The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker
The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker
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DOI：
10.1099/mic.0.2007/005959-0
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发表时间：
2007-11-01
期刊：
影响因子：
2.8
通讯作者：
Martinussen, Jan
中科院分区：
文献类型：
--
作者：
Defoor, Els;Kryger, Maj-Britt;Martinussen, Jan
从乳酸乳球菌（Lactococcus lactis subsp.）乳酸生物变种二乙酰乳酸菌株DB 0410。该质粒负责DB 0410对毒性嘧啶类似物5-氟乳清酸的敏感性。该基因的核苷酸序列为16404 bp。在15个开放阅读框中，发现3个是插入序列（IS）元件，分别被鉴定为2个IS 946和1个IS 982。两个ORF是不完整的，因为在它们的C-末端区域插入了一个IS元件。在IL 1403序列中发现了4个ORF的同源物：copB基因，编码铜-钾转运ATP酶B，以及ysbA、ysb B和ysbC基因。pDBORO复制区的结构组织在顺式（repA）和反式作用（repB和orfX）序列中与其他θ-复制质粒高度相似。通过质粒缺失分析和分子克隆，发现pDBORO上的单个位点赋予对5-氟乳清酸的敏感性。它被鉴定为ysbC，但为了反映其功能而重命名为oroP。oroP基因被认为是必不可少的乳清酸作为唯一的嘧啶来源，在嘧啶从头合成缺陷的菌株的利用。ORF编码的氨基酸序列显示了膜蛋白的特征。因此，oroP最有可能编码乳清酸转运蛋白。令人惊讶的是，oroP的同源物可以在两种L. lactis MG 1363和L. lactis IL 1403的菌株，尽管这些菌株不能显著利用乳清酸。oroP在大肠杆菌和枯草芽孢杆菌中的克隆表明乳清酸转运表型可以转化到这两种生物体中。研究结果表明，oroP可以作为一个强大的，食品级的选择/反选择标记在许多不同的生物体。
A new lactococcal plasmid, pDBORO, was isolated from the Lactococcus lactis subsp. lactis biovar diacetylactis strain DB0410. This plasmid is responsible for the sensitivity of DB0410 to the toxic pyrimidine analogue 5-fluoroorotate. The complete nucleotide sequence has been determined and amounts to 16 404 bp. Of 15 ORFs encountered, three were found to be insertion sequence (IS) elements, identified as two IS946 and one IS982. Two ORFs are incomplete due to the insertion of an IS element in their C-terminal region. Homologues for four ORFs were found in the IL1403 sequence: the copB gene, coding for a copper-potassium-transporting ATPase B, and the ysbA, ysbB and ysbC genes. The structural organization of the pDBORO replication region is highly similar to other theta-replicating plasmids in both the cis-(repA) and trans-acting (repB and orfX) sequences. By plasmid deletion analysis and molecular cloning, a single locus on pDBORO was found to confer sensitivity to 5-fluoroorotate. It was identified as ysbC, but renamed oroP in order to reflect its function. The oroP gene was found to be essential for the utilization of orotate as the sole pyrimidine source in a strain deficient in pyrimidine de novo synthesis. The amino acid sequence encoded by the ORF showed the characteristic features of a membrane protein. Therefore, oroP most probably encodes an orotate transporter. Surprisingly, homologues of oroP could be identified in the genomes of both L. lactis MG 1363 and L. lactis IL 1403 despite the fact that these strains were unable to significantly utilize orotate. Cloning of oroP in Escherichia coli and Bacillus subtilis showed that the orotate transport phenotype could be transformed to both organisms. The findings presented indicate that oroP can be used as a powerful, food-grade selection/counterselection marker in many different organisms.