A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1
A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1
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DOI：
10.1128/jb.178.17.5164-5173.1996
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发表时间：
1996-09-01
影响因子：
3.2
通讯作者：
Hammer, K
中科院分区：
文献类型：
--
作者：
Christiansen, B;Brondsted, L;Hammer, K
利用整合载体pBC 143（B.克里斯蒂安森，M。G. Johnsen，E. Stenby，F. K. Vogensen和K. Hammer，J. Bacteriol. 176：1069 - 1076，1994）。整合所需的噬菌体编码元件定位在包括噬菌体附着位点attP的2.8-kb NsiI-EcoRI片段上。对该片段进行DNA测序，序列分析显示三个质粒表达的开放阅读框，Orf 1、Orf 2和Orf 3。通过在orf1、orf2和orf3基因内引入突变，表明只有Orf1是整合过程所必需的。此外，还发现Orf 1、attP和orf 1基因上游425 bp的区域足以进行整合。Orf1含有485个氨基酸，位于attP的上游。Orf1的N-末端150至180个氨基酸显示38至44%的相似性的解离酶组的位点特异性整合酶，而没有相似性的已知蛋白质被发现在C-末端。因此，噬菌体TP 901 - 1含有一个独特的整合系统，该系统不类似于通常在温和噬菌体中发现的Int类位点特异性整合酶。构建的整合载体pBC 170非常有效地整合到染色体附着位点，并以相当于20%转化效率的频率形成稳定的转化体。
The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp, cremoris LM0230 and MG1363 with the use of deletion derivatives of the integration vector pBC143 (B. Christiansen, M. G. Johnsen, E. Stenby, F. K. Vogensen, and K. Hammer, J. Bacteriol. 176:1069-1076, 1994). The phage-encoded elements necessary for integration were localized on a 2.8-kb NsiI-EcoRI fragment including the phage attachment site, attP. This fragment was DNA sequenced, and sequence analysis revealed three putatively expressed open reading frames, Orf1, Orf2, and Orf3. By the introduction of mutations within the orf1, orf2, and orf3 genes, it was shown that only Orf1 was necessary for the integration process. Furthermore, it was found that Orf1, attP, and a 425-bp region upstream of the orf1 gene are sufficient for integration. Orf1 contains 485 amino acids and is located just upstream of attP. The N-terminal 150 to 180 amino acids of Orf1 showed 38 to 44% similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end. Bacteriophage TP901-1 therefore contains a unique integration system that does not resemble the Int class of site-specific integrases usually found in temperate bacteriophages. The constructed integration vector, pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency corresponding to 20% of the transformation efficiency.