Rella M, Mercenier A, Haas D
Gene. 1985;33(3):293-303. doi: 10.1016/0378-1119(85)90237-9.
For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.
为了对铜绿假单胞菌进行插入诱变，构建了卡那霉素抗性（KmR）转座子Tn5的衍生物（Tn5-751），该衍生物携带来自质粒R751的甲氧苄啶抗性（TpR）决定簇作为附加标记。对KmR和TpR进行双重选择可避免分离在铜绿假单胞菌中高频出现的自发氨基糖苷类抗性突变体。作为重组转座子的递送系统，宽宿主范围质粒RP1的衍生物质粒pME305被证明是有效的；pME305在43℃时对维持在大肠杆菌和铜绿假单胞菌中具有温度敏感性，并且缺失了IS21以及KmR和引发酶基因。在与携带pME9（= pME305::Tn5-751）的大肠杆菌供体进行交配时，在43℃下以约5×10⁻⁷/供体的频率回收了铜绿假单胞菌PAO的转座子插入突变体。在Tn5-751插入突变体中，0.9%是营养缺陷型。recA样基因座rec-102附近的thr::Tn5-751突变对于构建重组缺陷型菌株很有用。可以分离出几个arc::Tn5-751突变体，它们在将精氨酸作为能源进行厌氧利用方面存在缺陷。从其中三个突变体中，将arc基因区域克隆到大肠杆菌载体质粒中。由于Tn5-751在TpR和KmR基因之间有一个单一的EcoRI位点，携带任一抗性决定簇加相邻染色体DNA的EcoRI产生的片段可以在大肠杆菌中分别进行选择。因此，构建了arc区域的限制性图谱，并通过杂交实验进行了验证。arc基因紧密聚集，证实了早期的遗传学证据。
