Abstract
Novel self-transmissible plasmids were exogenously captured from environmental samples by triparental matings with pBBR1MCS-2 as a mobilizable plasmid and Pseudomonas resinovorans as a recipient. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. The whole nucleotide sequences of six plasmids were determined, including one IncP-1 plasmid (pSN1104-59), four PromA-like plasmids (pSN1104-11, pSN1104-34, pSN0729-62, and pSN0729-70), and one novel plasmid (pSN1216-29), whose incompatibility group has not been previously identified. No previously known antibiotic resistance genes were found in these plasmids. In-depth phylogenetic analyses showed that the PromA-like plasmids belong to subgroups of PromA (designated as PromAγ and PromAδ) different from previously proposed subgroups PromAα and PromAβ. Twenty-four genes were identified as backbone genes by comparisons with other PromA plasmids. The nucleotide sequences of pSN1216-29 share high identity with those found in clinical isolates. A minireplicon of pSN1216-29 was successfully constructed from repA encoding a replication initiation protein and oriV. All the captured plasmids were found to have a broad host range and could be transferred to and replicated in different classes of Proteobacteria. Notably, repA and oriV of pSN1216-29 showed high similarity with one of two replication systems of pSRC119-A/C, known as a plasmid with multidrug resistance genes found in Salmonella enterica serovar Senftenberg. Our findings suggest that these “cryptic” but broad-host-range plasmids may be important for spreading several genes as “vehicles” in a wider range of bacteria in natural environments.
Highlights
Conjugative plasmids are major mobile genetic elements transmitting various accessory genes that confer antibiotic and/or heavy-metal resistance, pathogenicity, and metabolic pathways onto their hosts (Hulter et al, 2017)
The isolates with putative environmental plasmids were classified by Polymerase chain reaction (PCR) with the specific primer sets to detect different Inc groups including IncA and IncC (Carattoli et al, 2005), IncW (Carattoli et al, 2005), IncP-1 (Bahl et al, 2009), and PromA (Zhang et al, 2015)
PCR products were obtained with IncP-1– and PromA-specific primers for four and 90 isolates (“IncP-1–positive” or “PromA-positive,” whereas both products were obtained from two isolates), respectively, whereas 180 isolates yielded no amplicons with any primer set (“PCRnegative,” Supplementary Table S3)
Summary
Conjugative plasmids are major mobile genetic elements transmitting various accessory genes that confer antibiotic and/or heavy-metal resistance, pathogenicity, and metabolic pathways onto their hosts (Hulter et al, 2017). Triparental mating using a donor with a mobilizable plasmid is an efficient method for collecting conjugative plasmids in environments without any marker genes, including antibiotic or heavy-metal resistance genes or metabolic genes (Smalla et al, 2015). This method is dependent on the mobilizing ability of a self-transmissible plasmid from environmental samples, which can act as a “helper” plasmid for a previously known mobilizable plasmid (Hill et al, 1992; Top et al, 1994). This finding implies that the triparental method involving pBBR1MCS vectors is appropriate for collecting new types of self-transmissible plasmids from environmental samples
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