Unraveling the IS Transposition-Mediated Anti-Phage Mechanism in Mycobacteria

This study is led by Prof. Zheng-Guo He (Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University,China). In a screening on a TM4-inserted mutant M. smegmatis library to characterize anti-phage growing mutant strain, the team unexpectedly found that the IS1096 transposition into the lsr2 gene, encoding a histone-like nucleoid-associated protein, occurs at high frequencies under the pressure of phage infection. Thereafter, they confirmed that lsr2 deletion resulted in a broad-spectrum anti-phage phenotype of M. smegmatis.

Next, a lipooligosaccharides synthesis-related gene cluster composed of 11 genes, designated as the LOS gene island,was found to significantly contribute to the lsr2 deletion-triggered resistance to infection of multiple clusters of mycobacterial phages.

Exactly, three upstream regulatory sequences of the gene island are required for the lsr2 inactivation-triggered anti-phage activity, and Lsr2 can directly bind to these regulatory sequences and negatively regulates expression of the island genes.

The team further found that the lsr2 gene plays a critically important role in maintaining the mycobacterial lipid hemostasis, and the LOS gene island triggers abnormal PIM accumulation in the cell envelope of lsr2 deletion strain. The lsr2 deletion resulted in phage K4JX5 absorption defect and the gene island is required for maintaining the defect phenotype of the lsr2 deletion strain.

Interestingly, a mutant phage has been successfully evolved, which can escape from the defense of lsr2 deletion strain by repeatedly spotting K4JX5 on the double-layer plate containing lsr2 deletion strain. A further sequencing assay found that K4JX5-mut had evolved several mutations in its two tail-filament genes, gp12 and gp14, which led to changes of 5 amino acids residues. The mutant phage re-obtained a good absorption capacity.

Very few anti-phage mechanisms have been clearly characterized in mycobacterial species although these bacterial genomes encode potential adaptive immune systems such as CRISPR-Cas system and some innate immune systems. This study uncovered a new signal pathway of IS transposition-triggered anti-phage immunity in M. smegmatis.

This may represent a new acquired defense strategy different from the previously reported CRISPR anti-phage mechanism."

Prof. Zheng-Guo He, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, China