Likely role of promoter reconstitution in Mpr-mediated D29 resistance by <i>Mycobacterium smegmatis</i>

Abstract The m ulti-copy p hage resistance gene ( mpr ) of M. smegmatis is a major factor in resistance to the lytic mycobacteriophage D29. Mpr is a membrane-bound exonuclease that cleaves phage DNA post injection, hence blocking downstream stages in the phage infection cycle. The mechanism of resistance allows for adsorption, is non-abortive and independent of any mutation in the gene. Rather, it depends on overexpression of a wild type copy of the gene. However, the underlying factor behind mpr overexpression in spontaneous D29-resistant mutants of M. smegmatis remained elusive. Here, we report that D29 infection triggers insertion sequence (IS) rearrangements, including the transposition and integration of IS6120 directly upstream of mpr . Mutants with IS6120 integration upstream of mpr show highly elevated Mpr expression. Whole genome sequence analysis reveals that IS6120 introduced a putative transcription factor-binding site and a canonical -35 promoter element at the integration site, hence reconstituting a fuller promoter ( rcp ) than the original promoter ( wtp ) at the site. Promoter reporter assays suggest that rcp is a far stronger promoter than wtp , implying that elevated mpr expression in D29-resistant mutants with this transposition event could be due to promoter reconstitution. While strains with this transposition event appear to grow normally, rcp -driven, vector-borne Mpr overexpression appears to be toxic as it barely allows for colony formation on agar plates. This study reports a previously unknown factor likely behind mpr regulation in M. smegmatis , adding to the existing knowledge of mycobacterial anti-phage defense mechanisms and guiding rational phage engineering efforts for therapeutic applications.