Diverse broad-host-range plasmids from freshwater carry few accessory genes

Abstract

Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid's ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities.

Fig 1

Illustration of the triparental mating method. Environmental bacteria containing plasmids (P) are mixed with a donor bacterium, which carries a mobilizable plasmid (M) with a specific marker, such as antibiotic resistance (Ab^R), and a recipient bacterium, which has a second selectable marker, such as rifampin resistance (Rif^R). When plated on both antibiotics, only recipient strains that carry a mobilizable plasmid can grow, and since only the environmental plasmid (P) can move the mobilizable plasmid into the recipient strain, the environmental plasmid is sometimes present too. One pathway by which transconjugants can be formed is shown by the arrows.

Fig 2

Alignment of newly sequenced plasmids showing their relationship to each other and to well-studied members of their incompatibility group. (a) IncP-1 plasmids. (b) IncU plasmids. Coding regions are shown as colored arrows; putative functions are indicated by the color key. The triphenylmethane reductase gene is labeled as tmr on pMBUI8. The degree of similarity between plasmids (percent nucleotide identity [nt id] of alignments performed using ClustalW) is indicated by gray scale-shaded regions; the darker the shading between two segments, the higher their similarity as shown in the heat key. Sites of mini-Tn21 insertion in the tagged plasmids are marked with a filled black triangle.

Fig 3

Genetic maps of 4 of the 10 plasmids isolated from Moscow, ID: the IncN plasmid pDS2, the IncW-like plasmid pMBUI4, and two plasmids whose Inc groups are unknown, pMBUI2 and pMBUI6. The gene modules are shown as colored arrows representing different functions (as indicated by the color key). In pMBUI6, the genes named “orf” are most closely related to transfer genes of different BHR plasmids. Genes with the same names on different plasmids are not necessarily homologous due to gene naming conventions for plasmids. Sites of mini-Tn21 insertion in the tagged plasmids are marked with a filled black triangle.

Fig 4

Phylogenetic relationship of pDS1 to other IncP-1 plasmid subgroups. The conserved 3′ end of the trfA gene was used to infer the phylogeny of 45 IncP-1 plasmids using the maximum-likelihood method (base frequencies, gamma shape parameter, proportion of invariant sites, and rate matrix [a b c c b a] were estimated from the data). The incompatibility subgroup of each clade is indicated on the right.

Fig 5

Unique plasmid restriction fragment patterns of captured plasmids generated by EcoRI digestion. (A) Plasmids isolated from Paradise Creek (PCP). (B) Plasmids isolated from Moscow, ID, WWTP (WW). All plasmid DNA samples were completely digested, as indicated by the linearized mobilizable plasmid at 4 kb (pBBR1MCS) or 6.2 kb (pSU4814), and were run on a 0.5% agarose gel for 18 h at 30 V. Plasmids captured using pBBR1MCS contain BB in their names while those captured with pSU4814 contain SU.