细菌整合子捕获与表达耐药性基因盒调控机制的研究
|
摘要
随着抗生素的广泛使用,细菌在抗生素的选择压力下,耐药菌株不断产生。其中通过基因的水平转移,获得外源性耐药基因是加快临床耐药菌株产生与播散的重要原因。整合子通过位点特异性重组捕获外源基因盒并使之表达,同时整合子可位于质粒上,或自身作为转座子的一个组成部分而参与转移,使耐药基因发生播散。目前在整合子中发现的基因盒已超过80种,大部分是耐药基因盒,编码产物可赋予细菌对几乎全部临床常用药物种类产生耐药性,同一个整合子可变区可带有多个耐药基因盒,使宿主菌对多种药物产生耐药性。整合子因其在细菌耐药基因水平转移中的重要作用而受到研究者的关注。
有研究表明细菌在不利于其生长的环境中,可通过SOS反应系统调控整合酶蛋白的表达,从而影响整合子捕获基因盒的效率,同时到目前为止,整合酶蛋白催化的基因盒位点特异性重组反应体系在体外无法成功构建,表明整合子捕获与表达外源性基因盒存在着某种调控机制。由于整合子可变区基因盒中耐药基因的表达关系到宿主细菌的耐药表型,而整合子对耐药性基因盒的捕获则关系到新耐药菌株的产生与播散,因此对整合子捕获与表达耐药性基因盒的调控机制进行研究,可加深对整合子这一细菌适应外界环境变化的进化平台的理解,进而有可能开发出相关的药物来下调或阻止整合子中耐药基因盒的表达,降低整合子对外源耐药基因盒的捕获频率,从而减少耐药菌株的产生与播散。
基于上述原因,本研究以第1类整合子作为研究对象,通过分子流行病学调查了解整合子在临床菌株中的分布情况；由于位于质粒上的基因盒的表达水平还与质粒的拷贝数有关,本研究建立了一种快速确定第1类整合子是否位于质粒上的方法；然后从整合子自身结构,主要是不同可变区启动子,对整合子捕获与表达耐药性基因盒的调控机制进行研究；同时建立一种适用于转座子插入突变文库筛选的、高通量的检测整合酶介导的基因盒剪切频率的方法,以便用于进一步寻找宿主细菌中影响整合子捕获耐药性基因盒的因素。
为明确整合子在临床菌株中的分布情况,同时为后续研究提供适当的整合子、基因盒等材料,本研究首先对临床菌株整合子进行分子流行病学调查。运用PCR筛查临床菌株中的第1、2、3类整合子,第1类整合子阳性菌株对整合子可变区序列进行分析。结果176株临床菌株中第1、2、3类整合子阳性率分别为76%(134株)、1%(2株)、0%(0株),134株第1类整合子阳性菌株中有76株成功扩增出可变区,可变区中最常见的基因盒及排列为dfrA17-aadA5(34株)及dfrA12-orfF-aadA2(26株),同时在一株临床菌株中发现并命名了一种新的甲氧苄啶耐药基因盒dfrA27。表明第1类整合子在临床菌株中分布广泛,其可变区中的基因盒均为赋予宿主菌对甲氧苄啶、氯霉素及早期使用的氨基糖苷类抗生素耐药,提示整合子对耐药性基因盒的捕获与播散需一定的抗生素选择压力和时间。
整合子位于质粒上,可随质粒的移动而移动,从而加快了耐药菌株的产生以及耐药基因的播散,同时位于质粒上的整合子中基因盒的表达还与质粒的拷贝数有关,因此,确定整合子及耐药基因是否位于质粒上,对于耐药基因播散机制的研究及感染控制均十分重要。现有的基因定位方法均费时且费用高,本研究建立了一种基于实时定量PCR的方法,对临床菌株中整合子基因定位进行快速鉴定。对于同一细菌标本,分别抽提质粒、全基因组DNA或制备菌体煮沸裂解模板,运用实时定量PCR检测第1类整合酶基因(intIl)与16S rDNA在上述3种模板中的拷贝数,如果整合子位于染色体上,那么intIl拷贝数与16S rDNA拷贝数的比值在不同模板中应是相同的,相反,如果整合子位于质粒上,质粒模板中intIl拷贝数与16S rDNA拷贝数的比值(Rp)要高于全基因组DNA或菌体煮沸裂解模板中intIl拷贝数与16S rDNA拷贝数的比值(Rg或Rb)。为提高检测的特异性,将Rp/Rg或Rp/Rb大于4作为判断整合子位于质粒上的标准。运用该方法对12株临床菌株中的整合子进行定位,结果该12株细菌中Rp/Rg或Rp/Rb均大于4(5.42-24.48),推测其均有整合子位于质粒上。用PFGE结合探针杂交对12株细菌中整合子基因定位进行确认,结果该12株细菌中整合子均位于质粒上质粒大小为48.5-240 kbp不等。为进一步确认该方法可对位于染色体上的基因进行定位,用该方法对10株lacZ基因(位于染色体上)阳性菌株中lacZ基因进行定位,结果该10株细菌中Rp/Rg或Rp/Rb均小于2(0.51-1.77)。与其它基因定位方法相比,该方法具有快速、简便、费用低、适用范围广、可计算整合子所在质粒的拷贝数等优点,可广泛应用于对其它基因或DNA序列进行快速定位。
由于市场上缺乏相关的研究抗体,有关基因盒中基因在翻译水平的研究的报导较少。aadA2基因是第1类整合子基因盒中最常见的基因之一,编码产物为29 kDa的氨基糖苷-3"-腺苷酰基转移酶[AAD(3")],赋予细菌对链霉素和大观霉素耐药。aadA2基因读码框起始部位有两个起始密码子ATG和GTG,密码子GTG上游有核糖体结合位点被认为是主要或唯一的起始密码子,但从密码子ATG起始也能合成一条完整的多肽链,但至今并没有证据表明在细菌中aadA2基因可从ATG密码子起始翻译并合成有功能的蛋白。本研究通过制备抗AAD(3")蛋白特异性多克隆抗体,对含有不同起始密码子的aadA2基因翻译产物进行检测,结果证实当位于第1类整合子可变区第1位基因盒中,aadA2基因在细菌中可从上游不具有核糖体结合位点的ATG密码子起始翻译并合成有功能的AAD(3”)蛋白,这一特性使得通过第1类整合酶介导整合入attI位点的基因盒,不需带有核糖体结合位点即可起始基因盒中相应读码框的翻译,这更有利于第1类整合子表达从外界环境中捕获的基因。
第1类整合子中基因盒上游可变区启动子是影响基因盒转录水平的最主要因素之一。国内外对于基因盒中基因在翻译水平的研究大多用报告基因替代原有基因,研究结果并不能完全代表自然情况下整合子中基因盒在翻译水平的表达情况。本研究通过构建8种含有不同可变区启动子的整合子,运用第三部分制备的抗AAD(3")多克隆抗体,观察其对下游aadA2基因盒中aadA2基因在转录和翻译水平的影响。结果强Pant启动子联合有活性的P2启动子下游aadA2基因的表达水平最高,比表达水平最低的弱Pant启动子下游aadA2基因在转录水平要高出约200倍,在翻译水平要高出约15倍,并首次检测了杂合2型Pant启动子联合有活性的P2启动子下游基因盒中aadA2基因的表达水平,发现其仅次于强Pant启动子联合有活性的P2启动子及强Pant启动子联合无活性的P2启动子。
