study we will focus on class 1 integron, we first conduct a molecular epidemiology investigation of integron to understand the distribution of integron in clinical stains. As the copy number of plasmid can influence the expression level of gene cassttes that located on it, we develop a rapid method to idertify class 1 integron whether located on plasmid. Then we investigate the regulation mechanism for integron capturing and expressing antibiotic resistance gene cassettes in bacteria by focusing on the structure of class 1 integron itself, especially on the different promoters of variable region. Meanwhile we develop a high-flux method that can be used in screen transposon mutanted library to measure the excision efficiency of gene cassettes that catalyzed by integron integrase, so as to find out the host factors in bacteria that can impact the capture efficiency of exogenous antibiotic resistance genes by integron.In order to understand the distribution of integrons in clinical strains and to provide research materials such as integrons and gene cassettes for further investigation, we first conducted a molecular epidemiology investigation of integron in clinical strains. PCR was used to screen the class 1,2 and 3 integrons in clinical strains, and the sequences of the variable regions of class 1 integrons were analysis. The result revealed that the positive percent of class 1,2 and 3 integrons in 176 clinical strains were 76%(134/176),1%(2/176) and 0%(0/176) respectively. The variable regions of 76 out of 134 class 1 integron positive strains were successfully amplified and the most popular gene cassette arrays were dfr A17-aadA5(34/76) and dfrAl2-orfF-aadA2(26/76). A new trimethoprim resistance gene has been found in a stain and named dfrA27. These manifested that class 1 integron was widespreaded in clinical strains. The gene cassettes in variable regions of integron conferred host bacteria resistant to trimethoprim, chloramphenicol and aminoglycosides antibiotics that has been used early. These indicated that the selective pressure of antibiotics as well as the exposure time are necessary for integron to capture and disseminate antibiotic resistance genes.Integron can located on plasmid and transfer along with plasmid, this facilitate the emerging and dissemination of antibiotic resistant strains. Meanwhile the expression level of the cassette-encoded gene can be influenced by the copy number of the plasmids on which the gene cassettes located. Thus to determine integron and gene cassettes whether located on plasmid is very important in understanding the mechanism for the dissemination of antibiotic resistance genes as well as in infection controls. However the available methods that has been used in determining the genetic location of certain gene were all time consuming and costly. In this study, we developed a quantitative real-time PCR based method to rapidly determine the genetic location of integron in bacteria. Different template materials (plasmid DNA, genomic DNA or the supernatant of boiled bacteria) were prepared from the same strain, and the copy number of the class 1 integron integrase gene(intIl) and 16S rDNA in different template materials were measured by using quantitative real-time PCR. If integron was located chromosomally, the ratios of the copy number of intIl to that of 16S rDNA would be similar in different template materials prepared from the same strain. In contrast, if integron was located on plasmid, the ratios of the copy number of intll to that of 16S rDNA in plasmid(Rp) would be higher than that in genomic DNA(Rg) or in the supernatant of boiled bacteria(Rb) that prepared from the same strain. To raise specificity, we definited that Rp/Rg or Rp/Rb was more than 4 when integron was located on plasmid in a strain. The genetic locations of class 1 integron in 12 clinical strains were determined by using this method, the result revealed that Rp/Rg or Rp/Rb in these strains were all more than 4(5.42-24.48), indicated that there must be integron that located on plasmid in these strains. Pulsed-field gel electrophoresis (PFGE) combined with Southern blotting were used to determine the genetic location of class 1 integron in these strains, the results verified that the class 1 integrons were all located on plasmids with their size varies from 48.5 kbp to 240 kbp. To further confirm that this method can be used to determin certain gene that was not located on plasmid, the genetic locations of lacZ gene(located chromosomally) in 10 lacZ gene positive strains were determined by using this method. The results indicated that Rp/Rg or Rp/Rb was all less than 2(0.51 to 1.77) in these 10 lacZ gene positive strains. This method, with the advantages of rapidly, easy to handle, cost efficient, widely applicable and the copy number of the plasmid on which integron located on can be calculated at the same time, is predicted to be widely used in identifying genetic locations for other genes or DNA sequences in bacteria.Few studies have dealt with the expression of the cassette-encoded gene at the translational level as there was no respective commercial antibody available. aadA2 gene, as one of the most popular gene within the gene cassettes in class 1 integron, codes for aminoglycoside-3"-adenyltransferase(AAD(3")) and confers bacteria resistance to streptomycin and spectinomycin. There are two start codons, ATG and GTG, at the initial position of the open reading frame(ORF) of the aadA2 gene. The codon GTG was presumed to be the main or the unique start codon of the aadA2 gene as there is a rebosome binding site (RBS) upstream of it. But initiation of translation from the ATG triple can also lead to a complete polypeptide, and up to now, there is no evidence for that the aadA2 