整合子捕获基因盒效率调控机制研究
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摘要
目前细菌的耐药性非常严重,尤其是多重耐药菌株的出现是对临床细菌感染性疾病治疗的一个巨大挑战。耐单种抗生素的菌株的出现可能在人们的预料之中,可能是基因突变造成的结果,那么在同一菌株中同时出现对多种抗生素的耐药性,就很难用基因突变来解释。现在研究证明遗传物质在菌种内以及菌种间的水平传播,是细菌获得新的耐药性的重要手段。在对一组来自社区并且至少在一个月未服用抗生素的健康人群进行研究,所分离到的大肠杆菌中整合子的出现频率为15%。Jones等人的研究表明携带有整合子的菌株要比不携带整合子的菌株更容易产生对多种抗生素的耐药性。到目前为止,已发现70多种耐药性基因盒。这些资料说明整合子在耐药基因的水平传播以及多重耐药菌株的出现中起重要作用。
整合子在某些情况下通过捕获有利的基因盒,比如耐药性基因盒而获得生存优势,但也有可能捕获某些对宿主产生毒性的基因盒而造成不利的影响,或者过度捕获基因盒而对宿主菌的繁殖造成一定负担。因此,细菌的整合子在捕获外来基因盒的过程中,一定有一套完整而又精细的调节机制,但这一机制仍未明了。对于这一机制的研究,有助于加深理解整合子在耐药基因的水平传播中的作用,对于临床上开发有效的药物来阻断或延缓这一过程提供有益的帮助。同时,为利用整合子捕获基因盒定点、定向的特性,将其开发为一种基因重组工具奠定基础。为此,我们将从四个方面研究影响整合子捕获基因盒的因素,分别是宿主因子、基因盒本身,主要是基因盒长度、外界环境因素,主要是抗生素的浓度、整合酶表达水平。
1.测定整合子捕获基因盒效率的新方法的建立
以前广泛使用的测定整合酶所催化的重组反应的效率,是利用接合实验用表型筛选的办法来进行。这种方法操作麻烦、费时、相对的精密度低,不便于整合子捕获基因盒机制的深入研究。因此我们建立了一种基于荧光定量PCR技术,从分子水平来测定整合频率的方法。首先我们用pUC19为载体构建了高表达整合酶的重组质粒pUCINT,随后我们在pACYC184载体的不同位置分别插入了整合子以及aadA2基因盒,该重组质粒命名为pACINAD,将这两种相容的重组质粒转化大肠杆菌BL21(DE3),过夜培养后用荧光定量PCR技术测定发生整合作用的整合子的拷贝数和总的整合子的拷贝数,前者除以后者即为整合频率。结果在大肠杆菌BL21(DE3)宿主中所测得的整合频率为1.87×10~(-4),CV值为24.6%,而在没有整合酶高表达的情况下的背景整合频率低于5.23×10~(-8)。该方法的建立为后续深入研究整合子捕获基因盒的调控机制奠定了基础。
2.宿主因子对于整合效率影响的研究
现有资料提示,整合子在捕获基因盒的过程中可能还有其它宿主因子的参与,目前还没有这方面的具体研究。为此我们利用转座子,对同时含有重组质粒pUCINT和pACINAD的大肠杆菌BL21(DE3),建立插入突变文库。利用前述方法对该突变文库进行筛选,找到一株整合频率较其相应野生株提高26倍的突变株,将其命名为TIM。利用大肠杆菌基因芯片对该突变株和其相应野生株表达有差异的基因进行筛选,结果检测到表达有差异的基因共有52个,其中上调基因33个,下调基因19个。涉及到原噬菌体DLP12共有8个,这些基因表达均上调,它们分别为ybcT、peaD′、ybcW、exoD′、ybcX、nohB、renD′、ybcV。这引起了我们的重视,因为现有资料也表明存在于众多细菌染色体上的原噬菌体,与我们所研究的整合子同样是细菌基因组进化的重要工具。利用荧光定量PCR技术对上述部分基因的表达水平进行验证,结果与基因芯片的检测数据相符。进一步的研究表明ybcV、ybcW和nohB基因对整合反应具有一定的促进作用。
3.基因盒长度对于整合效率的影响
