Mahairas G G, Lyon B R, Skurray R A, Pattee P A
Department of Microbiology, Iowa State University, Ames 50011.
J Bacteriol. 1989 Jul;171(7):3968-72. doi: 10.1128/jb.171.7.3968-3972.1989.
Tn4001, a 4.5-kilobase composite transposon with IS256 ends that confers resistance to gentamicin (Gmr), tobramycin, and kanamycin in Staphylococcus aureus, can transpose to diverse chromosomal sites in S. aureus. Chromosomal insertions of Tn4001 were isolated either after UV irradiation of transducing lysates carrying pII147::Tn4001 or by selection for thermoresistant Gmr isolates with strains containing thermosensitive derivatives of plasmids pI258 and pII147 carrying Tn4001. Frequent integration of the entire delivery plasmid occurred under these selective conditions in recombination-proficient hosts. When selection for thermoresistant Gmr isolates was done with these plasmids in recombination-deficient hosts, 99% or more of the Gmr isolates resulted from transposition of Tn4001 in the absence of plasmid integration. Efficient isolation of Tn4001 insertions near markers of interest and the isolation of insertional auxotrophs were achieved. Reversion frequencies of insertional auxotrophs were between 10(-6) and 10(-7) (higher than those observed with Tn551 and Tn917). About 50% of the prototrophic revertants were Gms, and these are attributed to precise excision of Tn4001. The Gmr prototrophic revertants were due to intergenic suppression.
Tn4001是一个4.5千碱基对的复合转座子，两端为IS256，可使金黄色葡萄球菌对庆大霉素（Gmr）、妥布霉素和卡那霉素产生抗性，它能转座到金黄色葡萄球菌的不同染色体位点。Tn4001的染色体插入可通过对携带pII147::Tn4001的转导裂解物进行紫外线照射后分离得到，也可通过用含有携带Tn4001的质粒pI258和pII147的热敏衍生物的菌株筛选耐热Gmr分离株来获得。在这些选择条件下，完整的传递质粒在重组能力强的宿主中频繁整合。当在重组缺陷宿主中用这些质粒筛选耐热Gmr分离株时，99%或更多的Gmr分离株是在没有质粒整合的情况下由Tn4001转座产生的。实现了在感兴趣标记附近高效分离Tn4001插入以及分离插入型营养缺陷型。插入型营养缺陷型的回复频率在10^(-6)到10^(-7)之间（高于用Tn551和Tn917观察到的频率）。大约50%的原养型回复株是Gms，这些归因于Tn4001的精确切除。Gmr原养型回复株是由于基因间抑制。
