Multiple gene products and sequences required for excision of the mobilizable integrated Bacteroides element NBU1
Multiple gene products and sequences required for excision of the mobilizable integrated Bacteroides element NBU1
复制标题
DOI：
10.1128/jb.182.4.928-936.2000
复制
发表时间：
2000-02-01
影响因子：
3.2
通讯作者：
Salyers, AA
中科院分区：
文献类型：
--
作者：
Shoemaker, NB;Wang, GR;Salyers, AA
NBU1是一个集成的10.3 kbp拟杆菌元件，它可以切除并转移到拟杆菌或大肠杆菌受体，在那里它整合到受体基因组中。NBU1依赖于大的60 kbp共轭转座子来触发切除因子和向受体动员环状结构。先前，我们发现一个整合酶基因intN1是将NBU1整合到拟杆菌或大肠杆菌基因组上的靶位点所必需和充分的。我们现在表明，切除NBU1需要一个出乎意料的大区域。除intN1外，该区域还包括四个开放阅读框，以及第四个基因prmN1下游的一个大区域。这个下游序列被命名为XRS，即“切除所需序列”。XRS包含环状NBU1的oriT和相邻的动员基因mobN1的约三分之二。这是第一次有一个参与环形偶联转移的oriT参与了切除，intN1， orf2的直接下游基因被破坏，完全取消了切除，下一个开放阅读框orf2x因为太小而无法被破坏，所以我们仍然不知道它是否在切除反应中起作用。orf2x， orf3和prmN1下游的两个开放阅读框均有缺失。这两种缺失都消除了切除，表明这些基因也是切除所必需的。尝试补充切除区域的各种突变使我们意识到，当在多拷贝质粒（每个细胞8到10个拷贝）上提供时，携带prmN1的部分切除区域和部分XRS （XRSHIII）抑制了切除。然而，携带prmN1、XRS和整个动员基因mobN1的片段没有这种效果。较小的片段可能通过吸引完整的NBU1产生的蛋白质，从而将它们从切除复合体中移除，从而干扰切除。我们的研究结果清楚地表明，切除是一个复杂的过程，涉及多种蛋白质和包括口部在内的顺式作用区（XRS）。我们认为，这种复杂的切除机制可能是必要的，可以使NBU1在切除和循环之前协调口部末端的切口和配偶转移期间的切口，以防止在NBU1切除和循环之前过早地在口部切口。
NBU1 is an Integrated 10.3-kbp Bacteroides element, which can excise and transfer to Bacteroides or Escherichia coli recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene, intN1, was necessary and sufficient for integration of NBU1 into its target site on the Bacteroides or E. coli genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to intN1, four open reading frames plus a large region downstream of the fourth gene, prmN1. This downstream sequence was designated XRS, for "excision required sequence." XRS contains the oriT of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene, mobN1. This is the first time an oriT, which is involved in conjugal transfer of the circular form, has been implicated in excision, Disruption of the gene immediately downstream of intN1, orf2, completely abolished excision, The next open reading frame, orf2x, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of orf2x, orf3 and prmN1. Both of these deletions abolished excision, indicating that these genes are also essential for excision, Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying prmN1 and part of the XRS (XRSHIII) inhibited excision when provided in trails on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying prmN1, XRS, and the entire mobilization gene, mobN1, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a cis-acting region (XRS) which includes the oriT, We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the oriT during conjugal transfer, to prevent premature nicking at the oriT before NBU1 has excised and circularized.