Mayer Oren, Jain Paras, Weisbrod Torin R, Biro Daniel, Ho Libby, Jacobs-Sera Deborah, Hatfull Graham F, Jacobs William R
Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA.
Department of Systems & Computational Biology, Albert Einstein College of Medicine, New York, New York, USA.
J Bacteriol. 2016 Nov 4;198(23):3220-3232. doi: 10.1128/JB.00592-16. Print 2016 Dec 1.
Mycobacteriophage DS6A is unique among the more than 8,000 isolated mycobacteriophages due to its ability to form plaques exclusively on mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Speculation surrounding this specificity has led to unsupported assertions in published studies and patents that nontuberculous mycobacteria (NTM) are wholly resistant to DS6A infection. In this study, we identified two independent nonessential regions in the DS6A genome and replaced them with an mVenus-expressing plasmid to generate fluorescent reporter phages ΦGFP12 and ΦGFP13. We show that even though DS6A is able to form plaques only on MTBC bacteria, infection of various NTM results in mVenus expression in transduced cells. The efficiency of DS6A in delivering DNA varied between NTM species. Additionally, we saw a striking difference in the efficiency of DNA delivery between the closely related members of the Mycobacterium abscessus complex, M. abscessus and Mycobacterium massiliense We also demonstrated that TM4 and DS6A, two phages that do not form plaques on M. massiliense, differ in their ability to deliver DNA, suggesting that there is a phage-specific restriction between mycobacterial species. Phylogenetic analysis reveals that the DS6A genome has a characteristically mosaic structure but provided few insights into the basis for the specificity for MTBC hosts. This study demonstrates that the inability of the MTBC-specific phage DS6A to form plaques on NTM is more complex than previously thought. Moreover, the DS6A-derived fluorophages provide important new tools for the study of mycobacterial biology.
The coevolution of bacteria and their infecting phages involves a constant arms race for bacteria to prevent phage infection and phage to overcome these preventions. Although a diverse array of phage defense systems is well characterized in bacteria, very few phage restriction systems are known in mycobacteria. The DS6A mycobacteriophage is unique in the mycobacterial world in that it forms plaques only on members of the Mycobacterium tuberculosis complex. However, the novel DS6A reporter phages developed in this work demonstrate that DS6A can infect nontuberculous mycobacteria at various efficiencies. By comparing the abilities of DS6A and another phage, TM4, to infect and form plaques on various mycobacterial species, we can begin to discern new phage restriction systems employed within the genus.
分枝杆菌噬菌体DS6A在已分离的8000多种分枝杆菌噬菌体中独一无二，因为它仅能在属于结核分枝杆菌复合群（MTBC）的分枝杆菌上形成噬菌斑。围绕这种特异性的猜测导致已发表的研究和专利中出现未经证实的断言，即非结核分枝杆菌（NTM）对DS6A感染完全具有抗性。在本研究中，我们在DS6A基因组中鉴定出两个独立的非必需区域，并用表达mVenus的质粒替换它们，以产生荧光报告噬菌体ΦGFP12和ΦGFP13。我们表明，尽管DS6A仅能在MTBC细菌上形成噬菌斑，但各种NTM的感染会导致转导细胞中mVenus表达。DS6A传递DNA的效率在不同的NTM物种之间有所不同。此外，我们在脓肿分枝杆菌复合群的密切相关成员脓肿分枝杆菌和马赛分枝杆菌之间观察到DNA传递效率存在显著差异。我们还证明，在马赛分枝杆菌上不形成噬菌斑的两种噬菌体TM4和DS6A在传递DNA的能力上有所不同，这表明分枝杆菌物种之间存在噬菌体特异性限制。系统发育分析表明，DS6A基因组具有典型的镶嵌结构，但对于其对MTBC宿主特异性的基础提供的见解很少。本研究表明，MTBC特异性噬菌体DS6A在NTM上无法形成噬菌斑比以前认为的更为复杂。此外，源自DS6A的荧光噬菌体为分枝杆菌生物学研究提供了重要的新工具。
细菌与其感染噬菌体的共同进化涉及细菌防止噬菌体感染和噬菌体克服这些预防措施的持续军备竞赛。尽管细菌中各种各样的噬菌体防御系统已得到充分表征，但分枝杆菌中已知的噬菌体限制系统却很少。DS6A分枝杆菌噬菌体在分枝杆菌世界中独一无二，因为它仅在结核分枝杆菌复合群的成员上形成噬菌斑。然而，本研究中开发的新型DS6A报告噬菌体表明，DS6A可以以不同效率感染非结核分枝杆菌。通过比较DS6A和另一种噬菌体TM4感染各种分枝杆菌物种并形成噬菌斑的能力，我们可以开始识别该属内采用的新的噬菌体限制系统。
