Defects in glycopeptidolipid biosynthesis confer phage I3 resistance in Mycobacterium smegmatis

Mycobacteriophages have played an important role in the development of genetic tools and diagnostics for pathogenic mycobacteria including Mycobacterium tuberculosis. However, despite the isolation of numerous phages that infect mycobacteria, the mechanisms of mycobacteriophage infection remain poorly understood and knowledge about phage receptors is minimal. In an effort to identify the receptor for phage I3, we screened a library of Mycobacterium smegmatis transposon mutants for phage resistant strains. All four phage I3-resistant mutants isolated were found to have transposon insertions in genes located in a cluster involved in the biosynthesis of the cell wall-associated glycopeptidolipid (GPL) and consequently did not synthesize GPLs. The loss of GPLs co-related specifically with phage I3 resistance as all mutants retained sensitivity to two other mycobacteriophages, D29 and BxZ1. In order to define the minimal receptor for phage I3 we then tested the phage sensitivity of previously described GPL-deficient mutants of M. smegmatis that accumulated biosynthesis intermediates of GPLs. The results indicated that, while the removal of most sugar residues from the fatty acyl tetrapeptide core (FATP) of GPL did not affect sensitivity to phage I3, a single methylated rhamnose, transferred by the rhamnosyl transferase Gtf2 to the FATP core, was critical for phage binding.