到目前为止,有关整合酶介导的对基因盒的切除和整合作用的确切机制仍未明了。整合子中存在临床上所使用的绝大多数抗生素耐药性基因,然而其中有关的基因盒长度很少超过1kb。因此我们假设长度不超过1 kb的基因盒是整合酶所催化的重组反应的最适底物。为此我们构建了一系列携带有相同attC位点但长度不同的基因盒的重组质粒。在论文第一部分所建立的方法基础之上,测定了整合酶所催化的对上述不同长度的基因盒的切除和整合频率。结果显示切除频率波动在3.08×10~(-2)和1.08×10~(-2)之间,而没有整合酶高表达的背景频率低于2.38×10~(-7)。通过对PCR产物的序列测定表明,切除是通过两个attC位点的重组反应而发生。这些数据显示整合酶介导的对于不同长度的基因盒的切除频率差别不大。对于在质粒pACZC上携带的基因盒的切除频率仅为在质粒pSA24上携带的基因盒的切除频率的2.4倍,而后者基因盒的长度大约是前者基因盒的长度的7倍。反映出整合酶所催化的切除反应,对于基因盒长度变化不敏感。而对于上述不同长度基因盒的整合频率波动在8.18×10~(-5)和1.39×10~(-6)之间,随着基因盒长度的增加,整合频率明显地降低,基因盒的长度对整合酶所催化的整合效率影响较大。这些研究结果提示,在整合子捕获基因盒的过程中,整合作用可能为其限速步骤。同时我们证明随着基因盒长度的增加,基因盒的切除频率仅轻度降低,而整合频率则大幅度降低。反映了由整合酶所催化的分子内重组反应要比分子间重组反应容易的多,过长的基因盒可能影响整合酶和其DNA底物之间所形成的复合物的稳定性。
4.抗生素浓度对于整合效率的影响
整合子捕获基因盒具有随机性,在环境的强大选择压力下,最终获得最有利于宿主菌生存的基因盒排列和组成方式的细菌被保留下来。在临床中所分离到的整合子中的绝大多数基因盒,都是编码对已知抗生素的抗性,这可能与抗生素在临床上的广泛使用有关。但是到目前为止还没有整合酶催化的重组反应的效率和抗生素浓度的关系的报道。为此,我们把在第三位置含有一aadA2基因盒的整合子克隆到质粒pACYC184。该重组质粒转化高表达整合酶的大肠杆菌BL21(DE3)后,加入不同浓度的链霉素过夜培养后,利用论文第一部分所建立的方法测定aadA2基因盒在attI位点的重组效率。结果显示在没有整合酶高表达的情况下的背景频率低于1.75×10~(-7)。在高表达整合酶的情况下,aadA2基因盒能够在整合子的attI位点发生重组,但重组频率变化较大,随着链霉素浓度的增加整合频率从1.97×10~(-3)增加到1.32。表明一定浓度的抗生素能够影响整合频率。用Western blotting进一步证实,aadA2基因盒在attI位点的表达水平大约是其在整合子的第三位置表达水平的10倍。
5.整合酶表达水平对于整合效率的影响
如前所述在一定范围内提高整合酶的表达水平可以使整合频率显著提高,但是在此基础上再进一步提高整合酶的表达水平,是否会使整合频率进一步提高呢?我们测定了克隆在pUC19质粒上的整合酶基因在不同浓度的IPTG诱导下,所催化的对基因盒的整合频率。测定结果显示在不同的诱导剂浓度下,整合频率并未见显著的变化。为了更进一步证实这一现象,我们也测定了克隆在pET28a质粒上的整合酶基因在不同浓度的IPTG诱导下,所催化的对基因盒的整合频率。测定结果显示在不同的诱导剂浓度下,整合频率同样未见明显的变化。SDS-PAGE显示克隆在pET28a质粒上的整合酶基因经IPTG诱导后,表达明显提高,最后用Western blotting也证实了这点。这说明整合酶表达水平并不是决定其所介导的整合反应效率的唯一因素,也可能其它诸如基因盒attC位点以单链形式存在的整体水平、整合酶四聚体的稳定性等方面,对于整合反应也有一定的影响作用。
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 frequency
Lateral 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 frequency
Most 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 integrase
1.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.
