Cloning vectors, mutagenesis, and gene disruption (ermR) for the erythromycin-producing bacterium Aeromicrobium erythreum

Abstract

Genetic systems for study of Aeromicrobium erythreum, a gram-positive, G+C-rich (72%) bacterium with the capacity for erythromycin biosynthesis, are described. High-copy-number plasmids suitable as gene cloning vectors include derivatives of the Streptomyces plasmids pIJ101, pVE1, and pJV1. pIJ101 derivatives with missense substitutions at the rep gene BamHI site do not replicate in A. erythreum. Ethyl methanesulfonate treatment generated several amino acid auxotrophs and non-erythromycin-producing (Ery-) strains. Using the Ery- strain AR1807 as a recipient for plasmid-directed integrative recombination, the chromosomal ermR gene (encoding 23S rRNA methyltransferase) was disrupted. Phenotypic characterizations demonstrated that ermR is the sole determinant of macrolide antibiotic resistance in A. erythreum.