School of Science, RMIT University, Bundoora, Victoria, Australia.
PLoS One. 2020 Aug 12;15(8):e0232806. doi: 10.1371/journal.pone.0232806. eCollection 2020.
There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as potential protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against WCFS1 (pTRKH2) as the indicator strain, showing that functional expression of plantacyclin B21AG had been achieved. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 ± 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 ± 0.05 and 25.19 ± 2.68 copies respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 results in similar killing activity being observed in both the wild type and recombinant hosts despite the lower copy number of pCycB21.
消费者对加工最少、无防腐剂和微生物安全的食品的需求日益增加。这些因素，再加上对抗生素耐药性的风险，使得人们对乳酸菌（LAB）产生的细菌素产生了兴趣，将其作为天然食品防腐剂和潜在的蛋白质治疗药物。我们之前报道了植物环素 B21AG 的发现，这是一种由植物乳杆菌 B21 产生的环形细菌素。在这里，我们描述了在益生菌植物乳杆菌 WCFS1 中克隆和表达细菌素基因簇。基因组测序表明，细菌素编码在一个 20kb 的天然质粒上，命名为 pB21AG01。七个开放阅读框（ORFs）推测参与细菌素的产生、分泌和免疫被克隆到一个大肠杆菌/乳杆菌穿梭载体 pTRKH2 中。得到的质粒 pCycB21 转化到植物乳杆菌 WCFS1 中。当测试对抗指示菌株 WCFS1（pTRKH2）时，B21 和 WCFS1（pCycB21）的无细胞上清液（CFS）均显示出 800 AU/mL 的抗菌活性，表明植物环素 B21AG 的功能表达已经实现。实时 PCR 分析显示，在植物乳杆菌 B21 中，pB21AG01 的相对拷贝数为 7.60 ± 0.79，而在 WCFS1 中，pCycB21 和 pTRKH2 的拷贝数分别为 0.51 ± 0.05 和 25.19 ± 2.68。这表明细菌素基因簇位于植物乳杆菌 B21 中的一个高度稳定的低拷贝数质粒 pB21AG01 上。包含来自 pB21AG01 的细菌素操纵子的天然启动子，导致在野生型和重组宿主中观察到相似的杀伤活性，尽管 pCycB21 的拷贝数较低。
