New vector tools with a hygromycin resistance marker for use with opportunistic pathogens.

The ability of many bacterial strains to tolerate antibiotics can limit the number of molecular tools available for research of these organisms. To help address this problem, we have modified a diverse set of vectors to include a broadly expressed hygromycin resistance (HmR) marker. Hygromycin B is an aminoglycoside antibiotic not used to treat infections in humans and has antimicrobial activity against a wide range of microorganisms. Vectors with four replication origins are represented, with potential applications including general cloning, allelic replacement, and transcriptional analysis. We show that vectors with the broad host range pBBR1-replicon conferred HmR to Achromobacter xylosoxidans, Acinetobacter baumannii, Pseudomonas aeruginosa, and Serratia marcescens, and a pC194-based vector was able to confer HmR to Francisella tularensis. We also used a subset of these plasmids to manipulate the genome of S. marcescens. Each vector has an origin of transfer for conjugation, and is also able to replicate in Saccharomyces cerevisiae to take advantage of the powerful yeast recombineering system.