第1类整合子可变区启动子位于基因盒重组位点attI上游,可变区基因盒在可变区启动子的作用下进行转录时需经过attI位点,并使attI位点双链解开,可能影响整合酶蛋白与双链attI位点结合,从而对整合酶介导的基因盒位点特异性重组产生影响。因此本研究对第1类整合子中不同可变区启动子对基因盒整合频率的影响进行研究。运用实时定量PCR检测高表达整合酶的条件下,基因盒插入不同可变区启动子下游attI位点的整合频率,结果发现含弱Pant启动子的整合子中基因盒的整合频率最高,是整合频率最低的强Pant启动子联合有活性P：启动子的整合子中基因盒整合频率的74倍,同时发现整合子中基因盒的整合频率与P2启动子种类密切相关,在含有无活性P2启动子的整合子中基因盒的整合频率明显高于含有有活性P2启动子的整合子,在含有相同P2启动子的整合子中基因盒的整合频率与整合子可变区基因盒的表达水平成负相关。提示整合子需要基因盒高表达以适应外界环境时,可通过降低整合频率以维持整合子结构的相对稳定。
研究表明细菌处于应激状态下,整合频率会大大提高,表明不同的外界环境可影响整合子捕获基因盒的效率,而外界环境的变化是通过细菌体内不同基因表达水平的变化而影响整合子捕获基因盒的效率的,因此,宿主菌中可能存在着影响整合子捕获基因盒效率的因素。转座子插入突变文库是比较成熟的筛选细菌中影响某一表型或生物学性状的基因的方法,但现有的检测整合子中基因盒剪切与整合频率的方法都不适用于对大规模转座子插入突变文库进行高通量筛选。本研究建立一种利用“乳头状试验”检测整合子中基因盒剪切频率的方法。lacα基因的读码框由于基因盒aadA5的插入而不能翻译成有功能的β-半乳糖苷酶的α片段,当lacα基因读码框内插入的基因盒aadA5在整合酶蛋白的作用下被剪切下来,lacα基因读码框能翻译成有功能的β-半乳糖苷酶的α片段,在大肠杆菌JM109中能发生α互补现象,由于基因盒aadA5的剪切是在细菌生长过程中发生的,因此在含X-gal与IPTG的平板上,在长出的白色菌落中会出现基因盒aadA5发生剪切的细菌生长所形成的蓝色斑点,而蓝色斑点出现的早晚、数量的多少与剪切频率有关,可通过观察蓝色斑点出现的早晚以及计数白色菌落中蓝色斑点的数量来评价剪切频率。该方法具有简便、高通量、费用低等优点,可运用以对转座子插入突变文库进行高通量筛选以寻找宿主菌中影响整合酶介导的基因盒剪切频率的因素。
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.
有研究表明细菌在不利于其生长的环境中,可通过SOS反应系统调控整合酶蛋白的表达,从而影响整合子捕获基因盒的效率,同时到目前为止,整合酶蛋白催化的基因盒位点特异性重组反应体系在体外无法成功构建,表明整合子捕获与表达外源性基因盒存在着某种调控机制。由于整合子可变区基因盒中耐药基因的表达关系到宿主细菌的耐药表型,而整合子对耐药性基因盒的捕获则关系到新耐药菌株的产生与播散,因此对整合子捕获与表达耐药性基因盒的调控机制进行研究,可加深对整合子这一细菌适应外界环境变化的进化平台的理解,进而有可能开发出相关的药物来下调或阻止整合子中耐药基因盒的表达,降低整合子对外源耐药基因盒的捕获频率,从而减少耐药菌株的产生与播散。
基于上述原因,本研究以第1类整合子作为研究对象,通过分子流行病学调查了解整合子在临床菌株中的分布情况；由于位于质粒上的基因盒的表达水平还与质粒的拷贝数有关,本研究建立了一种快速确定第1类整合子是否位于质粒上的方法；然后从整合子自身结构,主要是不同可变区启动子,对整合子捕获与表达耐药性基因盒的调控机制进行研究；同时建立一种适用于转座子插入突变文库筛选的、高通量的检测整合酶介导的基因盒剪切频率的方法,以便用于进一步寻找宿主细菌中影响整合子捕获耐药性基因盒的因素。
为明确整合子在临床菌株中的分布情况,同时为后续研究提供适当的整合子、基因盒等材料,本研究首先对临床菌株整合子进行分子流行病学调查。运用PCR筛查临床菌株中的第1、2、3类整合子,第1类整合子阳性菌株对整合子可变区序列进行分析。结果176株临床菌株中第1、2、3类整合子阳性率分别为76%(134株)、1%(2株)、0%(0株),134株第1类整合子阳性菌株中有76株成功扩增出可变区,可变区中最常见的基因盒及排列为dfrA17-aadA5(34株)及dfrA12-orfF-aadA2(26株),同时在一株临床菌株中发现并命名了一种新的甲氧苄啶耐药基因盒dfrA27。表明第1类整合子在临床菌株中分布广泛,其可变区中的基因盒均为赋予宿主菌对甲氧苄啶、氯霉素及早期使用的氨基糖苷类抗生素耐药,提示整合子对耐药性基因盒的捕获与播散需一定的抗生素选择压力和时间。
整合子位于质粒上,可随质粒的移动而移动,从而加快了耐药菌株的产生以及耐药基因的播散,同时位于质粒上的整合子中基因盒的表达还与质粒的拷贝数有关,因此,确定整合子及耐药基因是否位于质粒上,对于耐药基因播散机制的研究及感染控制均十分重要。现有的基因定位方法均费时且费用高,本研究建立了一种基于实时定量PCR的方法,对临床菌株中整合子基因定位进行快速鉴定。对于同一细菌标本,分别抽提质粒、全基因组DNA或制备菌体煮沸裂解模板,运用实时定量PCR检测第1类整合酶基因(intIl)与16S rDNA在上述3种模板中的拷贝数,如果整合子位于染色体上,那么intIl拷贝数与16S rDNA拷贝数的比值在不同模板中应是相同的,相反,如果整合子位于质粒上,质粒模板中intIl拷贝数与16S rDNA拷贝数的比值(Rp)要高于全基因组DNA或菌体煮沸裂解模板中intIl拷贝数与16S rDNA拷贝数的比值(Rg或Rb)。为提高检测的特异性,将Rp/Rg或Rp/Rb大于4作为判断整合子位于质粒上的标准。运用该方法对12株临床菌株中的整合子进行定位,结果该12株细菌中Rp/Rg或Rp/Rb均大于4(5.42-24.48),推测其均有整合子位于质粒上。用PFGE结合探针杂交对12株细菌中整合子基因定位进行确认,结果该12株细菌中整合子均位于质粒上质粒大小为48.5-240 kbp不等。为进一步确认该方法可对位于染色体上的基因进行定位,用该方法对10株lacZ基因(位于染色体上)阳性菌株中lacZ基因进行定位,结果该10株细菌中Rp/Rg或Rp/Rb均小于2(0.51-1.77)。与其它基因定位方法相比,该方法具有快速、简便、费用低、适用范围广、可计算整合子所在质粒的拷贝数等优点,可广泛应用于对其它基因或DNA序列进行快速定位。
