TRANSPOSON VECTORS CONTAINING NON-ANTIBIOTIC RESISTANCE SELECTION MARKERS FOR CLONING AND STABLE CHROMOSOMAL INSERTION OF FOREIGN GENES IN GRAM-NEGATIVE BACTERIA
TRANSPOSON VECTORS CONTAINING NON-ANTIBIOTIC RESISTANCE SELECTION MARKERS FOR CLONING AND STABLE CHROMOSOMAL INSERTION OF FOREIGN GENES IN GRAM-NEGATIVE BACTERIA
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DOI：
10.1128/jb.172.11.6557-6567.1990
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发表时间：
1990-11-01
影响因子：
3.2
通讯作者：
TIMMIS, KN
中科院分区：
文献类型：
--
作者：
HERRERO, M;DELORENZO, V;TIMMIS, KN
一个简单的程序克隆和稳定插入外源基因的染色体的革兰氏阴性真细菌的开发相结合的两套质粒（i）转座功能的Tn 10和Tn 5;（ii）的抗性除草剂双丙氨膦，汞盐和有机汞化合物，和亚砷酸盐，和（iii）自杀交付性能的R6 K为基础的质粒pGP 704。所得到的结构包含独特的NotI或SfiI位点，位于Tn 10或Tn 5反向重复序列的内部。这些位点很容易在两个额外的专门克隆质粒pUC 18 Not和pUC 18 Sfi的帮助下用于克隆DNA片段。新衍生的构建体只能在产生R6 K-特异性π的供体宿主菌株中维持。蛋白，其是R6 K和由其衍生的质粒的必需复制蛋白。将含有杂合转座子的供体质粒转化到具有染色体整合的RP 4的特化λ pir溶原性大肠杆菌菌株中，所述RP 4提供宽宿主范围的接合转移功能。通过与靶菌株交配完成将供体质粒递送到选定的宿主细菌中。杂合转座子从递送的自杀质粒转座到靶细胞中的复制子由在转座子外部的位点处的血浆上编码的同源转座酶介导。由于转座酶功能在靶细胞中不被维持，因此这些细胞对进一步的转座轮没有免疫力。因此，在同一菌株中的多个插入仅受不同选择标记的可用性的限制。该系统的效用被证明与卡那霉素抗性基因作为一个模型外源插入到恶臭假单胞菌和黑色素基因从链霉菌到肺炎克雷伯氏菌。由于克隆载体的功能部分的模块化性质，它们可以容易地被修饰，并且可以掺入进一步的选择标记。这里描述的克隆系统将特别适用于构建稳定保持插入基因的杂交细菌，可能在竞争情况下（例如，在开放系统和自然环境中），并且不携带大多数可用克隆载体的抗生素抗性标记特征（如目前肝细菌疫苗所需的）。
A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified .pi. protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized .lambda.pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasma at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of liver bacterial vaccines).