Zylstra G J, Olsen R H, Ballou D P
Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor 48109-0620.
J Bacteriol. 1989 Nov;171(11):5915-21. doi: 10.1128/jb.171.11.5915-5921.1989.
The locations of the genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.11.3) on a 9.5-kilobase-pair PstI fragment cloned from the Pseudomonas cepacia DBO1 chromosome were determined. This was accomplished through the construction of several subclones into the broad-host-range cloning vectors pRO2317, pRO2320, and pRO2321. The ability of each subclone to complement mutations in protocatechuate 3,4-dioxygenase (pcaA) was tested in mutant strains derived from P. cepacia, Pseudomonas aeruginosa, and Pseudomonas putida. These complementation studies also showed that the two subunits were expressed from the same promoter. The nucleotide sequence of the region encoding for protocatechuate 3,4-dioxygenase was determined. The deduced amino acid sequence matched that determined by N-terminal analysis of regions of the isolated enzyme. Although over 400 nucleotides were sequenced before the start of the genes, no homology to known promoters was found. However, a terminator stem-loop structure was found immediately after the genes. The deduced amino acid sequence showed extensive homology with the previously determined amino acid sequence of protocatechuate 3,4-dioxygenase from another Pseudomonas species.
确定了从洋葱伯克霍尔德氏菌DBO1染色体克隆的一个9.5千碱基对的PstI片段上原儿茶酸3,4-双加氧酶（EC 1.13.11.3）α和β亚基基因的位置。这是通过构建几个亚克隆到广泛宿主范围的克隆载体pRO2317、pRO2320和pRO2321中来实现的。在源自洋葱伯克霍尔德氏菌、铜绿假单胞菌和恶臭假单胞菌的突变菌株中测试了每个亚克隆对原儿茶酸3,4-双加氧酶（pcaA）突变的互补能力。这些互补研究还表明，这两个亚基是从同一启动子表达的。测定了编码原儿茶酸3,4-双加氧酶区域的核苷酸序列。推导的氨基酸序列与通过对分离酶区域的N端分析确定的序列相匹配。尽管在基因开始前测序了400多个核苷酸，但未发现与已知启动子的同源性。然而，在基因之后立即发现了一个终止子茎环结构。推导的氨基酸序列与先前确定的另一种假单胞菌属原儿茶酸3,4-双加氧酶的氨基酸序列显示出广泛的同源性。