由于市场上缺乏相关的研究抗体,有关基因盒中基因在翻译水平的研究的报导较少。aadA2基因是第1类整合子基因盒中最常见的基因之一,编码产物为29 kDa的氨基糖苷-3"-腺苷酰基转移酶[AAD(3")],赋予细菌对链霉素和大观霉素耐药。aadA2基因读码框起始部位有两个起始密码子ATG和GTG,密码子GTG上游有核糖体结合位点被认为是主要或唯一的起始密码子,但从密码子ATG起始也能合成一条完整的多肽链,但至今并没有证据表明在细菌中aadA2基因可从ATG密码子起始翻译并合成有功能的蛋白。本研究通过制备抗AAD(3")蛋白特异性多克隆抗体,对含有不同起始密码子的aadA2基因翻译产物进行检测,结果证实当位于第1类整合子可变区第1位基因盒中,aadA2基因在细菌中可从上游不具有核糖体结合位点的ATG密码子起始翻译并合成有功能的AAD(3”)蛋白,这一特性使得通过第1类整合酶介导整合入attI位点的基因盒,不需带有核糖体结合位点即可起始基因盒中相应读码框的翻译,这更有利于第1类整合子表达从外界环境中捕获的基因。
第1类整合子中基因盒上游可变区启动子是影响基因盒转录水平的最主要因素之一。国内外对于基因盒中基因在翻译水平的研究大多用报告基因替代原有基因,研究结果并不能完全代表自然情况下整合子中基因盒在翻译水平的表达情况。本研究通过构建8种含有不同可变区启动子的整合子,运用第三部分制备的抗AAD(3")多克隆抗体,观察其对下游aadA2基因盒中aadA2基因在转录和翻译水平的影响。结果强Pant启动子联合有活性的P2启动子下游aadA2基因的表达水平最高,比表达水平最低的弱Pant启动子下游aadA2基因在转录水平要高出约200倍,在翻译水平要高出约15倍,并首次检测了杂合2型Pant启动子联合有活性的P2启动子下游基因盒中aadA2基因的表达水平,发现其仅次于强Pant启动子联合有活性的P2启动子及强Pant启动子联合无活性的P2启动子。
第1类整合子可变区启动子位于基因盒重组位点attI上游,可变区基因盒在可变区启动子的作用下进行转录时需经过attI位点,并使attI位点双链解开,可能影响整合酶蛋白与双链attI位点结合,从而对整合酶介导的基因盒位点特异性重组产生影响。因此本研究对第1类整合子中不同可变区启动子对基因盒整合频率的影响进行研究。运用实时定量PCR检测高表达整合酶的条件下,基因盒插入不同可变区启动子下游attI位点的整合频率,结果发现含弱Pant启动子的整合子中基因盒的整合频率最高,是整合频率最低的强Pant启动子联合有活性P：启动子的整合子中基因盒整合频率的74倍,同时发现整合子中基因盒的整合频率与P2启动子种类密切相关,在含有无活性P2启动子的整合子中基因盒的整合频率明显高于含有有活性P2启动子的整合子,在含有相同P2启动子的整合子中基因盒的整合频率与整合子可变区基因盒的表达水平成负相关。提示整合子需要基因盒高表达以适应外界环境时,可通过降低整合频率以维持整合子结构的相对稳定。
研究表明细菌处于应激状态下,整合频率会大大提高,表明不同的外界环境可影响整合子捕获基因盒的效率,而外界环境的变化是通过细菌体内不同基因表达水平的变化而影响整合子捕获基因盒的效率的,因此,宿主菌中可能存在着影响整合子捕获基因盒效率的因素。转座子插入突变文库是比较成熟的筛选细菌中影响某一表型或生物学性状的基因的方法,但现有的检测整合子中基因盒剪切与整合频率的方法都不适用于对大规模转座子插入突变文库进行高通量筛选。本研究建立一种利用“乳头状试验”检测整合子中基因盒剪切频率的方法。lacα基因的读码框由于基因盒aadA5的插入而不能翻译成有功能的β-半乳糖苷酶的α片段,当lacα基因读码框内插入的基因盒aadA5在整合酶蛋白的作用下被剪切下来,lacα基因读码框能翻译成有功能的β-半乳糖苷酶的α片段,在大肠杆菌JM109中能发生α互补现象,由于基因盒aadA5的剪切是在细菌生长过程中发生的,因此在含X-gal与IPTG的平板上,在长出的白色菌落中会出现基因盒aadA5发生剪切的细菌生长所形成的蓝色斑点,而蓝色斑点出现的早晚、数量的多少与剪切频率有关,可通过观察蓝色斑点出现的早晚以及计数白色菌落中蓝色斑点的数量来评价剪切频率。该方法具有简便、高通量、费用低等优点,可运用以对转座子插入突变文库进行高通量筛选以寻找宿主菌中影响整合酶介导的基因盒剪切频率的因素。
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.
引文
[1]Hall RM, Collis CM. Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination [J].Mol Microbiol,1995,15:593-600.
[2]Cameron FH, Groot Obbink DJ,Ackerman VP,et al. Nucleotide sequence of AAD(2") aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfr Ⅱ in R388 [J].Nucl Acids Res,1986,14:8625-8635.
[3]Mazel D. Integrons:agents of bacterial evolution [J].Nat Rev Microbiol,2006,4: 608-620.
[4]Hall RM, Stokes HW. Integrons:novel DNA elemens which capture genes by site-specific recombination [J].Genetica,1993,90:115-132.
