Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon
Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon
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pMC11（一种从干酪乳杆菌 MCJ 中分离出的具有双复制起点的质粒）的表征以及带有每个复制子的穿梭载体的构建
DOI：
10.1007/s00253-014-5649-z
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发表时间：
2014-07-01
影响因子：
5
通讯作者：
Liang, Yunxiang
中科院分区：
文献类型：
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作者：
Chen, Zhengjun;Lin, Jinzhong;Liang, Yunxiang
许多乳酸菌携带不同的质粒，特别是那些通过θ机制复制的质粒。在这里，我们描述了干酪乳杆菌MCJ（CCTCC AB 20130356），一种含有pMC 11的新分离物，携带两种不同的θ型复制子。每个复制子在复制起点（oriV 1或oriV 2）中含有一个iteron，并含有一个编码复制酶的基因（RepA_1或RepB_1），这两个基因都是质粒复制所必需的。用每个复制子构建大肠杆菌/乳杆菌穿梭载体，分别产生基于oriV 2和oriV 1复制子的pEL 5.7和pEL 5.6。这些质粒表现出不同的性质：pEL 5.7能够在L.干酪MCJΔ1和德氏乳杆菌亚种（Lactobacillus delbrueckii subsp. pEL 5.6在乳酸杆菌LBCH-1中复制，但在另外两种测试的乳酸杆菌菌株中没有复制，而在三种不同的菌株中复制，包括L. casei MCJΔ1，L. casei NJ、Lactobacillus paracasei LPC-37和L.德氏乳酸LBCH-1。研究了质粒的稳定性：pEL 5.6和pEL 5.7在L. casei，因为每代损失率低于1%。pEL 5.7在L.德氏乳酸LBCH-1，损失率估计为3%。使用来自嗜酸乳杆菌的S层蛋白SlpA的启动子，这些载体用于表达绿色荧光蛋白（GFP）。GFP在不同菌株中的表达存在生长时相调控。总之，这些穿梭载体为乳酸杆菌的DNA克隆和异源基因表达提供了有效的遗传工具。
Many lactic acid bacteria carry different plasmids, particularly those that replicate via a theta mechanism. Here we describe Lactobacillus casei MCJ(CCTCC AB20130356), a new isolate that contains pMC11, carrying two distinct theta-type replicons. Each replicon contained an iteron in the origin of replication (oriV1 or oriV2) and a gene coding for the replicase (RepA_1 or RepB_1), both of which are essential for plasmid replication. Escherichia coli/Lactobacillus shuttle vectors were constructed with each replicon, yielding pEL5.7 and pEL5.6 that are based on oriV2 and oriV1 replicons, respectively. These plasmids showed distinct properties: pEL5.7 was capable of replicating in L. casei MCJΔ1 and Lactobacillus delbrueckii subsp. lactic LBCH-1 but failed to do so in two other tested lactobacilli strains whereas pEL5.6 replicated in three different strains, including L. casei MCJΔ1, L. casei NJ, Lactobacillus paracasei LPC-37 and L. delbrueckii subsp. lactic LBCH-1. Plasmid stability was studied: pEL5.6 and pEL5.7 were very stably maintained in L. casei, as the loss rate was lower than 1 % per generation. pEL5.7 was also stable in L. delbrueckii subsp. lactic LBCH-1 with the loss rate estimated to be 3 %. These vectors were employed to express a green fluorescent protein (GFP) using the promoter of S-layer protein SlpA from Lactobacillus acidophilus. And a growth-phase regulated expression of GFP was observed in different strains. In conclusion, these shuttle vectors provide efficient genetic tools for DNA cloning and heterologous gene expression in lactobacilli.