College of Life Science, South-Central Minzu University, Wuhan, China.
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Appl Environ Microbiol. 2023 Feb 28;89(2):e0165222. doi: 10.1128/aem.01652-22. Epub 2023 Feb 7.
pTAND672-2, a 144-kb resident plasmid of Bacillus thuringiensis serovar strain TAND672, was sequenced and characterized. This extrachromosomal element carries mosquitocidal toxin-, conjugation-, and recombinase-encoding genes, together with a putative arbitrium system, a genetic module recently discovered in temperate phages controlling lysogeny-lysis transition and in mobile genetic elements (MGEs) where its function remains clarified. Using conjugation experiments, pTAND672-2 is shown to be a novel integrative and conjugative element (ICE), which can horizontally transfer from B. thuringiensis serovar to Lysinibacillus sphaericus, another mosquitocidal bacterium, where it integrates into the chromosome. Its integration and circularization are reversible and involve a single-cross recombination between 33-bp specific sites, in the chromosome of and in pTAND672-2. CDS143, coding for the putative tyrosine integrase Int143 distantly related to site-specific tyrosine Xer recombinases and phage integrases, can mediate the integration of pTAND672-2 to . The B. thuringiensis mosquito-killing genes carried by pTAND672-2 are efficiently transcribed and expressed in , displaying a slight increased toxicity in this bacterium against Aedes albopictus larvae. The occurrence of pTAND672-2-like plasmids within the Bacillus cereus group was also explored and indicated that they all share a similar genetic backbone with diverse plasmid sizes, ranging from 58 to 225 kb. Interestingly, among them, the pEFR-4-4 plasmid of Bacillus paranthracis EFR-4 and p5 of B. thuringiensis BT-59 also display conjugative capability; moreover, like pTAND672-2 displays a chimeric structure between the pCH_133-e- and pBtoxis-like plasmids, pBTHD789-3 also appears to be mosaic of two plasmids. Horizontal transfer of mobile genetic elements carrying mosquitocidal toxin genes may play a driving role in the diversity of mosquitocidal bacteria. Here, the 144-kb mosquitocidal toxin-encoding plasmid pTAND672-2 is the first verified integrative and conjugative element (ICE) identified in Bacillus thuringiensis serovar . The key tyrosine integrase Int143, involved in the specific integration, is distantly related to other tyrosine recombinases. The study also reports the occurrence and potential interspecies transmission of pTAND672-2-like plasmids with varied sizes in B. thuringiensis, Bacillus paranthracis, and Bacillus wiedmannii isolates belonging to the Bacillus cereus group. This study is important for further understanding the evolution and ecology of mosquitocidal bacteria, as well as for providing new direction for the genetic engineering of biopesticides in the control of disease-transmitting mosquitoes.
pTAND672-2 是苏云金芽孢杆菌血清型菌株 TAND672 的一个 144kb 的驻留质粒,已被测序并进行了特征描述。这个染色体外元件携带有杀蚊毒素、 conjugation、和 recombinase 编码基因,以及一个假定的 arbitrium 系统,这是一个最近在控制溶原性-裂解转换的温和噬菌体和移动遗传元件 (MGE) 中发现的遗传模块,其功能仍有待澄清。通过 conjugation 实验,证明 pTAND672-2 是一种新型的整合和共轭元件 (ICE),可以从苏云金芽孢杆菌血清型菌株 TAND672 横向转移到另一种杀蚊细菌-球形芽孢杆菌中,在那里它整合到染色体中。其整合和环化是可逆的,涉及到染色体上 33-bp 特异位点和 pTAND672-2 中的 之间的单交叉重组。编码假定的酪氨酸整合酶 Int143 的 CDS143 与位点特异性酪氨酸 Xer 重组酶和噬菌体整合酶关系较远,可介导 pTAND672-2 整合到 中。pTAND672-2 携带的苏云金芽孢杆菌杀蚊基因在 中高效转录和表达,对 Aedes albopictus 幼虫表现出轻微增加的毒性。还探索了 pTAND672-2 样质粒在蜡状芽孢杆菌群中的存在情况,并表明它们都具有相似的遗传骨架,质粒大小不同,范围从 58 到 225kb。有趣的是,其中蜡状芽孢杆菌 EFR-4 的 pEFR-4-4 质粒和苏云金芽孢杆菌 BT-59 的 p5 也显示出接合能力;此外,与 pTAND672-2 一样,显示出 pCH_133-e- 和 pBtoxis-like 质粒之间的嵌合结构,pBTHD789-3 似乎也是两个质粒的嵌合体。携带杀蚊毒素基因的移动遗传元件的水平转移可能在杀蚊细菌的多样性中发挥了驱动作用。在这里,携带 144kb 杀蚊毒素基因的质粒 pTAND672-2 是首次在苏云金芽孢杆菌血清型菌株中鉴定到的整合和共轭元件 (ICE)。涉及特定整合的关键酪氨酸整合酶 Int143 与其他酪氨酸重组酶关系较远。该研究还报告了在属于蜡状芽孢杆菌群的苏云金芽孢杆菌、蜡状芽孢杆菌和魏氏芽孢杆菌分离株中,存在大小不同的 pTAND672-2 样质粒,并具有潜在的种间传播。该研究对于进一步了解杀蚊细菌的进化和生态学具有重要意义,并为利用生物农药控制传播疾病的蚊子的遗传工程提供了新的方向。