title: Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon.
abstract: 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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Zhengjun
authors.initials: Z
authors.lastname: Chen
authors.forename: Jinzhong
authors.initials: J
authors.lastname: Lin
authors.forename: Chengjie
authors.initials: C
authors.lastname: Ma
authors.forename: Shumiao
authors.initials: S
authors.lastname: Zhao
authors.forename: Qunxin
authors.initials: Q
authors.lastname: She
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Liang
mesh_terms: D004261:DNA Replication; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D042822:Genomic Instability; D007780:Lacticaseibacillus casei / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000302:isolation & purification*; D018741:Replication Origin; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase