Tam Christina, Glass Elizabeth M, Anderson Deborah M, Missiakas Dominique
Department of Microbiology, The University of Chicago, IL 60637, USA.
Plasmid. 2006 Jul;56(1):74-7. doi: 10.1016/j.plasmid.2006.01.002. Epub 2006 Mar 13.
Bacillus anthracis, a spore forming Gram-positive microbe, is the causative agent of anthrax. Although plasmid encoded factors such as lethal toxin (LeTx), edema toxin (EdTx), and gamma-poly-d-glutamic acid (PGA) capsule are known to be required for disease pathogenesis, B. anthracis genes that enable spore invasion, phagosomal escape and macrophage replication are still unknown. To establish transposon mutagenesis as a tool for the characterization of anthrax genes, we employed the mariner-based mini-transposon Bursa aurealis in B. anthracis strain Sterne 7702. B. aurealis carrying an erythromycin resistance cassette and its cognate transposase were delivered by transformation of two plasmids. B. aurealis transposition can be selected for by temperature shift to prevent plasmid replication and by screening colonies for erythromycin resistance. Using inverse polymerase chain reaction, DNA fragments of 129 random erythromycin-resistant transposon mutants were amplified and submitted to DNA sequence analysis. These studies demonstrate that B. aurealis inserts randomly into the genome of B. anthracis and can therefore be employed for finding genes involved in virulence.
炭疽芽孢杆菌是一种形成芽孢的革兰氏阳性微生物，是炭疽病的病原体。虽然已知诸如致死毒素（LeTx）、水肿毒素（EdTx）和γ-聚-d-谷氨酸（PGA）荚膜等质粒编码因子是疾病发病机制所必需的，但使芽孢侵入、吞噬体逃逸和巨噬细胞复制的炭疽芽孢杆菌基因仍然未知。为了将转座子诱变确立为一种用于表征炭疽基因的工具，我们在炭疽芽孢杆菌菌株Sterne 7702中使用了基于水手座的微型转座子金黄色ursa。携带红霉素抗性盒及其同源转座酶的金黄色ursa通过两种质粒的转化来传递。可以通过温度转换来选择金黄色ursa转座，以防止质粒复制，并通过筛选对红霉素具有抗性的菌落来进行。使用反向聚合酶链反应，扩增了129个随机的红霉素抗性转座子突变体的DNA片段，并进行了DNA序列分析。这些研究表明，金黄色ursa随机插入炭疽芽孢杆菌的基因组中，因此可用于寻找与毒力相关的基因。
