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丁香假单胞菌致病决定因子iaaM的克隆与特性分析

Cloning characterization of iaaM, a virulence determinant of Pseudomonas savastanoi.

作者信息

Comai L, Kosuge T

出版信息

J Bacteriol. 1982 Jan;149(1):40-6. doi: 10.1128/jb.149.1.40-46.1982.

DOI:10.1128/jb.149.1.40-46.1982
PMID:6274847
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC216589/
Abstract

Genes for indoleacetic acid production (iaaM and iaaH) are necessary for gall induction by the olive pathogen Pseudomonas savastanoi. 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 by using plasmid RSF1010 as vector. We isolated a recombinant plasmid encoding iaaM, the locus for tryptophan 2-monooxygenase. 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 we showed 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 Tn/mutagenesis. The promoter-distal end of iaaM was mapped between coordinates at 1.7 and 2.15 kilobase pairs of the cloned segment.

摘要

吲哚乙酸生成基因（iaaM和iaaH）是橄榄病原体野油菜黄单胞菌诱导形成虫瘿所必需的。在2009菌株中，这些决定簇位于质粒pIAA1上。为了对这些基因进行定位和表征，用EcoRI核酸内切酶处理产生的pIAA1片段以质粒RSF1010为载体克隆到大肠杆菌中。我们分离到了一个编码iaaM（色氨酸2-单加氧酶基因座）的重组质粒。这个名为pLUC1的质粒通过限制性核酸内切酶水解进行了表征。它含有pIAA1的一个2.75千碱基对的片段。通过将该片段克隆到pBR328和pBRH3B的EcoRI位点，我们发现iaaM的有效表达取决于其相对于载体启动子的方向，从而确定了转录方向。为了更精确地定位iaaM并确认转录方向，对质粒pLUC1进行了转座子Tn/m诱变。iaaM的启动子远端位于克隆片段1.7至2.15千碱基对的坐标之间。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/ce324ad7b7d3/jbacter00260-0063-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/c188d19f14ac/jbacter00260-0060-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/ef3e56d0d1a6/jbacter00260-0061-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/6245a767fb36/jbacter00260-0061-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/ce324ad7b7d3/jbacter00260-0063-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/c188d19f14ac/jbacter00260-0060-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/ef3e56d0d1a6/jbacter00260-0061-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/6245a767fb36/jbacter00260-0061-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ace7/216589/ce324ad7b7d3/jbacter00260-0063-a.jpg

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