Isolation of Plasmid from a New Strain of Enterococcus faecium and its Construction for Use as Shuttle Cloning Vector

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Introduction: Enterococci are natural inhabitants of the human gastrointestinal tract and contain plasmids, which are often used to generate plasmid vectors. RGK7, a strain of Enterococcus faecium isolated from neonatal faeces, natively contains a 1935 bp plasmid pRGK7d. Objective: This study focused on the molecular characterization of the Enterococcus faecium plasmid pRGK7d and its exploitation for the generation of shuttle cloning vectors. Methods and Results: The isolate RGK7 was identified as Enterococcus faecium by phenotypic and genotypic methods, including 16S rRNA sequencing. Phylogenetic analysis also confirmed that the strain belongs to the group of E. faecium. BLAST analysis of the pRGK7d plasmid sequence revealed the highest homology with E. faecium genes for replication protein. ORF Finder analysis of the 1.9 kb plasmid pRGK7d showed a total of 17 ORFs in multiple overlapping frames, and the biggest ORF sequence showed 97% homology with the Rep protein of E. faecium. In order to make it an E. coli-Enterococcus shuttle vector, the plasmid was first cloned in pUC18 and then recloned in T vector by incorporating antibiotic selection markers ampicillin resistance (ampr) and erythromycin resistance (eryr) for its selectivity in Gram-negative E. coli and Gram-- positive E. faecium. The shuttle vector was reduced in size and made more effective by cloning only the Rep protein instead of the whole plasmid along with the eryr gene. Conclusion: An E. coli-Enterococcus shuttle vector was generated utilizing the plasmid pRGK7d, which, on transformation, conferred ampicillin and erythromycin resistance to the E. coli and Enterococcus, respectively, that was otherwise sensitive. The shuttle vector was found to be stable in both organisms.