[5]Stokes HW, Hall RM. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions:integrons [J].Mol Microbiol, 1989,3:1669-1683.
[6]Collis CM, Hall RM. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase [J].J Bacteriol,1992,174: 1574-1585.
[7]Collis CM, Kim MJ, Stokes HW, et al. Binding of the purified integron DNA integrase IntI1 to integron-and cassette-associated recombination sites [J]. Mol Microbiol,1998,29:477-490.
[8]Gravel A, Fournier B, Roy PH. DNA complexes obtained with the integron integrase IntI1 at the attll site [J].Nucl Acids Res,1998,26:4347-4355.
[9]Francia MV, Zabala JC, de la Cruz F, et al. The IntI1 integron integrase preferentially binds single-stranded DNA of the attC site [J].J Bacteriol,1999, 181:6844-6849.
[10]Collis CM, Hall RM. Expression of antibiotic resistance genes in the integrated cassettes of integrons [J]. Antimicrob Agents Chemother,1995,39:155-162.
[11]Beaber JW, Hochut B, Waldor MK. SOS response promotes horizontal dissemination of antibiotic resistance genes [J].Nature,2004,427:72-74.
[12]Guerin E, Cambray G, Sanchez-Alberola N, et al. The SOS response controls integron recombination [J].Science,2009,324:1034.
[13]MacDonald D, Demarre G, Bouvier M, et al. Structural basis for broad DNA-specificity in integron recombination [J].Nature,2006,440:1157-1162.
[14]Walsh TR. Combinatorial genetic evolution of multiresistance [J]. Curr Opin Microbiol,2006,9:476-482.
[15]Skold O. Resistance to trimethoprim and sulfonamides [J].Vet Res,2001,32: 261-273.
[16]Agersφ Y, Peirano G, Aarestrup FM. dfrA25, a novel trimethoprim resistance gene from Salmonella Agona isolated from a human urine sample in Brazil [J].J. AntimicrobChemother,2006,58:1044-1047.
[17]Poirel L, Naas T, Guibert M, et al. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene [J].Antimicrob Agents Chemother,1999,43: 573-581.
[18]Clowes RC. Molecular structure of bacterial plasmids [J].Bacteriol Rev,1972,36: 361-405.
[19]Rodriguez I, Martin MC, Mendoza MC, et al. Class 1 and class 2 integrons in non-prevalent serovars of Salmonella enterica: structure and association with transposons and plasmids [J].J Antimicrob Chemother,2006,58:1124-1132.
[20]Ahmer BMM, Tran M, Heffron F. The virulence plasmid of Salmonella typhinurium is self-transmissible [J].J Bacteriol,1999,181:1364-1368.
[21]Polland S, Llosa M, Avila P, et al. General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids [J]. J Bacteriol,1990,172:5795-5802.
[22]Chamier B, Lorenz MG, Wackernagel W. Natural transformation of Acinetobacter calcoaceticus by plasmid DNA adsorbed on sand and groundwater aquifer material [J].Appl Environ Microbiol,1993,59:1662-1667.
[23]Schafer A, Kalinowski J, Simon R, et al. High-frequency conjugal plasmid transfer from Gram-negative Escherichia coli to various Gram-positive coryneform bacteria [J].J Bacteriol,1990,172:1663-1666.
[24]Poirel L, Menuteau O, Agoli N, et al. Outbreak of extended-spectrum-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French Hospital [J]. J Clin Microbiol,2003,41:3542-3547.
[25]Yoshida N, Sato M. Plasmid uptake by bacteria:a comparison of methods and efficiencies [J].Appl Microbiol Biotechnol,2009,83:791-798.
[26]Zong Z, Partridge SR, Iredell JR. A blaVEB-1 variant, blaVEB-6, associated with repeated elements in a complex genetic structure [J].Antimicrob Agents
Chemother,2009,53:1693-1697.
[27]Ohse M, Takahashi K, Kadowaki Y, et al. Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation [J].Biosci Biotechnol Biochem,1995,59:1433-1437.
[28]Szostkova M, Horakova D. The effect of plasmid DNA sizes and other factors on electrotransformation of Escherichia coli JM109 [J].Bioelectrochemistry,1998,47: 319-323.
[29]Jiang X, Ni Y, Jiang Y, et al. Outbreak of infection caused by Enterobacter cloacae producing the novel VEB-3 beta-lactamase in China [J].J Clin Microbiol, 2005,43:826-831.
[30]Barton BM, Harding GP, Zuccarelli AJ. A general method for detecting and sizing large plasmids [J].Analytical Biochemistry,1995,226:235-240.
[31]Avila P, de la Cruz F. Physical and genetic map of the IncW plasmid R388. [J].Plasmid,1988,20:155-157.
[32]Provedenti MA, O'Brien JM, Ewing RJ, et al. The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR [J].J Microbiol Methods,2006,65:476-487.
[33]Bito A, Susani M. Revised analysis of aadA2 gene of plasmid pSa [J]. Antimicrob Agents Chemother,1994,38:1172-1175.
[34]Martinez E, de la Cruz F. Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes [J].Embo J,1990;9:1275-1281.
[35]Levesque C, Brassard S, Lapointe J, et al. Diversity and relative strength of tandem promoters for antibiotic-resistance genes of several integrons [J].Gene, 1994,142:49-54.
[36]Hanau-Ber(?)ot B, Podglajen I, Casin I, et al. An intrinsic control element for translational initiation in class 1 integrons [J].Mol Microbiol,2002,44:119-130.
[37]Jacquier H, Zaoui C, Pors MS, et al. Translation regulation of integron gene cassette expression by the attC site [J].Mol Microbiol,2009,76:1475-1486.
[38]Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosome [J].Mol Cell Biol,1987,7:3438-3445.
[39]Rowe-Magnus DA, Guerout AM, Mazel D. Bacterial resistance evolution by recruitment of super-integron gene cassettes [J].Mol Microbiol,2002,43: 1657-1669.
[40]Blattner FR, Plunkett Ⅲ G, Bloch CA, Perna NT, Burland V, Riley M, et al. The complete genome sequence of Escherichia coli K-12 [J].Science,1997,277: 1453-1462.
[41]Chalut C, Egly J-M. AUC is used as a start codon in Escherichia coli [J].Gene 1995,156:43-45.
[42]Papagiannitsis CC, Tzouvelekis LS, Miriagou V. Relative strength of the class 1 integron promoter hybrid 2 and the combinations of strong and hybrid 1 with an active P2 promoter [J]. Antimicrob Agents Chemother,2009,53:277-280.
[43]Dayn A, Malkhosyan S, Mirkin SM. Transcriptionally driven cruciform formation in vivo [J].Nucl Acids Res,1992,20:5991-5997.
