SITE-SPECIFIC INTEGRATION IN SACCHAROPOLYSPORA-ERYTHRAEA AND MULTISITE INTEGRATION IN STREPTOMYCES-LIVIDANS OF ACTINOMYCETE PLASMID PSE101
SITE-SPECIFIC INTEGRATION IN SACCHAROPOLYSPORA-ERYTHRAEA AND MULTISITE INTEGRATION IN STREPTOMYCES-LIVIDANS OF ACTINOMYCETE PLASMID PSE101
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DOI：
10.1128/jb.170.5.2287-2295.1988
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发表时间：
1988-05-01
影响因子：
3.2
通讯作者：
KATZ, L
中科院分区：
文献类型：
--
作者：
BROWN, DP;CHIANG, SJD;KATZ, L
Saccharopolyspora erythraea NRRL 2338中有一个11.3kb碱基对的pSE101，它以整合序列(PSE101int)的形式存在于唯一的染色体位置，以自由形式存在，平均每10条染色体不到1个拷贝。红假单胞菌NRRL 2359中缺少该质粒序列。PSE101的游离型和整合型限制性内切酶图谱显示点对点对应。将pSE101、pBR322和硫代链霉菌抗性基因(TSR)连接，构建成重组表达载体pECT2。通过聚乙二醇介导法将其导入红链霉菌NRRL 2359的原生质体中，所有抗硫链球菌素的再生植株均在同一染色体上携带一份整合态的pECT2。菌株NRRL 2359(AttB)中pECT2整合的染色体位置与菌株NRRL 2338中pSE101int的染色体位置相对应。在携带pSE101int的宿主中，将质粒交叉位点(AttP)定位到与质粒序列中断位置相对应的质粒酶切位点上，表明发生了定点整合重组。在菌株NRRL 2338和NRRL 2359中也观察到了与pSE101片段同源的另外一个2.8kb的染色体序列。在将pECT2导入变铅青链霉菌后，大约一半的转化子被发现携带作为稳定的、自主复制的元件的质粒。另一半携带单个拷贝的pECT2作为一个完整的序列，但pECT2int在变青链霉菌中的位置在不同的转化子之间是不同的。在每种情况下，集成交叉使用的都是attP站点。提出了一个模型来解释pSE101在变铅青链霉菌中的特殊状态。
An 11.3-kilobase-pair plasmid, designated pSE101, exists in Saccharopolyspora erythraea NRRL 2338 as an integrated sequence (pSE101int) at a unique chromosomal location and in the free form in less than an average of 1 copy per 10 chromosomes. The plasmid sequence is missing from S. erythraea NRRL 2359. Restriction maps of the free and integrated forms of pSE101 showed point-to-point correspondence. Plasmid pECT2 was constructed by ligation of pSE101, pBR322, and the gene for thiostrepton resistance (tsr). When introduced by polyethylene glycol-mediated transformation into protoplasts of S. erythraea NRRL 2359, all thiostrepton-resistant regenerants examined were found to carry a single copy of pECT2 in the integrated state at a single chromosomal site. The chromosomal site of pECT2 integration in strain NRRL 2359 (attB) corresponded to the chromosomal location of pSE101int in strain NRRL 2338. The plasmid crossover site (attP) was mapped to the plasmid site that corresponded to the site of of interruption of the plasmid sequence in the host carrying pSE101int, indicating that site-specific integrative recombination had occurred. An additional 2.8-kilobase-pair chromosomal sequence homologous to a segment of pSE101 was also observed in strains NRRL 2338 and NRRL 2359. After introduction of pECT2 into Streptomyces lividans, approximately half of the transformants examined were found to carry the plasmid as a stable, autonomously replicating element. The other half carried a single copy of pECT2 as an integrated sequence, but the location of pECT2int in Streptomyces lividans varied from one transformant to another. In each case, integrative crossover used the attP site. A model is proposed to account for the determination of the particular state of pSE101 in Streptomyces lividans.