Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
PLoS One. 2014 Apr 16;9(4):e94951. doi: 10.1371/journal.pone.0094951. eCollection 2014.
Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of M. chelonae and M. abscessus is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for M. abscessus, genetic tools are lacking for M. chelonae. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.
龟分枝杆菌是一种快速生长的分枝杆菌机会性病原体，与脓肿分枝杆菌密切相关，可引起人类角膜、皮肤和软组织感染。尽管龟分枝杆菌和新兴的分枝杆菌病原体脓肿分枝杆菌长期以来被认为属于同一物种，但这两种微生物在最佳生长温度、药物敏感性、致病性和引起的感染类型方面存在显著差异。龟分枝杆菌和脓肿分枝杆菌临床分离株的全基因组测序为旨在了解这些病原体生物学并阐明其致病性和抗消毒剂性的分子基础的比较研究开辟了道路。然而，验证该方法将提出的众多假设的关键是提供遗传工具，允许在这些物种中表达和靶向基因突变。虽然最近已经为脓肿分枝杆菌描述了同源重组系统，但龟分枝杆菌缺乏遗传工具。我们在这里表明，两种不同的等位基因替换方法，一种基于分枝杆菌噬菌体编码的重组酶，另一种基于携带可选择标记 sacB 的温度敏感质粒，可以有效地在该物种中破坏基因。使用这两种方法中的一种构建了龟分枝杆菌 ATCC 35752 的三个孔蛋白基因中的每一个的敲除突变体，其中一种表现出葡萄糖摄取率显著降低，与孔蛋白表达减少一致。
