Pseudomonas aeruginosa dihydroorotases:: a tale of three pyrCs
Pseudomonas aeruginosa dihydroorotases:: a tale of three pyrCs
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DOI：
10.1007/s00203-004-0687-z
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发表时间：
2004-09-01
影响因子：
2.8
通讯作者：
O'Donovan, GA
中科院分区：
文献类型：
--
作者：
Brichta, DM;Azad, KN;O'Donovan, GA
铜绿假单胞菌PAO1被证明含有三个pyrC序列。其中两个基因，分别命名为pyrC（PA3527）和pyrC2（PA5541），编码具有二氢乳清酸酶（DHOase）活性的多肽，而第三个基因pyrC'（PA0401）编码一种类似DHOase的多肽，该多肽缺乏DHOase活性，但对天冬氨酸氨甲酰转移酶（ATCase）的结构和功能是必需的。pyrC和pyrC2都被克隆并能互补大肠杆菌pyrC突变体。此外，通过与各自突变的等位基因同源交换，在铜绿假单胞菌中分别使pyrC和pyrC2失活。所得到的突变菌株是原养型的。还构建了一个pyrC、pyrC2双突变体，该菌株对嘧啶有绝对需求。利用lacZ启动子融合来测量pyrC和pyrC2的转录活性。发现pyrC是组成型表达的，而pyrC2仅在pyrC突变体背景下表达。使用体外转录/翻译系统来估计pyrC2基因产物的大小。所表达的多肽约为47 kDa，这与理论分子量48 kDa相符，使其成为迄今为止鉴定出的最大的原核生物DHOase多肽。据我们所知，这是关于铜绿假单胞菌中真正的DHOase突变体的首次报道，也是对pyrC2编码具有DHOase活性的多肽的首次确认。
Pseudomonas aeruginosa PAO1 was shown to contain three pyrC sequences. Two of these genes, designated pyrC (PA3527) and pyrC2 (PA5541), encode polypeptides with dihydroorotase (DHOase) activity, while the third, pyrC' (PA0401), encodes a DHOase-like polypeptide that lacks DHOase activity, but is necessary for the structure and function of ATCase. Both pyrC and pyrC2 were cloned and complemented an Escherichia colipyrC mutant. In addition, pyrC and pyrC2 were individually inactivated in P. aeruginosa by homologous exchange with a mutated allele of each. The resulting mutant strains were prototrophic. A pyrC, pyrC2 double mutant was also constructed, and this strain had an absolute requirement for pyrimidines. The transcriptional activity of pyrC and pyrC2 was measured using lacZ promoter fusions. While pyrC was found to be constitutively expressed, pyrC2 was expressed only in the pyrC mutant background. An in vitro transcriptional/translational system was used to estimate the size of the pyrC2 gene product. The expressed polypeptide was approximately 47 kDa, which is in keeping with the theoretical molecular mass of 48 kDa, making it the largest prokaryotic DHOase polypeptide identified to date. To our knowledge, this is the first report of a true DHOase mutant in P. aeruginosa and also the first confirmation that pyrC2 encodes a polypeptide with DHOase activity.