Int-B13, an unusual site-specific recombinase of the bacteriophage P4 integrase family, is responsible for chromosomal insertion of the 105-kilobase clc element of Pseudomonas sp. strain B13
Int-B13, an unusual site-specific recombinase of the bacteriophage P4 integrase family, is responsible for chromosomal insertion of the 105-kilobase clc element of Pseudomonas sp. strain B13
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DOI：
10.1128/jb.180.21.5505-5514.1998
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发表时间：
1998-11-01
影响因子：
3.2
通讯作者：
van der Meer, JR
中科院分区：
文献类型：
--
作者：
Ravatn, R;Studer, S;van der Meer, JR
假单胞菌菌株B13在自身可传递的105-kb clc元件上携带编码氯儿茶酚降解酶的clcRABDE基因。该元件将位点和方向特异性地整合到各种细菌受体的染色体中，其中甘氨酸tRNA结构基因（glyV）作为整合位点。我们在这里报告的整合酶基因的定位和核苷酸序列和整合酶基因产物的活性介导的位点特异性整合。整合酶基因（int-B13）位于clc元件的右端附近。该基因的开放阅读框（ORF）为1,971 bp，编码657个氨基酸，经过表达和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，得到全长蛋白（74 kDa），其N端430个氨基酸与噬菌体P4家族整合酶的氨基酸序列基本相似，Int-B13的C端220个氨基酸与铜绿假单胞菌PaK 1萘降解基因簇侧翼的ORF同源。与噬菌体phi R73和P4类似，de元件整合到靶tRNA基因的3'端。从clc元件整合到其中的四种不同受体菌株中表征该靶位点，显示整合的序列特异性。在假单胞菌属菌株B13中，可以检测到环状形式的clc元件，其携带与glyV的3 '端部分相同的18-bp DNA序列作为其附着位点（attP）的一部分。将clc元件整合到染色体上的一个细菌附着位点（attB）后，在该元件的右端重建了一个功能性的glyV。在RecA缺陷型大肠杆菌中，用两个重组质粒证明了该整合过程，一个质粒携带int-B13基因和attP位点，另一个质粒携带恶臭假单胞菌F1的attB位点。
Pseudomonas sp. strain B13 carries the clcRABDE genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb clc element. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (glyV) as the integration site. We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. The integrase gene (int-B13) was located near the right end of the clc element. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa), The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation, The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases, The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in Pseudomonas aeruginosa PaK1. Similar to the bacteriophages phi R73 and P4, the de element integrates into the 3' end of the target tRNA gene. This target site was characterized from four different recipient strains into which the clc element integrated, showing sequence specificity of the integration, In Pseudomonas sp. strain B13, a circular form of the clc element, which carries an 18-bp DNA sequence identical to the 3'-end portion of glyV as part of its attachment site (attP), could be detected. Upon chromosomal integration of the clc element into a bacterial attachment site (attB), a functional glyV was reconstructed at the right end of the element, The integration process could be demonstrated in RecA-deficient Escherichia coli with two recombinant plasmids, one carrying the int-B13 gene and the attP site and the other carrying the attB site of Pseudomonas putida F1.