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.
Plasmids are known to spread among different bacteria. However, which plasmids spread among environmental samples and in which environments they are present is still poorly understood.
Strain or plasmidRelevant characteristics References Bacterial strains Escherichia coliDH5α F − , 80dlacZ M15, (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(r K − , m K + ), phoA, supE44, λ − , thi-1, gyrA96, relA1DH5α, Tc r This study S17-1 λpir RK2 tra regulon; host for pir-dependent plasmids; recA thi pro hsdR M RP4-2-Tc:Mu-Km:Tn7λpir Tp r Sm r Simon et al., 1983 Pseudomonas putida KT2440 pWW0-free Pseudomonas putida mt-2 Bagdasarian et al., 1981 KT2440(pBBR1MCS-5) pBBR1MCS-5-harboring KT2440 This study KT2440(pSN1216-29, pBBR1MCS-5) pSN1216-29 and pBBR1MCS-5-harboring KT2440 This study SMDBS A dapB-deleted strain of SM1443, Rif r of KT2440 (KT2442) with mini-Tn5-lacI q cassette inserted into the chromosome Shintani et al., 2014 SMDBS(pSN1216-29:gfp) SMDBS bearing pSN1216-29:gfp This study Pseudomonas resinovorans CA10dm4RGFP CA10dm4R (spontaneous rifampicin-resistant CA10dm4), miniTn7(Gm) P A1/O4/O3 -gfp-a was inserted into chromosome (Gm r , Cm r ). Yanagiya et al., 2018 CA10dm4RGFP(pSN1216-29, pBBR1MCS-2) pSN1216-29 and pBBR1MCS-2-harboring CA10dm4RGFP Yanagiya et al., 2018 Plasmids pBBR1MCS-2 Km r , lacZα mob; compatible with IncP, IncQ, and IncW plasmids Kovach et al., 1995 pBBR1MCS-3 Tc r , lacZα mob; compatible with IncP, IncQ, and IncW plasmids Kovach et al., 1995 mini-pBBR1MCS-3 Self-ligated 3020-bp DNA region containing oriV, rep, and Tc r gene of pBBR1MCS-3 This study pBBR1MCS-5 Gm r , lacZα mob; compatible with IncP, IncQ, and IncW plasmids Kovach et al., 1995 pJBA28 Ap r , Km r ; delivery plasmid for mini-Tn5-Km-P A1/O4/O3 -RBSII-gfpmut3*-T 0 -T 1 Andersen et al., 1998 pSN1216-29 Conjugative, broad-host-range plasmid Yanagiya et al., 2018 pSN1216-29:gfp mini-Tn5-Km-P A1/O4/O3 -RBSII-gfpmut3*-T 0 -T 1 was inserted in the end of tivB7 gene (25,653 nt of pSN1216-29). This study Gel/PCR DNA fragments Extraction kit (RBC Bioscience, New Taipei City, Taiwan), NEBuilder Hifi DNA Assembly system (New England Biolabs, Ipswich, MA, United States), and competent E. coli DH5α cells (RBCBioscience) were employed for cloning of DNA fragments. The other procedures were performed according to standard methods (Sambrook and Russell, 2001).
The frequency of transconjugants were compared for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1 under aerobic and anaerobic conditions. Filter mating assays were performed with one donor strain and one recipient strain using different donors of Pseudomonas and recipient strains, including Pseudomonas, Pantoea, and Buttiauxella. Under anaerobic condition, frequencies of transconjugants for both plasmids were 101-103-fold lower than those under aerobic condition regardless of whether aerobically or anaerobically grown donors and recipients were used. To compare the transconjugant ranges under aerobic and anaerobic conditions, conjugation was performed between the donor of pBP136 and recipient bacteria extracted from environmental samples. Several transconjugants were uniquely obtained from each aerobic or anaerobic condition. Our findings indicate that a plasmid can differently spread among bacteria depending on the oxygen concentrations of the environment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.