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鉴定乳球菌乳亚种 UC317 质粒 pCI305 的最小复制子。

Identification of the Minimal Replicon of Lactococcus lactis subsp. lactis UC317 Plasmid pCI305.

机构信息

Department of Food Microbiology and National Food Biotechnology Centre, University College, Cork, Ireland.

出版信息

Appl Environ Microbiol. 1990 Jan;56(1):202-9. doi: 10.1128/aem.56.1.202-209.1990.

DOI:10.1128/aem.56.1.202-209.1990
PMID:16348092
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC183273/
Abstract

Replication functions of the stable, cryptic 8.7-kilobase (kb) plasmid pCI305 from multi-plasmid-containing Lactococcus lactis subsp. lactis UC317 were studied. Analysis of this replicon was facilitated by the construction of replication probe vectors that consisted of the pBR322 replication region, a pUC18-derived multiple cloning site, and either the cat gene of pC194 (pCI341; 3.1 kb) or the erm gene of pAMbeta1 (pCI3330; 4.0 kb). Plasmid pCI305 was introduced into plasmid-free L. lactis subsp. lactis MG1363Sm, a streptomycin-resistant derivative of MG1363, by a transformation procedure with the 75-kb lactose-proteinase plasmid pCI301 of UC317 as a marker plasmid. A combination of transposon Tn5 mutagenesis and subcloning in pCI341 and pCI3330 with individual Tn5 insertions around the replication region facilitated the identification of a 1.6-kb minimal replicon on pCI305. This region was separable into two domains: (i) a 1.3-kb region (repB) encoding a trans-acting function (in vitro transcription-translation studies suggested the involvement of a 48-kilodalton protein); and (ii) a 0.3-kb region (repA) sufficient to direct replication when provided with repB in trans and thus probably containing the origin of replication. Lactococcus-Escherichia coli shuttle vectors based on the pCI305 replication region were constructed.

摘要

研究了来自多质粒乳球菌乳亚种 UC317 的稳定、隐匿的 8.7 千碱基 (kb) 质粒 pCI305 的复制功能。通过构建复制探针载体，分析了这个复制子，这些载体由 pBR322 复制区域、pUC18 衍生的多克隆位点以及 pC194 的 cat 基因 (pCI341；3.1 kb) 或 pAMbeta1 的 erm 基因 (pCI3330；4.0 kb) 组成。通过用 UC317 的 75kb 乳糖蛋白酶质粒 pCI301 作为标记质粒的转化程序，将质粒 pCI305 引入无质粒乳球菌乳亚种 MG1363Sm，这是 MG1363 的链霉素抗性衍生物。转座子 Tn5 诱变和在 pCI341 和 pCI3330 中的亚克隆的组合，用围绕复制区域的单个 Tn5 插入，促进了在 pCI305 上识别 1.6kb 的最小复制子。该区域可分为两个区域：(i) 1.3kb 区域 (repB) 编码反式作用功能（体外转录-翻译研究表明涉及 48 千道尔顿的蛋白质）；和 (ii) 0.3kb 区域 (repA) 足以在提供 repB 时进行复制，因此可能包含复制起点。基于 pCI305 复制区域构建了乳球菌-大肠杆菌穿梭载体。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54bb/183273/981c0e4fc700/aem00066-0228-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54bb/183273/7405fee570ac/aem00066-0226-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54bb/183273/981c0e4fc700/aem00066-0228-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54bb/183273/7405fee570ac/aem00066-0226-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54bb/183273/981c0e4fc700/aem00066-0228-a.jpg

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