Control of SXT integration and excision
Control of SXT integration and excision
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DOI：
10.1128/jb.185.17.5045-5054.2003
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发表时间：
2003-09-01
影响因子：
3.2
通讯作者：
Waldor, MK
中科院分区：
文献类型：
--
作者：
Burrus, V;Waldor, MK
霍乱弧菌SXT元件是一种接合性自身传递染色体整合元件，编码对多种抗生素的抗性。SXT以位点特异性方式整合在prfC，并从染色体上切除，形成环状但非复制的染色体外形式。染色体整合和切除都依赖于SXT编码的重组酶Int。在这里，我们发现Int对于SXT整合是必要的和充分的，并且在受体细胞中的Int表达需要SXT激活剂SetC和SetI。虽然在SXT基因组中没有xis样基因被注释，但Int不足以介导有效的SXT染色体切除。我们发现了一种新的SXT Xis，它似乎起着重组方向性因子（RDF）的作用，促进SXT切除并抑制SXT整合。虽然与任何先前表征的RDF无关，但Xis类似于五种假设的蛋白质，它们一起可能构成一个新的RDF家族。使用实时定量PCR测定来研究SXT从染色体上切除，我们确定虽然SXT切除是SXT转移所需的，但含有切除的环状SXT的细胞的百分比似乎不是限制SXT转移的主要因素;即，我们发现大多数含有切除的环状SXT分子的细胞不作为SXT供体。在不存在prfC的情况下，SXT整合到几个二级附着位点中，但优先整合到pntB的5'端。从含有pntB：：SXT的供体中切除和转移SXT减少，表明SXT整合位点也可能影响元件的可传递性。
The Vibrio cholerae SXT element is a conjugative self-transmissible chromosomally integrating element that encodes resistance to multiple antibiotics. SXT integrates in a site-specific fashion at prfC and excises from the chromosome to form a circular but nonreplicative extrachromosomal form. Both chromosomal integration and excision depend on an SXT-encoded recombinase, Int. Here we found that Int is necessary and sufficient for SXT integration and that int expression in recipient cells requires the SXT activators SetC and SetI). Although no xis-like gene was annotated in the SXT genome, Int was not sufficient to mediate efficient SXT chromosomal excision. We identified a novel SXT Xis that seems to function as a recombination directionality factor (RDF), facilitating SXT excision and inhibiting SXT integration. Although unrelated to any previously characterized RDF, Xis is similar to five hypothetical proteins that together may constitute a new family of RDFs. Using real-time quantitative PCR assays to study SXT excision from the chromosome, we determined that while SXT excision is required for SXT transfer, the percentage of cells containing an excised circular SXT does not appear to be a major factor limiting SXT transfer; i.e., we found that most cells harboring an excised circular SXT molecule do not act as SXT donors. In the absence of prfC, SXT integrated into several secondary attachment sites but preferentially into the 5' end of pntB. SXT excision and transfer from a donor containing pntB::SXT were reduced, suggesting that the SXT integration site may also influence the element's transmissibility.