galK-based suicide vector mediated allelic exchange in Mycobacterium abscessus

Mycobacterium abscessus is a fast-growing environmental organism and an important emerging pathogen. It is highly resistant to many antibiotics and undergoes a smooth to rough colony morphology change that appears to be important for pathogenesis. Smooth environmental strains have a glycopeptidolipid (GPL) on the surface, while certain types of clinical strains are often rough and lack this GPL, due to mutations in biosynthetic genes or the mmpL4b transporter gene. We report here the development and evaluation of an allelic exchange system for unmarked alleles in M. abscessus ATCC19977, using a suicide vector bearing the E. coli galK gene and 2-deoxygalactose counterselection. We describe here two variant galK suicide vectors, and demonstrate their utility in constructing a variety of mutants with deletion alleles of the mmpL4b GPL transporter gene, the mbtH GPL biosynthesis gene, the known beta-lactamase gene MAB_2875 and a putative beta-lactamase gene, MAB_2833. We also show that a novel allele of the E. coli aacC4 gene, conferring apramycin resistance (aacC41), can be used as a selectable marker in M. abscessus ATCC19977 at single copy.