Cloning and expression of the Erwinia carotovora subsp carotovora gene encoding the low-molecular-weight bacteriocin carocin S1
Cloning and expression of the Erwinia carotovora subsp carotovora gene encoding the low-molecular-weight bacteriocin carocin S1
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DOI：
10.1128/jb.01090-06
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发表时间：
2007-01-01
影响因子：
3.2
通讯作者：
Wu, Huang-pin
中科院分区：
文献类型：
--
作者：
Chuang, Duen-yau;Chien, Yung-chei;Wu, Huang-pin
本研究的目的是克隆胡萝卜素 S1 基因，并在不产胡萝卜素的胡萝卜软腐欧文氏菌菌株中表达该基因。使用 H-rif-8-2（胡萝卜软腐欧文菌胡萝卜亚种 89-H-4 的自发利福平抗性突变体）通过 Tn5 插入诱变获得了突变体 TH22-10，它产生高分子量细菌素，但不产生低分子量细菌素。使用热不对称交错 PCR，确定了 Tn5 插入位点的 DNA 序列和连续 2,280 bp 区域的 DNA 序列。在序列片段内鉴定出两个完整的开放阅读框（ORF），命名为 ORF2 和 ORF3。 ORF2和ORF3被鉴定为carocin S1基因caroS1K (ORF2)和caroS1I (ORF3)，它们分别编码杀伤蛋白(CaroS1K)和免疫蛋白(CaroS1I)。这些基因与pyocin S3基因和pyocin AP41基因同源。 Carocin S1 在 E. carotovora subsp. 中表达。 carotovora Ea1068 并在 TH22-10 中复制，但不能在大肠杆菌 (JM101) 中表达，因为不存在类似 SOS 盒的共有序列。在起始密码子的上游发现了与大肠杆菌环 AMP 受体蛋白结合位点 (-312 bp) 的共有序列相似的推定序列。这种细菌素的产生也是由葡萄糖和乳糖诱导的。同源性检索结果表明carocin S1基因(bp 1078～bp 1704)与铜绿假单胞菌中的pyocin S3和pyocin AP41基因同源。这些基因编码具有核酸酶活性的蛋白质（结构域 4）。本研究发现carocin S1也具有核酸酶活性。
The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.