[44]Rahmouni AR, Wells RD. Direct evidence for the effect of transcription on local DNA supercoiling in vivo [J].J Mol Biol,1992,223:131-144.
[45]Krasilnokov AS, Podtelezhnikov A, Vologodskii A, et al. Large-scale effects of transcriptional DNA supercoiling in vivo [J] J Mol Biol,1999,292:1149-1160.
[46]Pananicolaou C, Ripley LS. An in vitro approach to identifying specificity determinants of mutagenesis mediated by DNA misalignments [J].J Mol Biol, 1991,221:805-821.
[47]Goryshin LY, and Reznikoff WS.Tn5 in vitro transposition [J].J Biol.Chem,1998, 273:7367-7374.
[48]Goryshin LY, Jendrisak J, Hoffman LM. Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes [J].Nature Biotech, 2000,6:796-800.
[49]Twiss E, Coros AM, Tavakoli NP, et al. Transposition is modulated by a diverse set of host factors in Escherichia coli and is stimulated by nutritional stress [J]. Mol Microbiol,2005,57:1593-1607.
[50]Zehua Y, Xiaofei J, Quhao W, et al. A novel and rapid method for determining integration frequency catalyzed by integron integrase intIl [J].J.Microbiol Methods,2009,76:97-100.
[51]Huisman O, Kleckner N. A new generalizable test for detection of mutations affecting Tn10 transposition [J].Genetics,1987,116:185-189.
[52]Swingle B, O'Carroll M, David Haniford D, et al. The effect of host-encoded nucleoid proteins on transposition:H-NS influences targeting of both IS903 and Tn10 [J].Mol Microbiol,2004,52:1055-1067.
[53]Coros AM, Twiss E, Tavakoli NP, et al. Genetic Evidence that GTP Is Required for Transposition of IS903 and Tn552 in Escherichia coli [J].J Bacteriol,2005, 187:4598-4606.
[54]Bouvier M, Demarre G, Mazel D. Integron cassette insertion:a recombination process involving a folded single strand substrate [J] Embo J,2005,24: 4356-4367.
[55]Bouvier M, Ducos-Galand M, Loot C, Bikard D, Mazel D. Structural features of single-stranded integron cassette attC sites and their role in strand selection [J]. PloS Genet,2009,5:e1000632.doi:10.1371/journal.pgen.1000632.
[56]Demarre G, Frumerie C, Gopaul DN, Mazel D. Identification of key structural determinants of the IntI1 integron integrase that influence attCXattll recombination efficiency [J].Nucleic Acids Res,2007,35:6475-6489.
[57]Frumerie C, Ducos-Galand M, Gopaul DN, Mazel D. The relaxed requirements of the integron cleavage site allow predictable changes in integron target specificity [J].Nucleic Acids Res,2010,38:559-569.
[58]Jove T, Da Re S, Denis F, et al. Inverse correlation between promoter strength and excision activity in class 1 integrons [J].PloS Genet,2010,6:e1000793. doi:10.1371/journal.pgen.1000793.
[1]Hall RM, Collis CM. Mobile gene cassettes and integrons:capture and spread of genes by site-specific recombination [J].Mol Microbiol,1995,15:593-600.
[2]Cameron FH, Groot Obbink DJ, Ackerman VP, et al.Nucleotide sequence of AAD(2") aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfr II in R388 [J].Nucl Acids Res,1986,14:8625-8635.
[3]Swedberg G. Organization of two sulfonamide resistance genes on plasmids of gram-negative bacteria [J].Antimicrob Agents Chemother,1987,31:306-311.
[4]Sundstrom L, Vinayagamoorthy T, Skold O. Novel type of plasmid-borne resistance to trimethoprim [J].Antimicrob Agents Chemother,1987,31:60-66.
[5]Stokes HW, Hall RM. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions:integrons [J].Mol Microbiol, 1989,3:1669-1683.
[6]Hall RM, Stokes HW. Integrons:novel DNA elemens which capture genes by site-specific recombination [J].Genetica,1993,90:115-132.
[7]Collis CM,Hall RM. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase [J].J Bacteriol,1992,174: 1574-1585.
[8]Fluit AC, Schmitz FJ. Class 1 integrons, gene cassettes,mobility and Epidemiology [J]. Eur J Clin Microbiol Infect Dis,1999,18:761-770.
[9]Recchia GD, Hall RM. Gene cassettes:a new class of mobile element [J]. Microbiology,1995,141:3015-3027.
[10]Hall RM, Collis CM. Mobile gene cassettes and integrons in evolution [J].Ann N Y Acad Sci,1999,18:68-80.
[11]Arakawa Y, Murakami M, Suzuki K, et al. A novel integron-like element carrying the metallo-β-lactamase gene blaIMP [J]. Antimicrob Agents Chemother,1995,39:1612-1615.
[12]Correia M, Boavida F, Grosso F, et al. Molecular characterization of a new class 3 integron in Klebsiella pneumoniae [J].Antimicrob Agents Chemother, 2003,47:2838-2843.
[13]Mazel D, Dychinco B, Webb VA, et al. A distinctive class of integron in the Vibrio chloerae genome [J].Science,1998,280:605-608.
[14]Mazel D. Integrons: agents of bacterial evolution [J].Nat Rev Microbiol, 2006,4:608-620.
[15]Stokes HW, O' Gorman DB, Recchia GD, et al. Structure and function of 59-base element recombination sites associated with mobile gene cassettes [J].Mol Microbiol,1997,26:731-745.
[16]Collis C M, Hall RM. Expression of antibiotic resistance genes in the integrated cassettes of integrons [J].Antimicrob Agents Chemother,1995,39: 155-162.
[17]Recchia GD, Hall RM. Origins of the mobile gene cassettes found in integrons [J].Trends Microbiol,1997,5:389-394.
[18]Stokes HW, Holmes AJ, Nield BS, et al.Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA [J].Appl Environ Microbiol,2001,67:5240-5246.
[19]Collis CM, Grammaticopoulos G, Briton J, et al. Site-specific insertion of gene cassettes into integrons [J].Mol Microbiol,1993,9:41-52.
[20]Collis, CM, Hall RM. Gene cassettes from the insert region of integrons are excised as covalently closed circles [J].Mol Microbiol,1992,6:2875-2885.
[21]Gravel A, Messier N, Roy PH. Point mutations in the integron integrase IntI1 that affect recombination and /or substrate recognition [J].J Bacteriol,1998, 180:5437-5442.
[22]Messier N, Roy PH. Integron integrases possess a unique additional domain necessary for activity [J].J Bacteriol,2001,183:6699-6706.
[23]Partridge SR, Recchia GD, Scaramuzzi C, et al. Definition of the attI1 site of class 1 integrons [J].Microbiology,2000,146:2855-2865.
