Characterization of Plasmid pML21 of Enterococcus faecalis ML21 from Koumiss

Abstract

Plasmid mobilization has been recognized as the major mechanism for horizontal gene transfer in the evolution of antibiotic-resistant Enterococcus faecalis strains capable of causing human infection [1]. An investigation of plasmid distribution in E. faecalis and an analysis of their role in the dissemination of antimicrobial resistance can contribute to revealing the differences in the properties of clinically relevant versus probiotic strains of E. faecalis. Although a large number of E. faecalis plasmids have been sequenced and characterized, very few are from probiotic strains [2]. Many native plasmids from other lactic acid bacteria, such as lactobacilli and lactococci, have been used for the design of molecular tools, especially food-grade cloning and expression vectors [3, 4]. Although many E. faecalis strains isolated from food harbor abundant natural plasmids [5], only a few have been developed into valuable tools for biotechnological applications. Enterococcus faecalis strain ML21 (CGMCC No. 6166) was originally isolated from the koumiss produced by nomadic families of the Ili pastoral area of Xinjiang Uyghur Autonomous Region, China. Susceptibility testing by the agar disk diffusion method showed that the isolate is susceptible to vancomycin and some other antibiotics (e.g., erythromycin, chloramphenicol, and ampicillin). This susceptibility is remarkably different from the vast majority of enterococci from clinical cases [6]. E. faecalis ML21 harbors a native plasmid, designated pML21. DNA sequencing on an ABI 3730XL DNA Analyzer (Applied Biosystems Inc., Foster City, CA) showed that this plasmid is 5,598 bp in size with 32.9 % G?C, and is predicted to carry five putative open reading frames (ORFs) (Fig. 1a). Sequence analysis of the replicon region (ORF1, ORF2, and the sequence upstream of ORF1) of pML21 revealed that it is a member of the pCI305 family of plasmids, belonging to the group of lactococcal theta-type replicons [8], which differ from the rep9 family replicon plasmids that are prevalent in clinical strains of E. faecalis [2]. We therefore speculated that horizontal gene transfer might have occurred between E. faecalis ML21 and lactococcal strains in this niche, as the fermented dairy product koumiss contains a complex microbial ecological community. The plasmid also contains an e antitoxin component of the e/f-type toxin–antitoxin system (encoded by ORF5) but interestingly, no cognate toxin gene was detected on this plasmid. Based on homology blast, pML21 ORF3 was predicted to encode an abortive infection phage resistance (AIPR) protein which is often found in restriction–modification system operons. ORF3 and ORF4 were found likely to be under the control of a specific bidirectional promoter in pML21. ORF4 was predicted to encode a hypothetical MerR family transcriptional regulator protein, but its function remains unknown. The relative copy number of this plasmid was estimated to be about 137.81 ± 3.20 copies per cell, as determined by quantitative real-time PCR [9], higher than that of other closely related plasmids [10, 11]. To develop molecular tools using pML21, an Escherichia coli lactic acid bacteria shuttle vector designated pML23e was constructed (Fig. 1b). This vector was able to transform all tested lactic acid bacteria strains, including E. faecalis ML21, Lactococcus lactis subsp. cremoris MG1363, F. Zuo X. Feng X. Sun C. Du S. Chen (&) Key Laboratory of Functional Dairy Science of Chinese Ministry of Education and Municipal Government of Beijing, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China e-mail: swchen@cau.edu.cn