gene can translated from the ATG triplet and synthesize a functional protein in bacteria. In this study, anti-AAD(3") specific antisera was prepared and was used to detect the translation products of aadA2 gene with different start codons. The results indicated that aadA2 gene can translate from the ATG triple(there is no RBS upstream of it) and synthesize a functional AAD(3") protein in bacteria when aadA2 gene was within the first gene cassette in class 1 integron. The character described above make class 1 integron translate the gene within the gene cassette that integrated into the attl site no matter there is a RBS upstream of the start codon of the gene or not. This make class 1 integron easy to express the exogenous genes that captured by integron.The promoter of the variable region is one of the most important factors that can impact the transcription of the gene cassettes in class 1 integron. However certain report gene was used instead of the original gene when investigating the translation of the gene within gene cassette, and the translation of the report gene may not represent the original gene itself. In this study,8 integrons with different promoters of the variable region were constructed. By using the anti-AAD(3") specific antisera that was prepared in partⅢ, the transcriptional and translational level of the aadA2 gene within gene cassette in integrons with different promoters of the variable region were detected. The results indicated that the transcriptional and translational level of the aadA2 gene that downstream of the strong Pant promoter combined with active P2 promoter, the most strong promoter, were about 200 folds and 15 folds higher than that downstream of the weak Pant promoter, the most weak promoter, respectively. The relative strength of the "hybrid 2" Pant promoter combined with active P2 promoter was also detected for the first time, and the results indicated its relative strength was only weaker than strong Pant promoter combined with active P2 promoter and strong Pant promoter.The promoter of the variable region of the class 1 integron is located preceding the attI site and the double strands attI site may be unwinding during the transcription of the downsteam cassette-encoded genes. These may impact on the binding of the integron integrase on the double strands attI site and thus impact on the site specific recombination catalyzed by the integron integrase. In this study, the impact of the different promoters of the variable region on the integration efficiency in class 1 integron was investigated. Under the condition that the integron integrase was expressed in sufficient, the integration efficiency of gene cassette into the attI site that downstream of different promoters of the variable region of the class 1 integron was detected by using quantitive real-time PCR. The results indicated that the integration efficiency in integron with weak Pant promoter, which has the highest integration efficiency, was 74 folds as high as that in integron with the strong Pant promoter combined with active P2 promoter, which has the lowest integration efficiency. Meanwhile, the integration efficiency was closely associated with the types of P2 promoter, the integration efficiency in integron with inactive P2 promoter was obviously higher than that with active P2 promoter respectively. With the same P2 promoter, there was an inverse correlation between promoter strength and the integration efficiency in class 1 integron. These indicated that bacteria can maintain the structure of the integron relative steady through down regulating the integration efficiency when the expression of the gene within gene cassette was necessary for bacteria to adapt the environment.It has been reported that the integration efficiency would be raised obviously when bacteria was under stringent state. This indicated that the environment can impact integron on its capture exogenous gene cassettes. However the change of the environment that caused the impact on integron is through the changes of the expression of genes in bacteria, thus there must be factors within bacteria that can impact the efficiency of integron capture gene cassettes. To construct transposon mutant library was a comparative maturity method to find out genes that may influence certain phenotype or biological character in bacteria. However the available methods that has been used in detecting excision efficiency and integration efficiency in integron can not be used in high-flux screen in transposon mutant library. In this study, we developed a "papillatiom assay" based method to detect the excision efficiency of gene cassette in class 1 integron. laca gene can not be translated to be a functionalαfragment ofβ-galactosidae due to the insertion of aadA5 gene cassette in its ORF. When the aadA5 gene cassette was excised by integron integrase, the ORF of laca gene can be read through and translated to be a functionalαfragment ofβ-galactosidae and can generateαcomplement phenomenon in E.coli JM109. As the excision of the aadA5 gene cassette was occurred during the growth of bacteria, there were blue spots that caused by the excision of the aadA5 gene cassette in white colony when growing on LB plate containing IPTG and X-gal. As the number and the occurring period of the blue spots were associated with the excision efficiency of gene cassette, we can estimate the excision efficiency by viewing the occurring period of the blue spots or calculating the number of blue spots in white colony. This method, with the advantages of convenient, high-flux and cost efficient, can be used in screen transposon mutant library to find out the host factors in bacteria that can impact the excision efficiency of exogenous gene cassettes in integron.