Leucine auxotrophic marker for genetic manipulation of <i>Mycobacterium abscessus</i>

ABSTRACT Mycobacterium abscessus is a clinically important multidrug-resistant pathogen for which genetic manipulation remains challenging. Here, we developed a second-generation genetic toolbox based on leucine auxotrophy that enables antibiotic-free positive selection. A Δ leuB mutant, lacking the gene encoding isopropyl malate dehydrogenase in the leucine biosynthesis pathway, was generated by targeted gene deletion. This mutant requires exogenous leucine for growth and can be complemented by plasmid-borne leuB , establishing a robust auxotrophy-based selection system. To support genetic manipulation, we constructed a suite of second-generation vectors, including a multicopy replicative vector (pRep-amp- leuB ), a single-copy integrative vector (pInt-amp- leuB ), and a suicide vector (pSuc-amp- leuB ) for allelic replacement. These vectors enable gene overexpression, complementation, and targeted gene deletion, respectively, without reliance on aminoglycoside resistance markers. Using this system, we demonstrate efficient transformation and functional complementation of the Δ leuB mutant, achieving high transformation efficiencies and near-zero background growth under leucine selection. In contrast to antibiotic-based systems, this approach eliminates nonspecific background and avoids activation of stress response pathways, such as the whiB7 regulon. Overall, this auxotrophy-based toolbox provides a versatile platform for precise genetic manipulation in M. abscessus , improving selection stringency and enabling antibiotic-free functional genomics approaches, as demonstrated by deleting aac(2 ′), eis2, and a 19-kb fragment of the gpl locus. IMPORTANCE The development of a leucine auxotroph-based genetic system for Mycobacterium abscessus addresses critical challenges in mycobacterial genetics. By avoiding established antibiotic resistance markers, this approach reduces selective pressure for antibiotic-resistant transformants, supports antibiotic stewardship, and minimizes costly disposal from laboratory waste. It also avoids unintended activation of whiB7 , a master regulator of approximately 100 genes, particularly those involved in antibiotic stress responses, thereby improving the accuracy of phenotypic drug susceptibility testing. The versatile genetic toolbox developed here, including novel replicative, integrative, and suicide plasmids, provides precise control over functional studies, overexpression, complementation, and gene deletion. It significantly reduces dependency on antibiotics for genetic manipulation, aligning with the goals of sustainable research and offering new opportunities for studying this clinically significant pathogen. This approach represents a critical advance in microbial genetics, enhancing our capacity to explore the molecular basis of pathogenesis and drug resistance in M. abscessus .