申请人:The Board of Trustees of the University of illinois
公开号:EP1188829A2
公开(公告)日:2002-03-20
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. In some instances, the selected DNA is used to transform fresh populations of bacteria and the cycle of DNA selection and transformation is repeated as many times as needed for obtaining hypersusceptibility mutants. After the DNA of such a mutant is collected, purified and sequenced, the location of a selectable or screenable marker identifies the antibacterial hypersusceptibility locus. The proteins encoded by these loci can serve as targets for potentiators of an antibacterial agent.
本发明公开了利用可选择或可筛选标记对细菌群体进行随机插入诱变,用抗菌剂处理诱变的细菌群体,并选择受抗菌剂影响的细胞的 DNA,从而鉴定抗生素高敏感性突变位点的方法。在某些实施方案中,所选 DNA 是在抗菌处理过程中从裂解的细菌中释放出来的。被选中的 DNA 也可能是在接触非溶解性抗菌剂并结合一种或多种额外处理导致细菌裂解后释放出来的。在其他情况下,只有通过细菌基因工程插入裂解基因盒,才能从细菌中释放出所选 DNA。在某些情况下,选定的 DNA 被用于转化新的细菌群体,DNA 的选择和转化循环根据需要重复多次,以获得高敏感性突变体。在对突变体的 DNA 进行收集、纯化和测序后,可选择或可筛选标记的位置将确定抗菌高敏感性基因座。这些基因座编码的蛋白质可作为抗菌剂增效剂的靶标。