整合子在某些情况下通过捕获有利的基因盒,比如耐药性基因盒而获得生存优势,但也有可能捕获某些对宿主产生毒性的基因盒而造成不利的影响,或者过度捕获基因盒而对宿主菌的繁殖造成一定负担。因此,细菌的整合子在捕获外来基因盒的过程中,一定有一套完整而又精细的调节机制,但这一机制仍未明了。对于这一机制的研究,有助于加深理解整合子在耐药基因的水平传播中的作用,对于临床上开发有效的药物来阻断或延缓这一过程提供有益的帮助。同时,为利用整合子捕获基因盒定点、定向的特性,将其开发为一种基因重组工具奠定基础。为此,我们将从四个方面研究影响整合子捕获基因盒的因素,分别是宿主因子、基因盒本身,主要是基因盒长度、外界环境因素,主要是抗生素的浓度、整合酶表达水平。
1.测定整合子捕获基因盒效率的新方法的建立
以前广泛使用的测定整合酶所催化的重组反应的效率,是利用接合实验用表型筛选的办法来进行。这种方法操作麻烦、费时、相对的精密度低,不便于整合子捕获基因盒机制的深入研究。因此我们建立了一种基于荧光定量PCR技术,从分子水平来测定整合频率的方法。首先我们用pUC19为载体构建了高表达整合酶的重组质粒pUCINT,随后我们在pACYC184载体的不同位置分别插入了整合子以及aadA2基因盒,该重组质粒命名为pACINAD,将这两种相容的重组质粒转化大肠杆菌BL21(DE3),过夜培养后用荧光定量PCR技术测定发生整合作用的整合子的拷贝数和总的整合子的拷贝数,前者除以后者即为整合频率。结果在大肠杆菌BL21(DE3)宿主中所测得的整合频率为1.87×10~(-4),CV值为24.6%,而在没有整合酶高表达的情况下的背景整合频率低于5.23×10~(-8)。该方法的建立为后续深入研究整合子捕获基因盒的调控机制奠定了基础。
2.宿主因子对于整合效率影响的研究
现有资料提示,整合子在捕获基因盒的过程中可能还有其它宿主因子的参与,目前还没有这方面的具体研究。为此我们利用转座子,对同时含有重组质粒pUCINT和pACINAD的大肠杆菌BL21(DE3),建立插入突变文库。利用前述方法对该突变文库进行筛选,找到一株整合频率较其相应野生株提高26倍的突变株,将其命名为TIM。利用大肠杆菌基因芯片对该突变株和其相应野生株表达有差异的基因进行筛选,结果检测到表达有差异的基因共有52个,其中上调基因33个,下调基因19个。涉及到原噬菌体DLP12共有8个,这些基因表达均上调,它们分别为ybcT、peaD′、ybcW、exoD′、ybcX、nohB、renD′、ybcV。这引起了我们的重视,因为现有资料也表明存在于众多细菌染色体上的原噬菌体,与我们所研究的整合子同样是细菌基因组进化的重要工具。利用荧光定量PCR技术对上述部分基因的表达水平进行验证,结果与基因芯片的检测数据相符。进一步的研究表明ybcV、ybcW和nohB基因对整合反应具有一定的促进作用。
3.基因盒长度对于整合效率的影响
到目前为止,有关整合酶介导的对基因盒的切除和整合作用的确切机制仍未明了。整合子中存在临床上所使用的绝大多数抗生素耐药性基因,然而其中有关的基因盒长度很少超过1kb。因此我们假设长度不超过1 kb的基因盒是整合酶所催化的重组反应的最适底物。为此我们构建了一系列携带有相同attC位点但长度不同的基因盒的重组质粒。在论文第一部分所建立的方法基础之上,测定了整合酶所催化的对上述不同长度的基因盒的切除和整合频率。结果显示切除频率波动在3.08×10~(-2)和1.08×10~(-2)之间,而没有整合酶高表达的背景频率低于2.38×10~(-7)。通过对PCR产物的序列测定表明,切除是通过两个attC位点的重组反应而发生。这些数据显示整合酶介导的对于不同长度的基因盒的切除频率差别不大。