Application of a thermosensitive suicide vector for Streptococcus to construction of deletion mutants in Melissococcus plutonius, the causative agent of European foulbrood
Application of a thermosensitive suicide vector for Streptococcus to construction of deletion mutants in Melissococcus plutonius, the causative agent of European foulbrood
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DOI：
10.1080/00218839.2015.1103996
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发表时间：
2015-03-15
影响因子：
1.9
通讯作者：
Osaki, Makoto
中科院分区：
文献类型：
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作者：
Takamatsu, Daisuke;Yoshiyama, Mikio;Osaki, Makoto
斑纹夜蛾是一种革兰氏阳性披针形球菌，是引起蜜蜂幼虫欧洲污育病的重要病原菌。虽然最初对它的描述是在一个世纪前(White，1912)，但我们对它的生理学和发病机制的了解仍然非常有限。缺乏分子方法的工具是阻碍普氏支原体研究的一个因素。为了改善这种情况，我们最近开发了一种基因表达载体pMX2和一种针对普氏支原体的电穿孔协议(Takamatsu等人，2013年)。然而，基因敲除系统在这种细菌中仍然不可用。因此，在本研究中，我们选择了一个sortase A基因同源基因(srtA；MPD5_0048)[DDBJ/GenBank/EMBL登录号：AP012282])，并试图使用pSET6S产生框内srtA缺失突变体，pSET6S是一种具有氯霉素抗性基因(CAT)的温敏质粒载体，最初是为猪链球菌(Takamatsu，Osaki，&Sekizaki，2001)的基因替换而开发的，以研究它是否也可以用于M.plutonius。为了构建srtA缺失载体(pΔsrtA)，从Plutonius DAT561(Arai等人，2012年)扩增srtA的侧翼区，并使用iProof HF Master Mix(Bio-Rad)和引物(图1(A))通过重叠延伸聚合酶链式反应将其融合：
Melissococcus plutonius, a Gram-positive lanceolate coccal bacterium, is an important pathogen that causes European foulbrood disease in honeybee larvae. Although it was originally described a century ago (White, 1912), our understanding of its physiology and pathogenesis remains very limited. A lack of tools for molecular approaches was a factor that hampered studies of M. plutonius. To improve this situation, we recently developed a gene expression vector, pMX2, and an electroporation protocol for M. plutonius (Takamatsu et al., 2013). However, gene knockout systems are still unavailable in this bacterium. Therefore, in this study, we selected a sortase A gene homolog (srtA; MPD5_0048 [DDBJ/GenBank/EMBL accession No. AP012282]) of M. plutonius as an example and attempted to generate in-frame srtA deletion mutants using pSET6s, a thermosensitive plasmid vector, which has a chloramphenicol resistance gene (cat) and was originally developed for gene replacement in Streptococcus suis (Takamatsu, Osaki, & Sekizaki, 2001) to investigate if it could also be used in M. plutonius. To construct the srtA deletion vector (pΔSrtA), the flanking regions of srtA were amplified from M. plutonius DAT561 (Arai et al., 2012) and fused by overlap-extension PCR using iProof HF Master Mix (Bio-Rad) and the primers (Figure 1 (A)):-