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).

This study investigated general physicochemical properties of tea garden soils at the alluvial plain of Cong River in Tan Cuong commune, Vietnam. Four gardens were selected as study sites on three transect lines established perpendicularly to the river. Soil samples were collected from the surface (0-10 cm) and subsurface (20-30 cm). Soil texture classes varied from sandy loam to light clay, which was affected by different terrains along the transect lines as well as severe disturbance such as terracing and earth excavation. The levels of total C and total N were correlated with increasing garden age, suggesting the replenishment of soil organic matter pool by the addition of plant residue and manure. Meanwhile, the soils showed strongly acidic nature with the average pH(H 2 O) of 3.7 at the surface and 3.9 at the subsurface. The effective cation exchange capacity (ECEC) was low at 4.7 and 4.9 cmol c kg −1 , respectively, and dominated by exchangeable Al 3+ . Soil acidification was exacerbated with increasing garden age. However, a relatively large saturation of exchangeable calcium (Ca 2+ ), potassium (K + ), and magnesium (Mg 2+ ) on the ECEC was found in the surface soils. The levels of available P were high, occasionally exceeding 1000 and 500 mg kg −1 at the surface and subsurface, respectively. In spite of strongly acidic condition, ammonium (NH 4 -N) applied as fertilizer was converted to nitrate (NO 3 -N) to move down to deeper layers. The levels of the bases, P, and mineral N seem to be principally determined by management practices. Significant portion of these nutrients was likely to exist in water soluble forms without adsorption onto soils. It should be required to develop proper schemes and to educate the owners for adequate fertilizer managements.

Nucleotide sequence similarity, including k-mer plasmid composition, has been used for prediction of plasmid evolutionary host range, representing the hosts in which a plasmid has replicated at some point during its evolutionary history. However, the relationships between the bacterial taxa of experimentally identified transconjugants and the predicted evolutionary host ranges are poorly understood. Here, four different PromA group plasmids showing different k-mer compositions were used as model plasmids. Filter mating assays were performed with a donor harbouring plasmids and recipients of bacterial communities extracted from environmental samples. A broad range of transconjugants was obtained with different bacterial taxa. A calculation of the dissimilarities in k-mer compositions as Mahalanobis distance between the plasmid and its sequenced transconjugant chromosomes revealed that each plasmid and transconjugant were significantly more similar than the plasmid and other non-transconjugant chromosomes. These results indicate that plasmids with different k-mer compositions clearly have different host ranges to which the plasmid will be transferred and replicated. The similarity of the nucleotide compositions could be used for predicting not only the plasmid evolutionary host range but also future host ranges.