Wiater L A, Sadosky A B, Shuman H A
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Mol Microbiol. 1994 Feb;11(4):641-53. doi: 10.1111/j.1365-2958.1994.tb00343.x.
Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Tn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (KmR) and carries a 5' truncated 'lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cis-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. KmR LacZ+ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular KmR LacZ+ mutant, AB1156, does not produce the brown pigment (Pig-) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposon-linked mutation into a wild-type background results in a Pig- phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and beta-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.
对嗜肺军团菌致病性分子基础的研究，若有高效诱变剂将得到促进，该诱变剂不仅能标记基因组突变，还可用于反映巨噬细胞感染期间的基因表达。Tn903的衍生物Tn903dlllacZ在嗜肺军团菌中显示出高效转座。Tn903dlllacZ编码对卡那霉素的抗性（KmR），并携带一个5'截短的“lacZ基因，转座时可与嗜肺军团菌基因形成翻译融合。顺式作用的Tn903转座酶在Tn903dlllacZ之外提供，因此染色体整合的拷贝是稳定的。分离出嗜肺军团菌的KmR LacZ+插入突变体，通过DNA杂交表明其染色体在不同位置携带单个插入的Tn903dlllacZ。一个特定的KmR LacZ+突变体AB1156不产生军团菌属特有的棕色色素（Pig-）。Tn903dlllacZ导致这种表型，因为将转座子连锁突变重新引入野生型背景会导致Pig-表型。嗜肺军团菌色素产生通常在培养细胞的稳定期生长中观察到，从pig::lacZ融合体测量的β-半乳糖苷酶活性在对数期生长期间增加，并在稳定期开始时达到峰值。有趣的是，pig::lacZ表达在巨噬细胞感染期间也增加。然而，色素本身似乎不是嗜肺军团菌在宿主巨噬细胞内生长或杀死宿主巨噬细胞所必需的。
