Biskri Latefa, Bouvier Marie, Guérout Anne-Marie, Boisnard Stéphanie, Mazel Didier
Unité Postulante Plasticité du Génome Bactérien, CNRS URA 2171, Département Structure et Dynamique des Génomes, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris, France.
J Bacteriol. 2005 Mar;187(5):1740-50. doi: 10.1128/JB.187.5.1740-1750.2005.
Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.
超级整合子（SIs）和多重耐药整合子（MRIs）有两个主要的结构差异：（i）超级整合子平台是固定的，而多重耐药整合子平台通常与可移动DNA元件相关联；（ii）超级整合子基因盒簇的重组位点（attC）高度同源，而多重耐药整合子盒阵列的重组位点在长度和序列上高度可变。为了确定后一种差异是否与重组活性的差异相关，我们对1类多重耐药整合子（IntI1）和霍乱弧菌超级整合子（VchIntIA）的整合酶进行了比较研究。我们开发了两种检测方法，使我们能够独立测量同源attI位点上盒式缺失和整合的频率。我们证明，VchIntIA有效重组的attC位点范围比IntI1有效重组的attC位点范围窄。将突变引入霍乱弧菌重复序列（VCRs），即霍乱弧菌超级整合子盒式的attC位点，使我们能够将影响VchIntIA和IntI1活性的位置映射到不同程度。使用共整合检测，我们确定在大肠杆菌中，IntI1催化的attI1-x-VCR重组比VchIntIA催化的attIVch-x-VCR重组效率高2600倍。我们在霍乱弧菌中进行了相同的实验，并确定VchIntIA催化的attIVch-x-VCR重组比在大肠杆菌中测得的重组高2000倍。综上所述，我们的结果表明，在霍乱弧菌超级整合子中，VchIntIA介导的底物识别和重组反应可能不同于1类多重耐药整合子模式。
