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Characterization of a novel theta-type plasmid pSM409 of Enterococcus faecium RME isolated from raw milk
Tawsif Ahmed Kazi1, Suranjita Mitra1, Bidhan Chandra Mukhopadhyay1
1Department of Botany, Visva-Bharati University, Santiniketan 731235, West Bengal, India.
Abstract:
Enterococcal plasmids have generated renewed interest for their indispensable role in pathogenesis and dissemination of multidrug-resistance. Recently, a novel plasmid pSM409 (4303-bp, GC% = 33.6%), devoid of antibiotic-resistance and virulence genes, has been identified in Enterococcus faecium RME, isolated from raw milk by us. pSM409 contains six open reading frames encoding a replication initiator protein (RepB) and five accessory proteins: antitoxin epsilon, bacteriocin immunity protein, HsdS, and two hypothetical proteins. Comparative sequence analysis of pSM409 reveals a mosaic pattern of similarity with different loci obtained from different theta plasmids, which dictates the plasmid to be heterogeneous or mosaic, possibly due to recombination. The pSM409 comprised of a typical theta-type origin of replication with four and a half direct repeats (iterons) of 22 nucleotides. The pSM409-RepB shared 76-82% homology with the RepB of reported theta plasmids from different genera, with dissimilarities mostly in its DNA-binding and C-terminal domain. The RepB sequence-based phylogenetic tree revealed its distinct position relative to the reported ones. The RepB grouped in the same clade has identical DNA-binding domains and their cognate iterons, possibly due to their sequence-specific interaction to initiate plasmid replication. Comparative analysis of the pSM409-iteron reveals that the repeats markedly differed from their closest homologues. This clade-specific relationship provides a new concept of classifying theta plasmids. The theta-type replicon identified in pSM409 has been found to be unique to E. faecium RME, prompting us to further investigate its utility as a vector for genetic manipulation of enterococci for health and industry.
Insights
A novel Enterococcus faecium plasmid, pSM409, lacks resistance genes but possesses a unique theta-type replication system. Its distinct RepB protein and iteron sequences offer a new way to classify plasmids and explore their use in genetic engineering.
Area of Science:
- Microbiology
- Molecular Biology
- Plasmid Biology
Background:
- Enterococcal plasmids are crucial for spreading multidrug resistance and virulence.
- Novel plasmids lacking antibiotic resistance and virulence genes are of interest for understanding plasmid biology.
- Enterococcus faecium is an important opportunistic pathogen and a common inhabitant of raw milk.
Purpose of the Study:
- To characterize a novel plasmid, pSM409, identified in Enterococcus faecium RME.
- To analyze the genetic content and replication mechanism of pSM409.
- To explore the potential of pSM409 as a vector for genetic manipulation.
Main Methods:
- Plasmid isolation and sequencing.
- Open reading frame identification and protein analysis.
- Comparative sequence analysis and phylogenetic tree construction.
- Iteron analysis and comparison with homologous sequences.
Main Results:
- A 4303-bp plasmid, pSM409, was identified in Enterococcus faecium RME, lacking antibiotic resistance and virulence genes.
- pSM409 contains six open reading frames, including a replication initiator protein (RepB) and accessory proteins.
- The plasmid exhibits a mosaic structure with a theta-type origin of replication, characterized by unique iteron sequences.
- RepB shows homology to RepB proteins of other theta plasmids but with distinct DNA-binding and C-terminal domains, suggesting clade-specific interactions.
- Phylogenetic analysis reveals a unique position for pSM409-RepB, proposing a new classification system for theta plasmids.
Conclusions:
- The novel plasmid pSM409 from Enterococcus faecium RME possesses a unique theta-type replication system.
- The distinct RepB protein and iteron sequences of pSM409 offer a new perspective on theta plasmid classification.
- pSM409 represents a potentially valuable tool for developing vectors for genetic manipulation in enterococci for health and industrial applications.
