Mycobacteriophage TM4 requires XylR for successful infection in Mycobacterium smegmatis mc ² 155

Mycobacteriophages are viruses that infect mycobacteria, including Mycobacterium tuberculosis , and have emerged as promising alternatives to antibiotics in the face of increasing antimicrobial resistance. However, evolution of phage-resistance remains a major challenge to the clinical implementation of phage therapy. We describe a TM4 phage resistant mutant in M. smegmatis harboring an in-frame deletion in the xylR , transcriptional regulator implicated in lipid metabolism, and cell envelope homeostasis. Using spontaneous mutagenesis, transcriptomics and biochemical approaches, we identify a previously uncharacterized resistance mechanism mediated by cell-envelope remodeling that impedes productive phage infection. The xylR mutation disrupted phage DNA injection through enhanced recruitment of lipooligosaccharides to the cell surface, without inhibiting phage adsorption, genome replication, and virion assembly. Remodeling of the cell envelope was further enhanced by the induction of lipooligosaccharide biosynthesis upon TM4 infection, however, the phenotype can be reverted through chemical treatment, restoring phage sensitivity. Our study expands the paradigm of innate mechanisms underlying broad-spectrum phage resistance in mycobacteria.