CLONING AND CHARACTERIZATION OF IAAM, A VIRULENCE DETERMINANT OF PSEUDOMONAS-SAVASTANOI
CLONING AND CHARACTERIZATION OF IAAM, A VIRULENCE DETERMINANT OF PSEUDOMONAS-SAVASTANOI
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DOI：
10.1128/jb.149.1.40-46.1982
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发表时间：
1982-01-01
影响因子：
3.2
通讯作者：
KOSUGE, T
中科院分区：
文献类型：
--
作者：
COMAI, L;KOSUGE, T
IAA生产基因（iaaM和iaaH）是橄榄病原体P. savastanoi [P. gringae]诱导瘿所必需的。在菌株2009中，这些决定簇携带在质粒pIAA 1上。为了对基因进行图谱和表征，使用质粒RSF 1010作为载体，将通过EcoRI内切酶处理产生的pIAA 1片段克隆到大肠杆菌中。分离编码色氨酸2-单加氧酶基因座iaaM的重组质粒[EC 1.13.12.3]。该质粒称为pLUC 1，其特征在于限制性内切酶水解。它含有pIAA 1的2.75-双酶对片段。通过将该片段克隆到pBR 328和pBRH 3B的EcoRI位点，表明iaaM的有效表达依赖于相对于载体启动子的方向，从而决定了转录的方向。为了更精细地定位iaaM并确认转录方向，对质粒pLUC 1进行转座子Tn 1诱变。iaaM的启动子远端被定位在克隆片段的1.7和2.15个酶对的坐标之间。
Genes for IAA production (iaaM and iaaH) are necessary for gall induction by the olive pathogen P. savastanoi [P. syringae]. In strain 2009 these determinants are borne on plasmid pIAA1. To map and characterize the genes, fragments of pIAA1 generated by EcoRI endonuclease treatment were cloned in Escherichia coli using plasmid RSF1010 as vector. A recombinant plasmid encoding iaaM, the locus for tryptophan 2-monooxygenase [EC 1.13.12.3], was isolated. This plasmid, called pLUC1, was characterized by restriction endonuclease hydrolysis. It contained a 2.75-kilobase-pair segment of pIAA1. By cloning this segment in the EcoRI site of pBR328 and pBRH3B it was shown that efficient expression of iaaM was dependent on the orientation with respect to the vector promoters, and thus determined the direction of transcription. To more finely map iaaM and confirm the orientation of transcription, plasmid pLUC1 was subjected to transposon Tnl mutagenesis. The promoter-distal end of iaaM was mapped between coordinates at 1.7 and 2.15 kilobase pairs of the cloned segment.