Transposon mutagenesis in identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity.

() is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in by conducting transposon sequencing (TnSeq) on the reference strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in , we further characterized , a predicted penicillin-binding protein and hypothetical lipoprotein (PBP-lipo) that is essential in and non-essential in (). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the model organism, , indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 clinical isolates to several classes of antibiotics, including the β-lactams, ampicillin, and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study -specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat infections.