Department of Chemical Biology & Applied Chemistry, College of Engineering, Nihon University, 1 Nakagawara, Tokusada, Tamura-machi, Koriyama, Fukushima 963-8642, Japan.
Appl Microbiol Biotechnol. 2013 May;97(9):4039-48. doi: 10.1007/s00253-012-4491-4. Epub 2012 Oct 16.
Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ∼2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ∼10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ∼10-kbp DNA into the genome with an efficiency of ∼10(4) transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli "centromere" analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC-migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.
丝氨酸噬菌体整合酶分别在噬菌体和宿主细菌基因组中催化附着位点 attP 和 attB 之间的单向特异性重组；当这些整合酶和 DNA 靶位点被转移到异源细胞中时，它们的功能会非常有效。我们之前开发了一种基于肌噬菌体 TG1 整合酶的体内特异性基因组整合系统，该系统将约 2-kbp 的 DNA 导入插入到异源大肠杆菌基因组中的 att 位点。在这里，我们分析了 TG1 整合酶介导的含有 att 位点的约 10-kbp DNA 整合到预先插入到各种基因组位置的相应 att 位点的情况；此外，我们开发了一种将约 10-kbp DNA 导入基因组的系统，其效率约为 10(4)转化体/μg DNA。含有 attB 的 DNA 整合到含有 attP 的基因组中比含有 attP 的 DNA 整合到含有 attB 的基因组中更有效，并且靶向插入在复制起点 oriC 附近和大肠杆菌“着丝粒”类似物 migS 中的 attP 的整合比靶向基因组中其他区域中的 attP 的整合更有效。由于 oriC 和 migS 位点附近的基因组区域在染色体分离过程中位于细胞的极端极点，因此 oriC-migS 区域可能比大肠杆菌染色体的其他区域更容易暴露于细胞质中。因此，在异源细胞中，丝氨酸整合酶的整合效率可能与预插入的 attP 对含有 attB 的传入 DNA 的可及性有关。这些结果可能有助于开发基于丝氨酸整合酶的各种细菌基因组整合系统。
