-based suicide vector mediated allelic exchange in .

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 transporter gene. We report here the development and evaluation of an allelic exchange system for unmarked alleles in ATCC19977, using a suicide vector bearing the gene and 2-deoxygalactose counterselection. We describe here two variant suicide vectors, and demonstrate their utility in constructing a variety of mutants with deletion alleles of the GPL transporter gene, the GPL biosynthesis gene, the known β-lactamase gene and a putative β-lactamase gene, . We also show that a novel allele of the gene, conferring apramycin resistance (), can be used as a selectable marker in ATCC19977 at single copy.