[24]Collis CM, Kim MJ, Stokes HW, et al. Binding of the purified integron DNA integrase IntI1 to integron- and cassette-associated recombination sites [J]. Mol Microbiol,1998,29:477-490.
[25]Gravel A, Fournier B, Roy PH. DNA complexes obtained with the integron integrase IntI1 at the attI1 site [J].Nucl Acids Res,1998,26:4347-4355.
[26]Francia MV, Zabala JC, de la Cruz F, et al. The IntI1 integron integrase preferentially binds single-stranded DNA of the attC site [J].J Bacteriol,1999, 181:6844-6849.
[27]Johansson C, Kamali-Moghaddam M, Sundstrom L. Integron integrase binds to bulged hairpin DNA [J].Nucleic Acids Res,2004,32:4033-4043.
[28]Recchia GD, Stokes HW, Hall RM. Characterization of specific and secondary recombination sites recognized by the integron DNA integrase [J].Nucleic Acids Res,1994,22:2071-2078.
[29]Grape M, Farra A, Kronvall G, et al. Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram- negative bacteria [J].Clin Microbiol Infect,2005,11:185-192.
[30]Gombac F, Riccio ML, Rossolini GM, et al.Molecular Characterization of Integrons in Epidemiologically Unrelated Clinical Isolates of Acinetobacter baumannii from Italian Hospitals Reveals a Limited Diversity of Gene Cassette Arrays [J].Antimicrob Agents Chemother,2002,46:3665-3668.
[31]Su J, Shi L,Yang L, et al.Analysis of integrons in clinical isolates of Escherichia coli in China during the last six years [J].FEMS Microbiol Lett, 2006,254:75-80.
[32]Shi L, Zheng MP, Xiao ZH, et al.Unnoticed spread of class 1 integrons in gram-positive clinical strains isolated in Guangzhou,China [J].Microbiol Immunol,2006,50:463-467.
[33]Rosser SJ, Young HK. Identification and characterization of class 1 integrons in bacteria from an aquatic environment [J].J Antimicrob Chemother,1999,44: 11-18.
[34]Dillon B, Thomas L, Mohmand G, et al. Multiplex PCR for screening of integrons in bacterial lysates [J].J Microbiol Methods,2005,62:221-232.
[35]冯洁,石磊,肖增璜,等.细菌耐药整合子intl分类的多重PCR方法的建立及应用[J].中国抗生素杂志,2005,30：599-603.
[36]李心晖,石磊,杨维青,等.三类整合酶基因(intl)的简并引物PCR方法建立及应用[J].中华微生物学和免疫学杂志,2005,25：156-160.
[37]Maguire AJ, Brown DF, Gray JJ, et al. Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology [J].Antimicrob Agents Chemother,2001, 45:1022-1029.
[38]石磊,陈洵,肖增璜,等.多重耐药临床菌株中整合子结构的检测与分析[J].中国抗生素杂志,2007,32：43-48.
[39]张宏梅,石磊,李琳,等.第一类整合子整合酶基因intI1的定位分析[J].中国抗生素杂志,29：300-302.
[2]Cameron FH, Groot Obbink DJ,Ackerman VP,et al. Nucleotide sequence of AAD(2") aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfr Ⅱ in R388 [J].Nucl Acids Res,1986,14:8625-8635.
[3]Mazel D. Integrons:agents of bacterial evolution [J].Nat Rev Microbiol,2006,4: 608-620.
[4]Hall RM, Stokes HW. Integrons:novel DNA elemens which capture genes by site-specific recombination [J].Genetica,1993,90:115-132.
[5]Stokes HW, Hall RM. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions:integrons [J].Mol Microbiol, 1989,3:1669-1683.
[6]Collis CM, Hall RM. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase [J].J Bacteriol,1992,174: 1574-1585.
[7]Collis CM, Kim MJ, Stokes HW, et al. Binding of the purified integron DNA integrase IntI1 to integron-and cassette-associated recombination sites [J]. Mol Microbiol,1998,29:477-490.
[8]Gravel A, Fournier B, Roy PH. DNA complexes obtained with the integron integrase IntI1 at the attll site [J].Nucl Acids Res,1998,26:4347-4355.
[9]Francia MV, Zabala JC, de la Cruz F, et al. The IntI1 integron integrase preferentially binds single-stranded DNA of the attC site [J].J Bacteriol,1999, 181:6844-6849.
[10]Collis CM, Hall RM. Expression of antibiotic resistance genes in the integrated cassettes of integrons [J]. Antimicrob Agents Chemother,1995,39:155-162.
[11]Beaber JW, Hochut B, Waldor MK. SOS response promotes horizontal dissemination of antibiotic resistance genes [J].Nature,2004,427:72-74.
[12]Guerin E, Cambray G, Sanchez-Alberola N, et al. The SOS response controls integron recombination [J].Science,2009,324:1034.
[13]MacDonald D, Demarre G, Bouvier M, et al. Structural basis for broad DNA-specificity in integron recombination [J].Nature,2006,440:1157-1162.
[14]Walsh TR. Combinatorial genetic evolution of multiresistance [J]. Curr Opin Microbiol,2006,9:476-482.
[15]Skold O. Resistance to trimethoprim and sulfonamides [J].Vet Res,2001,32: 261-273.
[16]Agersφ Y, Peirano G, Aarestrup FM. dfrA25, a novel trimethoprim resistance gene from Salmonella Agona isolated from a human urine sample in Brazil [J].J. AntimicrobChemother,2006,58:1044-1047.
[17]Poirel L, Naas T, Guibert M, et al. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene [J].Antimicrob Agents Chemother,1999,43: 573-581.
[18]Clowes RC. Molecular structure of bacterial plasmids [J].Bacteriol Rev,1972,36: 361-405.
[19]Rodriguez I, Martin MC, Mendoza MC, et al. Class 1 and class 2 integrons in non-prevalent serovars of Salmonella enterica: structure and association with transposons and plasmids [J].J Antimicrob Chemother,2006,58:1124-1132.
[20]Ahmer BMM, Tran M, Heffron F. The virulence plasmid of Salmonella typhinurium is self-transmissible [J].J Bacteriol,1999,181:1364-1368.
[21]Polland S, Llosa M, Avila P, et al. General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids [J]. J Bacteriol,1990,172:5795-5802.
[22]Chamier B, Lorenz MG, Wackernagel W. Natural transformation of Acinetobacter calcoaceticus by plasmid DNA adsorbed on sand and groundwater aquifer material [J].Appl Environ Microbiol,1993,59:1662-1667.
