Identification of insertion sites for the integrative and conjugative element Tn916 in the Bacillus subtilis chromosome

Integrative and conjugative elements (ICEs) are found in many bacterial species and are mediators of horizontal gene transfer. Tn916, an ICE found in several Gram-positive species, can integrate into many sites in the host chromosome, in contrast to the many ICEs that preferentially integrate into a single site. The consensus integration motif for Tn916, based on analyses of approximately 200 independent insertions, is an ~16 bp AT-rich sequence. Here, we describe the identification and mapping of approximately 105 independent Tn916 insertions in the Bacillus subtilis chromosome. The insertions were distributed between 1554 chromosomal sites, and approximately 99% of the insertions were in 303 sites and 65% were in only ten sites. One region, between ykuC and ykyB (kre), was a 'hot spot' for integration with ~22% of the insertions in that single location. In almost all of the top 99% of sites, Tn916 was found with similar frequencies in both orientations relative to the chromosome and relative to the direction of transcription, with a few notable exceptions. Using the sequences of all insertion regions, we determined a consensus motif which is similar to that previously identified for Clostridium difficile. The insertion sites are largely AT-rich, and some sites overlap with regions bound by the nucleoid-associated protein Rok, a functional analog of H-NS of Gram-negative bacteria. Rok functions as a negative regulator of at least some horizontally acquired genes. We found that the presence or absence of Rok had little or no effect on insertion site specificity of Tn916. Transposon-Directed Insertion Site (TraDIS) sequencing pipeline was used to sequence a library of Tn916-chromosome junctions

整合接合元件（Integrative and conjugative elements, ICEs）广泛存在于多种细菌物种中，是水平基因转移的重要介导因子。Tn916是一种存在于数种革兰氏阳性菌中的整合接合元件，相较于偏好单一整合位点的多数整合接合元件，它可整合至宿主染色体的多个位点。基于约200个独立插入事件的分析，Tn916的保守整合基序为一段长约16 bp的AT富集序列。本研究报道了枯草芽孢杆菌（Bacillus subtilis）染色体中约105个独立Tn916插入位点的鉴定与定位结果。这些插入事件分布于1554个染色体位点，其中约99%的插入事件集中于303个位点，65%的插入事件仅发生于10个位点。其中ykuC与ykyB（kre）之间的区域为整合热点，约22%的插入事件均位于该单一位置。在排名前99%的插入位点中，几乎所有位点的Tn916取向与染色体方向、转录方向的分布频率均相近，仅存在少数显著例外。利用所有插入区域的序列，我们鉴定得到一段保守基序，其与此前在艰难梭菌（Clostridium difficile）中鉴定的基序相似。插入位点整体AT富集，部分位点与类核结合蛋白Rok的结合区域重叠——Rok是革兰氏阴性菌H-NS蛋白的功能同源物，可作为至少部分水平转移基因的负调控因子。我们发现，Rok的存在与否对Tn916的插入位点特异性几乎无影响。本研究采用转座子定向插入位点测序（Transposon-Directed Insertion Site sequencing, TraDIS）流程，对Tn916-染色体连接片段的文库进行了测序。