Efficient gene inactivation in Bacillus anthracis

A procedure for high-efficiency gene inactivation in Bacillus anthracis has been developed. It is based on a highly temperature-sensitive plasmid vector carrying kanamycin resistance cassette surrounded by DNA fragments flanking the desired insertion site. The approach was tested by constructing glutamate racemase E1 ( racE1), glutamate racemase E2 ( racE2) and comEC knock-out mutants of B. anthracis strain ΔANR. Allelic replacements were observed at high frequencies, ranging from ∼0.5% for racE2 up to 50% for racE1 and comEC. The system can be used for genetic validation of potential drug targets.