Transcriptional regulation of the ant operon, encoding two-component anthranilate 1,2-dioxygenase, on the carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10
Transcriptional regulation of the ant operon, encoding two-component anthranilate 1,2-dioxygenase, on the carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10
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DOI：
10.1128/jb.186.20.6815-6823.2004
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发表时间：
2004-10-01
影响因子：
3.2
通讯作者：
Nojiri, H
中科院分区：
文献类型：
--
作者：
Urata, M;Miyakoshi, M;Nojiri, H
树脂假单胞菌菌株CA10的咔唑降解质粒pCAR1具有carAaAaBaBbCAcAdDFE和antABC两个基因簇，分别参与咔唑转化为邻苯二甲酸酯和邻苯二甲酸酯转化为儿茶酚。我们通过Northern杂交和逆转录- pcr （RT-PCR）分析证明，编码双组分苯甲酸1,2-双加氧酶的antABC基因簇构成一个单一的转录单元。antA的转录起始点定位在其翻译起始点上游53 bp处，-10和-35框与保守的sigma（70）识别序列同源。因此，蚂蚁操纵子的启动子被命名为P-ant。使用荧光素酶作为报告基因的5'缺失分析表明，距离antA转录起始点至少70 bp的区域是激活P-ant所必需的。p -蚂蚁荧光素酶的表达是由邻氨基苯甲酸引起的，而不是由儿茶酚引起的。在pCAR1上发现的两个可能的AraC/ xyls型调控基因，开放阅读框22 （ORF22）和ORF23，同时位于antA基因上游3.2 kb处。我们发现ORF23的产物，被命名为AntR，对于刺激脓假单胞菌细胞中的P-ant是必不可少的。Northern杂交和RT-PCR分析显示，P-ant的另一个拷贝，被认为是由于ISPre1的转位而在carAa基因上游约2.1 kb的位置，实际上驱动carAa基因的转录，这表明AntR同时调节ant和car操纵子。
The carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10 has two gene clusters, carAaAaBaBbCAcAdDFE and antABC, which are involved in the conversions of carbazole to anthranilate and anthranilate to catechol, respectively. We proved that the antABC gene cluster, encoding two-component anthranilate 1,2-dioxygenase, constitutes a single transcriptional unit through Northern hybridization and reverse transcription-PCR (RT-PCR) analyses. The transcription start point of antA was mapped at 53 bp upstream point of its translation start point, and the -10 and -35 boxes were homologous to conserved sigma(70) recognition sequence. Hence the promoter of the ant operon was designated P-ant. 5' Deletion analyses using luciferase as a reporter showed that the region up to at least 70 bp from the transcription start point of antA was necessary for the activation of P-ant. Luciferase expression from P-ant was induced by anthranilate itself, but not by catechol. Two probable AraC/XylS-type regulatory genes found on pCAR1, open reading frame 22 (ORF22) and ORF23, are tandemly located 3.2 kb upstream of the antA gene. We revealed that the product of ORF23, designated AntR, is indispensable for the stimulation of P-ant in Pseudomonas putida cells. Northern hybridization and RT-PCR analyses revealed that another copy of P-ant, which is thought to be translocated about 2.1 kb upstream of the carAa gene as a consequence of the transposition of ISPre1, actually drives transcription of the carAa gene in the presence of anthranilate, indicating that both ant and car operons are simultaneously regulated by AntR.