[23]Schafer A, Kalinowski J, Simon R, et al. High-frequency conjugal plasmid transfer from Gram-negative Escherichia coli to various Gram-positive coryneform bacteria [J].J Bacteriol,1990,172:1663-1666.
[24]Poirel L, Menuteau O, Agoli N, et al. Outbreak of extended-spectrum-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French Hospital [J]. J Clin Microbiol,2003,41:3542-3547.
[25]Yoshida N, Sato M. Plasmid uptake by bacteria:a comparison of methods and efficiencies [J].Appl Microbiol Biotechnol,2009,83:791-798.
[26]Zong Z, Partridge SR, Iredell JR. A blaVEB-1 variant, blaVEB-6, associated with repeated elements in a complex genetic structure [J].Antimicrob Agents
Chemother,2009,53:1693-1697.
[27]Ohse M, Takahashi K, Kadowaki Y, et al. Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation [J].Biosci Biotechnol Biochem,1995,59:1433-1437.
[28]Szostkova M, Horakova D. The effect of plasmid DNA sizes and other factors on electrotransformation of Escherichia coli JM109 [J].Bioelectrochemistry,1998,47: 319-323.
[29]Jiang X, Ni Y, Jiang Y, et al. Outbreak of infection caused by Enterobacter cloacae producing the novel VEB-3 beta-lactamase in China [J].J Clin Microbiol, 2005,43:826-831.
[30]Barton BM, Harding GP, Zuccarelli AJ. A general method for detecting and sizing large plasmids [J].Analytical Biochemistry,1995,226:235-240.
[31]Avila P, de la Cruz F. Physical and genetic map of the IncW plasmid R388. [J].Plasmid,1988,20:155-157.
[32]Provedenti MA, O'Brien JM, Ewing RJ, et al. The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR [J].J Microbiol Methods,2006,65:476-487.
[33]Bito A, Susani M. Revised analysis of aadA2 gene of plasmid pSa [J]. Antimicrob Agents Chemother,1994,38:1172-1175.
[34]Martinez E, de la Cruz F. Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes [J].Embo J,1990;9:1275-1281.
[35]Levesque C, Brassard S, Lapointe J, et al. Diversity and relative strength of tandem promoters for antibiotic-resistance genes of several integrons [J].Gene, 1994,142:49-54.
[36]Hanau-Ber(?)ot B, Podglajen I, Casin I, et al. An intrinsic control element for translational initiation in class 1 integrons [J].Mol Microbiol,2002,44:119-130.
[37]Jacquier H, Zaoui C, Pors MS, et al. Translation regulation of integron gene cassette expression by the attC site [J].Mol Microbiol,2009,76:1475-1486.
[38]Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosome [J].Mol Cell Biol,1987,7:3438-3445.
[39]Rowe-Magnus DA, Guerout AM, Mazel D. Bacterial resistance evolution by recruitment of super-integron gene cassettes [J].Mol Microbiol,2002,43: 1657-1669.
[40]Blattner FR, Plunkett Ⅲ G, Bloch CA, Perna NT, Burland V, Riley M, et al. The complete genome sequence of Escherichia coli K-12 [J].Science,1997,277: 1453-1462.
[41]Chalut C, Egly J-M. AUC is used as a start codon in Escherichia coli [J].Gene 1995,156:43-45.
[42]Papagiannitsis CC, Tzouvelekis LS, Miriagou V. Relative strength of the class 1 integron promoter hybrid 2 and the combinations of strong and hybrid 1 with an active P2 promoter [J]. Antimicrob Agents Chemother,2009,53:277-280.
[43]Dayn A, Malkhosyan S, Mirkin SM. Transcriptionally driven cruciform formation in vivo [J].Nucl Acids Res,1992,20:5991-5997.
[44]Rahmouni AR, Wells RD. Direct evidence for the effect of transcription on local DNA supercoiling in vivo [J].J Mol Biol,1992,223:131-144.
[45]Krasilnokov AS, Podtelezhnikov A, Vologodskii A, et al. Large-scale effects of transcriptional DNA supercoiling in vivo [J] J Mol Biol,1999,292:1149-1160.
[46]Pananicolaou C, Ripley LS. An in vitro approach to identifying specificity determinants of mutagenesis mediated by DNA misalignments [J].J Mol Biol, 1991,221:805-821.
[47]Goryshin LY, and Reznikoff WS.Tn5 in vitro transposition [J].J Biol.Chem,1998, 273:7367-7374.
[48]Goryshin LY, Jendrisak J, Hoffman LM. Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes [J].Nature Biotech, 2000,6:796-800.
[49]Twiss E, Coros AM, Tavakoli NP, et al. Transposition is modulated by a diverse set of host factors in Escherichia coli and is stimulated by nutritional stress [J]. Mol Microbiol,2005,57:1593-1607.
[50]Zehua Y, Xiaofei J, Quhao W, et al. A novel and rapid method for determining integration frequency catalyzed by integron integrase intIl [J].J.Microbiol Methods,2009,76:97-100.
[51]Huisman O, Kleckner N. A new generalizable test for detection of mutations affecting Tn10 transposition [J].Genetics,1987,116:185-189.
[52]Swingle B, O'Carroll M, David Haniford D, et al. The effect of host-encoded nucleoid proteins on transposition:H-NS influences targeting of both IS903 and Tn10 [J].Mol Microbiol,2004,52:1055-1067.
[53]Coros AM, Twiss E, Tavakoli NP, et al. Genetic Evidence that GTP Is Required for Transposition of IS903 and Tn552 in Escherichia coli [J].J Bacteriol,2005, 187:4598-4606.
[54]Bouvier M, Demarre G, Mazel D. Integron cassette insertion:a recombination process involving a folded single strand substrate [J] Embo J,2005,24: 4356-4367.
[55]Bouvier M, Ducos-Galand M, Loot C, Bikard D, Mazel D. Structural features of single-stranded integron cassette attC sites and their role in strand selection [J]. PloS Genet,2009,5:e1000632.doi:10.1371/journal.pgen.1000632.
[56]Demarre G, Frumerie C, Gopaul DN, Mazel D. Identification of key structural determinants of the IntI1 integron integrase that influence attCXattll recombination efficiency [J].Nucleic Acids Res,2007,35:6475-6489.
[57]Frumerie C, Ducos-Galand M, Gopaul DN, Mazel D. The relaxed requirements of the integron cleavage site allow predictable changes in integron target specificity [J].Nucleic Acids Res,2010,38:559-569.
[58]Jove T, Da Re S, Denis F, et al. Inverse correlation between promoter strength and excision activity in class 1 integrons [J].PloS Genet,2010,6:e1000793. doi:10.1371/journal.pgen.1000793.
[1]Hall RM, Collis CM. Mobile gene cassettes and integrons:capture and spread of genes by site-specific recombination [J].Mol Microbiol,1995,15:593-600.
