Shoemaker N B, Wang G R, Salyers A A
Department of Microbiology, University of Illinois, Urbana 61801, USA.
J Bacteriol. 2000 Feb;182(4):928-36. doi: 10.1128/JB.182.4.928-936.2000.
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 (XRS(HIII)) inhibited excision when provided in trans 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.
NBU1是一个整合的10.3千碱基对的拟杆菌元件，它可以切除并转移到拟杆菌或大肠杆菌受体中，并整合到受体基因组中。NBU1依赖大于60千碱基对的接合转座子来提供触发切除的因子以及将环状形式转移到受体的因子。此前，我们表明单个整合酶基因intN1对于NBU1整合到拟杆菌或大肠杆菌基因组上的靶位点是必要且充分的。我们现在表明NBU1切除需要一个出乎意料的大区域。该区域除intN1外，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NBU1环状形式的oriT以及相邻转移基因mobN1的大约三分之二。这是首次发现参与环状形式接合转移的oriT与切除有关。intN1下游紧邻的基因orf2的破坏完全消除了切除。下一个开放阅读框orf2x太小无法破坏，所以我们仍然不知道它是否在切除反应中起作用。在orf2x下游的两个开放阅读框orf3和prmN1中分别进行了缺失。这两个缺失都消除了切除，表明这些基因对于切除也是必不可少的。对切除区域各种突变进行互补的尝试使我们意识到，携带prmN1和部分XRS（XRS(HIII)）的切除区域的一部分在多拷贝质粒（每个细胞8至10个拷贝）上反式提供时会抑制切除。然而，携带prmN1、XRS和整个转移基因mobN1的片段没有这种作用。较小的片段可能通过吸引完整NBU1产生的蛋白质从而将它们从切除复合物中移除来干扰切除。我们的结果清楚地表明切除是一个复杂的过程，涉及多种蛋白质和顺式作用区域（XRS），其中包括oriT。我们认为这种复杂的切除机制可能是必要的，以便NBU1在切除过程中协调末端切口和在接合转移过程中oriT处的切口，以防止在NBU1切除并环化之前oriT处过早切口。
