Broad-host-range plasmids from agricultural soils have IncP-1 backbones with diverse accessory genes

Abstract

Broad-host-range plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to antibiotics and heavy metals or degradation of pollutants. Although some broad-host-range plasmids have been extensively studied, their evolutionary history and genetic diversity remain largely unknown. The goal of this study was to analyze and compare the genomes of 12 broad-host-range plasmids that were previously isolated from Norwegian soils by exogenous plasmid isolation and that encode mercury resistance. Complete nucleotide sequencing followed by phylogenetic analyses based on the relaxase gene traI showed that all the plasmids belong to one of two subgroups (β and ε) of the well-studied incompatibility group IncP-1. A diverse array of accessory genes was found to be involved in resistance to antimicrobials (streptomycin, spectinomycin, and sulfonamides), degradation of herbicides (2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenoxypropionic acid), and a putative new catabolic pathway. Intramolecular transposition of insertion sequences followed by deletion was found to contribute to the diversity of some of these plasmids. The previous observation that the insertion sites of a Tn501-related element are identical in four IncP-1β plasmids (pJP4, pB10, R906, and R772) was further extended to three more IncP-1β plasmids (pAKD15, pAKD18, and pAKD29). We proposed a hypothesis for the evolution of these Tn501-bearing IncP-1β plasmids that predicts recent diversification followed by worldwide spread. Our study increases the available collection of complete IncP-1 plasmid genome sequences by 50% and will aid future studies to enhance our understanding of the evolution and function of this important plasmid family.

Fig. 1.

Phylogenetic tree showing the relationship of the 12 pAKD plasmids described in this study and pAKD4 (27) (bold) with other completely sequenced IncP-1 plasmids. A maximum likelihood tree was inferred from nucleotide sequences of the relaxase gene, traI. Bootstrap values of the deep branches are shown to the left of each node. The scale bar represents the probability of nucleotide substitutions per site. Accession numbers for the plasmids used to construct the phylogenetic tree are given in Materials and Methods.

Fig. 2.

Alignment of 12 pAKD plasmids showing their evolutionary relationship to each other and to the well-known IncP-1 plasmids R751 and pKJK5. Coding regions are shown as colored arrows; putative functions are indicated by the color key (top right). The degree of similarity between plasmids (percent nucleotide identity of alignments performed using ClustalW) is indicated by grayscale-shaded regions; the darker the shading between two segments, the higher their similarity, as shown in the heat key (middle right).

Fig. 3.

Alignment of accessory regions of five pAKD plasmids indicating deletions of various sizes relative to pAKD15. Arrows represent ORFs showing direction of transcription. The plasmids shown here have 100% identity along their backbone sequences but have deletions in their accessory regions, shown as black lines. All deletions are flanked by IS21 at the 3′ end, while sequences at the 5′ end are variable. The mercury resistance genes are carried on a Tn501-like transposon that is not intact. Also shown here is the putative novel catabolic pathway of pAKD15 (Table 3). Intergenic regions are not drawn to scale.

Fig. 4.

Hypothetical evolutionary pathway for the Tn501 group of IncP-1β plasmids.

Fig. 5.

Comparison of tfd gene regions from pAKD25 and pAKD26 with pEST4011. The black arrows show tfd genes, while the gray arrows show flanking sequences. The tfd gene order is conserved among all three plasmids, but pEST4011 has the most complete tfd gene cluster, while both pAKD25 and pAKD26 have been interrupted and are missing tfdF. clp, chloride channel protein; hyp, hypothetical protein; gtdA, gentisate 1,2-dioxygenase; iaah, indole acetamide hydrolase. Intergenic regions are not drawn to scale.

Fig. 6.

Antibiotic resistance-bearing Tn21-like transposon of pAKD1. This 11,920-bp transposon disrupted the resolvase (parA), resulting in separation of the 3′ and 5′ ends. It consists of transposition genes tnpA and tnpR, a class 1 integron, IS6100, a sulfate transporter (sulP), and a universal stress protein (uspA). The integron has an integrase (int1), an aminoglycoside adenyltransferase gene (aadA) encoding resistance to streptomycin and spectinomycin, a sulfonamide resistance gene (sul1), and a putative acetyltransferase (orf5).