Mobility and Generation of Mosaic Non-Autonomous Transposons by Tn3-Derived Inverted-Repeat Miniature Elements (TIMEs)

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.从波兰卢宾矿黑色页岩矿石及热拉兹尼莫斯特浮选尾矿中分离得到的多株假单胞菌属（Pseudomonas spp.）菌株的功能型转座因子（transposable elements, TEs），采用阳性选择陷阱质粒策略完成了鉴定。该策略成功捕获并表征了三类转座因子：（i）隶属于IS3、IS5、ISL3、IS30及IS1380共5个插入序列（insertion sequences, IS）家族的13条插入序列；（ii）两类Tn3家族转座子（Tn3-family transposons）的同工型——Tn5563a与Tn4662a（后者携带毒素-抗毒素系统）；以及（iii）结构多样、长度介于262 bp至3892 bp的非自主转座因子。这类非自主元件可转座至AT富集DNA区域，并在转座靶位点产生5 bp或6 bp的序列重复。尽管此类转座因子不编码转座酶，但它们携带有同源的38 bp末端反向重复序列（IRs），该序列在Tn5563a及众多其他Tn3家族转座子中高度保守。其中结构最简单的元件为262 bp的TIMEs，即Tn3家族来源的反向重复微型元件（Tn3 family-derived Inverted-repeat Miniature Elements, TIMEs），它们在假单胞菌属的两种天然质粒pZM1P1与pLM8P2中被发现。研究证实，TIMEs可介导质粒DNA片段的转座，进而生成结构类似插入序列驱动的复合转座子的更为复杂的非自主元件。这类类转座子元件的核心区域可携带包括质粒复制系统在内的多种功能遗传模块。另一种通过陷阱质粒捕获的非自主元件为携带预测解离酶基因与Tn3家族转座子典型res位点的TIME衍生物。可移动位点特异性重组系统的鉴定，进一步佐证了TIME家族非自主转座因子在细菌基因组遗传信息重排过程中的关键作用。此类嵌合元件的转座，不仅对转座子与质粒的多样性及进化具有显著影响，也可作用于其他类型的可移动遗传元件（mobile genetic elements）。