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Characterization of a novel rolling-circle replication plasmid pYSI8 from Lactobacillus sakei YSI8
Zhengyuan Zhai1, Yanling Hao, Sheng Yin
1College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qing Hua East Road, Hai Dian District, Beijing 100083, China.
Abstract:
A plasmid from Lactobacillus sakei YSI8, designated as pYSI8, was sequenced and characterized. It consisted of a 4973bp circular molecule with a G+C content of 35.6%. The plasmid pYSI8 was predicted to contain five putative ORFs, in which ORF1 shared 79% and 76% identity with Rep proteins of pLH2 and pLC2, members of rolling-circle replication (RCR) pMV158 family. Detection of single-stranded DNA (ssDNA) intermediates by Southern hybridization and mung bean nuclease treatment confirmed that pYSI8 replicated via the RCR mechanism. 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 (dsDNA). Furthermore, the copy number of pYSI8 was estimated to be 41.9+/-0.5 in each cell by real-time polymerase chain reaction.
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