[2]Cameron FH, Groot Obbink DJ, Ackerman VP, et al.Nucleotide sequence of AAD(2") aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfr II in R388 [J].Nucl Acids Res,1986,14:8625-8635.
[3]Swedberg G. Organization of two sulfonamide resistance genes on plasmids of gram-negative bacteria [J].Antimicrob Agents Chemother,1987,31:306-311.
[4]Sundstrom L, Vinayagamoorthy T, Skold O. Novel type of plasmid-borne resistance to trimethoprim [J].Antimicrob Agents Chemother,1987,31:60-66.
[5]Stokes HW, Hall RM. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions:integrons [J].Mol Microbiol, 1989,3:1669-1683.
[6]Hall RM, Stokes HW. Integrons:novel DNA elemens which capture genes by site-specific recombination [J].Genetica,1993,90:115-132.
[7]Collis CM,Hall RM. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase [J].J Bacteriol,1992,174: 1574-1585.
[8]Fluit AC, Schmitz FJ. Class 1 integrons, gene cassettes,mobility and Epidemiology [J]. Eur J Clin Microbiol Infect Dis,1999,18:761-770.
[9]Recchia GD, Hall RM. Gene cassettes:a new class of mobile element [J]. Microbiology,1995,141:3015-3027.
[10]Hall RM, Collis CM. Mobile gene cassettes and integrons in evolution [J].Ann N Y Acad Sci,1999,18:68-80.
[11]Arakawa Y, Murakami M, Suzuki K, et al. A novel integron-like element carrying the metallo-β-lactamase gene blaIMP [J]. Antimicrob Agents Chemother,1995,39:1612-1615.
[12]Correia M, Boavida F, Grosso F, et al. Molecular characterization of a new class 3 integron in Klebsiella pneumoniae [J].Antimicrob Agents Chemother, 2003,47:2838-2843.
[13]Mazel D, Dychinco B, Webb VA, et al. A distinctive class of integron in the Vibrio chloerae genome [J].Science,1998,280:605-608.
[14]Mazel D. Integrons: agents of bacterial evolution [J].Nat Rev Microbiol, 2006,4:608-620.
[15]Stokes HW, O' Gorman DB, Recchia GD, et al. Structure and function of 59-base element recombination sites associated with mobile gene cassettes [J].Mol Microbiol,1997,26:731-745.
[16]Collis C M, Hall RM. Expression of antibiotic resistance genes in the integrated cassettes of integrons [J].Antimicrob Agents Chemother,1995,39: 155-162.
[17]Recchia GD, Hall RM. Origins of the mobile gene cassettes found in integrons [J].Trends Microbiol,1997,5:389-394.
[18]Stokes HW, Holmes AJ, Nield BS, et al.Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA [J].Appl Environ Microbiol,2001,67:5240-5246.
[19]Collis CM, Grammaticopoulos G, Briton J, et al. Site-specific insertion of gene cassettes into integrons [J].Mol Microbiol,1993,9:41-52.
[20]Collis, CM, Hall RM. Gene cassettes from the insert region of integrons are excised as covalently closed circles [J].Mol Microbiol,1992,6:2875-2885.
[21]Gravel A, Messier N, Roy PH. Point mutations in the integron integrase IntI1 that affect recombination and /or substrate recognition [J].J Bacteriol,1998, 180:5437-5442.
[22]Messier N, Roy PH. Integron integrases possess a unique additional domain necessary for activity [J].J Bacteriol,2001,183:6699-6706.
[23]Partridge SR, Recchia GD, Scaramuzzi C, et al. Definition of the attI1 site of class 1 integrons [J].Microbiology,2000,146:2855-2865.
[24]Collis CM, Kim MJ, Stokes HW, et al. Binding of the purified integron DNA integrase IntI1 to integron- and cassette-associated recombination sites [J]. Mol Microbiol,1998,29:477-490.
[25]Gravel A, Fournier B, Roy PH. DNA complexes obtained with the integron integrase IntI1 at the attI1 site [J].Nucl Acids Res,1998,26:4347-4355.
[26]Francia MV, Zabala JC, de la Cruz F, et al. The IntI1 integron integrase preferentially binds single-stranded DNA of the attC site [J].J Bacteriol,1999, 181:6844-6849.
[27]Johansson C, Kamali-Moghaddam M, Sundstrom L. Integron integrase binds to bulged hairpin DNA [J].Nucleic Acids Res,2004,32:4033-4043.
[28]Recchia GD, Stokes HW, Hall RM. Characterization of specific and secondary recombination sites recognized by the integron DNA integrase [J].Nucleic Acids Res,1994,22:2071-2078.
[29]Grape M, Farra A, Kronvall G, et al. Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram- negative bacteria [J].Clin Microbiol Infect,2005,11:185-192.
[30]Gombac F, Riccio ML, Rossolini GM, et al.Molecular Characterization of Integrons in Epidemiologically Unrelated Clinical Isolates of Acinetobacter baumannii from Italian Hospitals Reveals a Limited Diversity of Gene Cassette Arrays [J].Antimicrob Agents Chemother,2002,46:3665-3668.
[31]Su J, Shi L,Yang L, et al.Analysis of integrons in clinical isolates of Escherichia coli in China during the last six years [J].FEMS Microbiol Lett, 2006,254:75-80.
[32]Shi L, Zheng MP, Xiao ZH, et al.Unnoticed spread of class 1 integrons in gram-positive clinical strains isolated in Guangzhou,China [J].Microbiol Immunol,2006,50:463-467.
[33]Rosser SJ, Young HK. Identification and characterization of class 1 integrons in bacteria from an aquatic environment [J].J Antimicrob Chemother,1999,44: 11-18.
[34]Dillon B, Thomas L, Mohmand G, et al. Multiplex PCR for screening of integrons in bacterial lysates [J].J Microbiol Methods,2005,62:221-232.
[35]冯洁,石磊,肖增璜,等.细菌耐药整合子intl分类的多重PCR方法的建立及应用[J].中国抗生素杂志,2005,30：599-603.
[36]李心晖,石磊,杨维青,等.三类整合酶基因(intl)的简并引物PCR方法建立及应用[J].中华微生物学和免疫学杂志,2005,25：156-160.
[37]Maguire AJ, Brown DF, Gray JJ, et al. Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology [J].Antimicrob Agents Chemother,2001, 45:1022-1029.
[38]石磊,陈洵,肖增璜,等.多重耐药临床菌株中整合子结构的检测与分析[J].中国抗生素杂志,2007,32：43-48.
[39]张宏梅,石磊,李琳,等.第一类整合子整合酶基因intI1的定位分析[J].中国抗生素杂志,29：300-302.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |