Stone B J, Brier A, Kwaik Y A
Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, Kentucky, 40536-0084, USA.
Microb Pathog. 1999 Dec;27(6):369-76. doi: 10.1006/mpat.1999.0311.
Transposon mutagenesis was performed using mTn 10phoA to identify Legionella pneumophila genes that are expressed under certain in vitro conditions, and are required for intracellular replication. Of the 1653 PhoA fusions examined, 19 PhoA(+)fusion mutants were isolated and screened for differential expression of fusion proteins after growth at 30 or 37 degrees C, in the presence of low iron, or increased magnesium concentrations. The mutants were examined for their cytopathogenicity and intracellular replication within U937 macrophage-like cells and the protozoan Hartmannella vermiformis. One of the mutants generated, BS10, was defective in its multiplication within U937 macrophage-like cells and H. vermiformis. The defect in BS10 was complemented with a cosmid clone containing the wild type locus. The open reading frame interrupted by the insertion was homologous to prpD of Salmonella typhimurium and mmgE of Bacillus subtilis.
利用mTn 10phoA进行转座子诱变,以鉴定嗜肺军团菌在特定体外条件下表达且在细胞内复制所必需的基因。在检测的1653个PhoA融合体中,分离出19个PhoA(+)融合突变体,并在30或37摄氏度、低铁存在或镁浓度增加的条件下生长后,筛选融合蛋白的差异表达。检测这些突变体在U937巨噬细胞样细胞和原生动物蠕虫状哈特曼氏阿米巴内的细胞致病性和细胞内复制情况。所产生的一个突变体BS10在U937巨噬细胞样细胞和蠕虫状哈特曼氏阿米巴内的增殖存在缺陷。BS10的缺陷通过含有野生型基因座的黏粒克隆得到互补。被插入中断的开放阅读框与鼠伤寒沙门氏菌的prpD和枯草芽孢杆菌的mmgE同源。