对于在质粒pACZC上携带的基因盒的切除频率仅为在质粒pSA24上携带的基因盒的切除频率的2.4倍,而后者基因盒的长度大约是前者基因盒的长度的7倍。反映出整合酶所催化的切除反应,对于基因盒长度变化不敏感。而对于上述不同长度基因盒的整合频率波动在8.18×10~(-5)和1.39×10~(-6)之间,随着基因盒长度的增加,整合频率明显地降低,基因盒的长度对整合酶所催化的整合效率影响较大。这些研究结果提示,在整合子捕获基因盒的过程中,整合作用可能为其限速步骤。同时我们证明随着基因盒长度的增加,基因盒的切除频率仅轻度降低,而整合频率则大幅度降低。反映了由整合酶所催化的分子内重组反应要比分子间重组反应容易的多,过长的基因盒可能影响整合酶和其DNA底物之间所形成的复合物的稳定性。
4.抗生素浓度对于整合效率的影响
整合子捕获基因盒具有随机性,在环境的强大选择压力下,最终获得最有利于宿主菌生存的基因盒排列和组成方式的细菌被保留下来。在临床中所分离到的整合子中的绝大多数基因盒,都是编码对已知抗生素的抗性,这可能与抗生素在临床上的广泛使用有关。但是到目前为止还没有整合酶催化的重组反应的效率和抗生素浓度的关系的报道。为此,我们把在第三位置含有一aadA2基因盒的整合子克隆到质粒pACYC184。该重组质粒转化高表达整合酶的大肠杆菌BL21(DE3)后,加入不同浓度的链霉素过夜培养后,利用论文第一部分所建立的方法测定aadA2基因盒在attI位点的重组效率。结果显示在没有整合酶高表达的情况下的背景频率低于1.75×10~(-7)。在高表达整合酶的情况下,aadA2基因盒能够在整合子的attI位点发生重组,但重组频率变化较大,随着链霉素浓度的增加整合频率从1.97×10~(-3)增加到1.32。表明一定浓度的抗生素能够影响整合频率。用Western blotting进一步证实,aadA2基因盒在attI位点的表达水平大约是其在整合子的第三位置表达水平的10倍。
5.整合酶表达水平对于整合效率的影响
如前所述在一定范围内提高整合酶的表达水平可以使整合频率显著提高,但是在此基础上再进一步提高整合酶的表达水平,是否会使整合频率进一步提高呢?我们测定了克隆在pUC19质粒上的整合酶基因在不同浓度的IPTG诱导下,所催化的对基因盒的整合频率。测定结果显示在不同的诱导剂浓度下,整合频率并未见显著的变化。为了更进一步证实这一现象,我们也测定了克隆在pET28a质粒上的整合酶基因在不同浓度的IPTG诱导下,所催化的对基因盒的整合频率。测定结果显示在不同的诱导剂浓度下,整合频率同样未见明显的变化。SDS-PAGE显示克隆在pET28a质粒上的整合酶基因经IPTG诱导后,表达明显提高,最后用Western blotting也证实了这点。这说明整合酶表达水平并不是决定其所介导的整合反应效率的唯一因素,也可能其它诸如基因盒attC位点以单链形式存在的整体水平、整合酶四聚体的稳定性等方面,对于整合反应也有一定的影响作用。
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 frequency
Lateral 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 frequency
Most 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 integrase
1.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.
引文
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