Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, Fujisawa-shi, Kanagawa, Japan.
Appl Microbiol Biotechnol. 2012 Jan;93(1):295-304. doi: 10.1007/s00253-011-3545-3. Epub 2011 Sep 2.
The serine-type phage integrase is an enzyme that catalyzes site-specific recombination between two attachment sites of phage and host bacterial genomes (attP and attB, respectively) having relatively short but distinct sequences without host auxiliary factor(s). Previously, we have established in vivo and in vitro site-specific recombination systems based on the serine-type integrase produced by actinophage TG1 and determined the minimal sizes of attP(TG1) and attB(TG1) sites required for the in vitro TG1 integrase reaction as 43- and 39-bp, respectively. Here, DNA databases were surveyed by FASTA program with the authentic attB(TG1) sequence of Streptomyces avermitilis as a query. As a result, possible attB(TG1) sequences were extracted from genomes of bacterial strains belonging to Class Alphaproteobacteria in addition to those of Class Actinobacteria. Those sequences extracted with a high similarity score and high sequence identity (we took arbitrarily more than 80% identity) turned out to be located within a conserved region of dapC or related genes encoding aminotransferases and proved to be actually recognized as the cognate substrate of attP(TG1) site by the in vitro TG1 integrase assay. Furthermore, the possible attB(TG1) site of Rhodospirillum rubrum revealed to be used actually as a native (endogenous) attachment site for the in vivo TG1-based integration system. These features are distinct from other serine-type phage integrases and advantageous for a tool of genome technology in varied industrially important bacteria belonging to Class Alphaproteobacteria.
丝氨酸噬菌体整合酶是一种酶，能够在噬菌体和宿主细菌基因组的两个附着位点（分别为 attP 和 attB）之间催化特异性重组，这些附着位点具有相对较短但独特的序列，不需要宿主辅助因子。以前，我们已经建立了基于肌动噬菌体 TG1 产生的丝氨酸整合酶的体内和体外特异性重组系统，并确定了体外 TG1 整合酶反应所需的最小 attP(TG1) 和 attB(TG1) 位点大小分别为 43 个和 39 个碱基。在这里，我们使用链霉菌avermitilis 的真实 attB(TG1) 序列作为查询，通过 FASTA 程序对 DNA 数据库进行了调查。结果，除了放线菌门的菌株基因组外，还从α变形菌纲的细菌菌株基因组中提取了可能的 attB(TG1)序列。那些具有高相似度得分和高序列同一性（我们任意取超过 80%的同一性）的序列被证明位于 dapC 或编码氨基转移酶的相关基因的保守区域内，并且实际上被体外 TG1 整合酶实验证明是 attP(TG1) 位点的同源底物。此外，红色螺旋菌的可能的 attB(TG1) 位点被证明实际上被用作体内基于 TG1 的整合系统的天然（内源性）附着位点。这些特征与其他丝氨酸噬菌体整合酶不同，并且有利于在属于α变形菌纲的各种工业上重要的细菌中作为基因组技术的工具。
