Panis Gaël, Méjean Vincent, Ansaldi Mireille
Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph Aiguier, Marseille 13402, Cedex 20, France.
J Biol Chem. 2007 Jul 27;282(30):21798-809. doi: 10.1074/jbc.M701827200. Epub 2007 May 31.
KplE1 is one of the 10 prophage regions of Escherichia coli K12, located at 2464 kb on the chromosome. KplE1 is defective for lysis, but it is fully competent for excisive recombination. In this study, we have mapped the binding sites of the recombination proteins, namely IntS, TorI, and IHF on attL and attR, and the organization of these sites suggests that the intasome is architecturally different from the lambda canonical form. We also measured the relative contribution of these proteins to both excisive and integrative recombination by using a quantitative in vitro assay. These experiments show a requirement of the TorI excisionase for excisive recombination and of the IntS integrase for both integration and excision. Moreover, we observed a strong influence of the supercoiled state of the substrates. The KplE1 recombination module, composed of the integrase and excisionase genes together with the attL and attR DNA regions, is highly similar to that of several phages infecting various E. coli strains as well as Shigella flexneri and Shigella sonnei. The in vitro recombination data reveal that HK620 and KplE1 att sequences are exchangeable. This study thus defines a new site-specific recombination module, and implications for the mechanism and regulation of recombination are discussed.
KplE1是大肠杆菌K12的10个原噬菌体区域之一，位于染色体上2464 kb处。KplE1的裂解功能存在缺陷，但它完全具备进行切除重组的能力。在本研究中，我们绘制了重组蛋白IntS、TorI和IHF在attL和attR上的结合位点，这些位点的组织方式表明整合体在结构上与λ噬菌体的典型形式不同。我们还通过定量体外试验测量了这些蛋白对切除重组和整合重组的相对贡献。这些实验表明，切除重组需要TorI切除酶，而整合和切除都需要IntS整合酶。此外，我们观察到底物超螺旋状态的强烈影响。由整合酶和切除酶基因以及attL和attR DNA区域组成的KplE1重组模块与几种感染不同大肠杆菌菌株以及弗氏志贺菌和宋内志贺菌的噬菌体的重组模块高度相似。体外重组数据表明HK620和KplE1的att序列是可交换的。因此，本研究定义了一个新的位点特异性重组模块，并讨论了其对重组机制和调控的意义。
