Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan.
J Biosci Bioeng. 2011 Feb;111(2):109-16. doi: 10.1016/j.jbiosc.2010.10.001. Epub 2010 Oct 29.
ϕRSM1, a filamentous phage infecting Ralstonia solanacearum, encodes an open reading frame (ORF14) that exhibits significant homology to members of the resolvase/invertase subfamily of site-specific serine recombinases. Similar prophages are found in the genomes of various strains of R. solanacearum, R. pickettii, and Burkholderia pseudomallei. We have characterized the integrative and excisive recombination reactions mediated by the ϕRSM1 integrase using in vivo assays. An E. coli plasmid containing the RSM1 orf14 and attP sequences (pT-orf14-attP) was shown to integrate into the attB sequence of R. solanacearum cells. Intermolecular recombination between pT-orf14-attP and pS-attB (a R. solanacearum plasmid containing attB) also occurred in R. solanacearum cells. The products of attP/attB recombination, i.e., attL and attR, were exactly identical to the sequences found in the prophage ϕRSM in R. solanacearum strains. Intramolecular recombination between the attL and attR sequences (excisive recombination) on the pRecomb plasmid was also shown to occur in an orf14-dependent manner. This is the first evidence that a small serine recombinase from the resolvase/invertase group may function in integrative and excisive recombination for filamentous phages.
φRSM1 是一种感染茄青枯雷尔氏菌的丝状噬菌体，它编码一个开放阅读框 (ORF14)，该框与位点特异性丝氨酸重组酶的解旋酶/反转酶亚家族的成员具有显著的同源性。类似的前噬菌体存在于各种茄青枯雷尔氏菌、R. pickettii 和 Burkholderia pseudomallei 菌株的基因组中。我们使用体内测定法对 φRSM1 整合酶介导的整合和切除重组反应进行了特征描述。含有 RSM1 orf14 和 attP 序列的大肠杆菌质粒（pT-orf14-attP）被证明可以整合到茄青枯雷尔氏菌细胞的 attB 序列中。pT-orf14-attP 和 pS-attB（一种含有 attB 的茄青枯雷尔氏菌质粒）之间的分子间重组也发生在茄青枯雷尔氏菌细胞中。attP/attB 重组的产物，即 attL 和 attR，与在茄青枯雷尔氏菌菌株中发现的噬菌体 φRSM 中的前噬菌体序列完全相同。pRecomb 质粒上 attL 和 attR 序列之间的分子内重组（切除重组）也以 orf14 依赖性的方式发生。这是第一个证据表明来自解旋酶/反转酶组的小丝氨酸重组酶可能在丝状噬菌体的整合和切除重组中发挥作用。
