Abstract: The present invention discloses methods for identifying loci of antibiotic hypersusceptibility mutations using random insertional mutagenesis of a bacterial population with a selectable or screenable marker, treatment of a mutagenized bacterial population with an antibacterial agent, and selection of DNA of cells affected by the antibacterial agents. In some embodiments, the DNA selected is released from bacteria lysed in response to antibacterial treatment. The selected DNA also may be released as a result of exposure to a non-lysing antibacterial agent in combination with one or more additional treatments that results in bacterial lysis. In other instances, selected DNA may be released from bacteria only as a result of insertion of a lysis gene cassette through genetic engineering of the bacteria.