Léonard C, Zekri O, Mahillon J
Laboratoire de Génétique Microbienne, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
Infect Immun. 1998 May;66(5):2163-9. doi: 10.1128/IAI.66.5.2163-2169.1998.
The genome structure of Bacillus cereus is relatively complex, its DNA being modulated between a size-varying chromosome and large plasmids. To study the genetic organization of the B. cereus type strain ATCC 14579, thermosensitive transposition vectors were designed on the basis of IS231A-derived cassettes containing uncommon restriction sites. A highly preferred insertion site for IS231A was detected in the chromosome by Southern blotting and pulsed-field gel electrophoresis (PFGE) analyses of independent insertion mutants. However, once this insertional hot spot was occupied, secondary IS231A insertions occurred randomly, as demonstrated by isolation of independent B. cereus auxotrophs at a frequency of approximately 0.6%. The hot-spot site, as well as several auxotrophic mutations, were mapped by using NotI, SfiI, and AscI PFGE restriction profiles. It was confirmed by sequencing that one of the insertions, generating an Ade- phenotype, had disrupted a gene of the purine synthesis pathway. These results showed that combined PFGE and sequencing analyses of mini-IS231A insertions enable the construction of integrated physical and genetic maps of B. cereus type strain. Moreover, the presence of the ultrarare I-SceI restriction site in the mini-IS231A allowed the isolation, in double-insertion mutants, of contiguous and nonoverlapping large chromosomal fragments, convenient for direct sequencing. The system detailed in this report is therefore a powerful tool for comparative genetic studies among members of the B. cereus group (i.e., B. cereus, B. thuringiensis, B. mycoides, and B. anthracis) and could also be applied to more distantly related gram-positive bacteria.
蜡样芽孢杆菌的基因组结构相对复杂，其DNA在大小可变的染色体和大质粒之间进行调控。为了研究蜡样芽孢杆菌模式菌株ATCC 14579的遗传组织，基于含有罕见限制酶切位点的IS231A衍生盒设计了热敏转座载体。通过对独立插入突变体的Southern杂交和脉冲场凝胶电泳（PFGE）分析，在染色体中检测到IS231A的一个高度优先插入位点。然而，一旦这个插入热点被占据，二级IS231A插入就会随机发生，从频率约为0.6%的独立蜡样芽孢杆菌营养缺陷型的分离情况可以证明这一点。利用NotI、SfiI和AscI PFGE限制酶切图谱对热点位点以及几个营养缺陷型突变进行了定位。通过测序证实，其中一个产生腺嘌呤缺陷型（Ade-）表型的插入破坏了嘌呤合成途径的一个基因。这些结果表明，对mini-IS231A插入进行PFGE和测序分析相结合，能够构建蜡样芽孢杆菌模式菌株的综合物理图谱和遗传图谱。此外，mini-IS231A中极罕见的I-SceI限制酶切位点的存在，使得在双插入突变体中能够分离出连续且不重叠的大染色体片段，便于直接测序。因此，本报告中详细描述的系统是蜡样芽孢杆菌组（即蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌和炭疽芽孢杆菌）成员间比较遗传研究的有力工具，也可应用于亲缘关系更远的革兰氏阳性菌。
