Novel Self-Transmissible and Broad-Host-Range Plasmids Exogenously Captured From Anaerobic Granules or Cow Manure

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.

Keywords: PromA; broad host range; conjugation; plasmid; replication.

FIGURE 1

Phylogenetic analyses of captured plasmids and other related plasmids. A phylogenetic tree was constructed from amino acid sequences of a replication initiation protein (TrfA for IncP-1 plasmid, Rep or RepA for the others) by the maximum likelihood method with bootstrap percentages indicated at the nodes (Tamura-Nei model). A scale bar (0.50) shows substitutions per amino acid position. Previously known Inc groups are highlighted in colors, and a previously unrecognized plasmid group contains pSN1216-29. The accession numbers of the references are as follows: pPRO2 (CP000484), R7K (AM901564), pIE321 (EF633507), pSa (U30471), R388 (NC_028464), pXV2 (AF201825), pRM21 (U10426), pXCAW19 (NZ_CP009038), pKB14400_4 (CP014678), pGX4 (NZ_CP004364), pGLE121P3 (KC542383), and pNCPPB880-40 (JQ418534). Other references of PromA, IncP-1, and previously unrecognized plasmid groups are listed in Supplementary Table S5.

FIGURE 2

Phylogenetic analyses of 24 concatenated genes conserved in other PromA group plasmids (left), and alignment of 12 plasmids (right). (Left) A phylogenetic tree was constructed from nucleotide sequences of 24 core genes by the maximum likelihood method with bootstrap percentages indicated at nodes (Tamura–Nei model). A scale bar (0.050) indicates substitutions per nucleotide position. (Right) Coding sequences of each plasmid are presented as colored arrows in accordance with their putative functions. Sequences of oriV and putative oriT are indicated in red circles. Accession numbers of similar plasmids are shown in Supplementary Table S5.

FIGURE 3

A circular map of plasmid pSN1216-29. Coding sequences are shown as arrows indicating their transcriptional direction. Colors indicate their putative functions: red, replication; orange, maintenance; green, conjugation; light blue, resolvase; gray, a hypothetical protein. The sequences of oriV and putative oriT are indicated by a rectangle.

FIGURE 4

Alignment of pSN1216-29 with similar plasmids. Coding sequences of each plasmid are presented as colored arrows on the basis of their putative functions including MOB, T4CP, and T4SS for conjugation. Putative oriV and oriT sequences are indicated in red circles. Accession numbers of similar plasmids are given in Supplementary Table S5.

FIGURE 5

Structure of repA and the oriV region of pSN1216-29 used to construct the serial miniplasmids (A). The red arrow indicates the repA gene, colored triangles show putative iterons (orange) and DnaA boxes with their direction. Capacity for replication of miniplasmids with the Tc^r gene in Escherichia coli JM109 is denoted by a plus (+) or minus (–) sign. Alignments of putative iterons (B) and DnaA boxes (C).