Yin Sheng, Hao Yanling, Zhai Zhengyuan, Li Ruoyu, Huang Ying, Tian Hongtao, Luo Yunbo
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
FEMS Microbiol Lett. 2008 Aug;285(2):183-7. doi: 10.1111/j.1574-6968.2008.01229.x. Epub 2008 Jun 28.
A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.
从鲜牛奶中分离得到的植物乳杆菌M4的一个隐蔽质粒，命名为pM4，进行了测序和特性分析。它长度为3320 bp，G+C含量为38.73 mol%。预测质粒pM4编码三个推定的开放阅读框（ORF），其中ORF1与已报道的滚环复制（RCR）pC194家族质粒pWCFS101和pF8801的Rep蛋白分别具有99%和98%的同源性。序列分析揭示了一个典型的pC194家族双链起源（dso）和一个位于rep基因上游的推定单链起源（sso）。绿豆核酸酶分析和Southern杂交证实了单链DNA（ssDNA）中间体的存在，表明pM4属于RCR pC194家族。利福平处理菌株中ssDNA的积累意味着宿主编码的RNA聚合酶参与了ssDNA向双链DNA的转化。此外，通过实时PCR估计pM4在每个细胞中的相对拷贝数约为25。这种新的RCR质粒在构建用于食品工业的克隆载体方面将具有重要价值。
