This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either σA- or σB-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kanr cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Ermr gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10−2) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a “TA” dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo− clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.
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机译:该报告描述了基于海洋的转座子系统的构建和表征,该系统设计用于枯草芽孢杆菌,但可能适用于其他革兰氏阳性细菌。创建了两个来自pUC19的质粒,其中包含Mariner-Himar1转座酶基因,在σ A sup>-或σ B sup>-的控制下进行了修饰,可在枯草芽孢杆菌中表达。依赖启动子。这两个质粒还包含一个转座因子(TnYLB-1),由一个被Himar1识别的反向末端重复序列括起来的Kan r sup>盒组成,以及对温度敏感的复制子和Erm r < / sup> pE194ts的基因。 TnYLB-1从任一质粒以高频率(10 -2 sup>)转座枯草芽孢杆菌染色体。对15个转座子的Southern杂交分析和其中10个转座子的插入位点的序列分析与随机转座一致,只需要“ TA”二核苷酸作为受体DNA的基本靶标即可。筛选了200个具有孢子形成能力和营养缺陷型的转座子,产生了5个Spo - sup>克隆,其中3个插入了已知的孢子形成基因(kinA,spoVT和yqfD),两个插入了功能未知的基因(ybaN和yubB)。在200个转座子中鉴定出两个营养缺陷型突变体,一个在lysA中插入,另一个在其功能未知的基因(yjzB)中插入。
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