Evolution of Plasmid Mobility: Origin and Fate of Conjugative and Nonconjugative Plasmids

Abstract

Conjugation drives the horizontal transfer of adaptive traits across prokaryotes. One-fourth of the plasmids encode the functions necessary to conjugate autonomously, the others being eventually mobilizable by conjugation. To understand the evolution of plasmid mobility, we studied plasmid size, gene repertoires, and conjugation-related genes. Plasmid gene repertoires were found to vary rapidly in relation to the evolutionary rate of relaxases, for example, most pairs of plasmids with 95% identical relaxases have fewer than 50% of homologs. Among 249 recent transitions of mobility type, we observed a clear excess of plasmids losing the capacity to conjugate. These transitions are associated with even greater changes in gene repertoires, possibly mediated by transposable elements, including pseudogenization of the conjugation locus, exchange of replicases reducing the problem of incompatibility, and extensive loss of other genes. At the microevolutionary scale of plasmid taxonomy, transitions of mobility type sometimes result in the creation of novel taxonomic units. Interestingly, most transitions from conjugative to mobilizable plasmids seem to be lost in the long term. This suggests a source-sink dynamic, where conjugative plasmids generate nonconjugative plasmids that tend to be poorly adapted and are frequently lost. Still, in some cases, these relaxases seem to have evolved to become efficient at plasmid mobilization in trans, possibly by hijacking multiple conjugative systems. This resulted in specialized relaxases of mobilizable plasmids. In conclusion, the evolution of plasmid mobility is frequent, shapes the patterns of gene flow in bacteria, the dynamics of gene repertoires, and the ecology of plasmids.

Keywords: bacteria; conjugation; genomes; horizontal gene transfer; plasmids.

Fig. 1.

Conjugative transfer and archetypal sets of conjugative transfer genes of conjugative and mobilizable mobile plasmids. (A) Different steps of the transfer by conjugation of conjugative and mobilizable plasmids. pCONJ, conjugative plasmid; pMOB, mobilizable plasmid. The red rectangle represents the relaxase. (B) Archetypal sets of transfer genes for conjugative plasmids (F and pTi), decayed conjugative plasmids (pdCONJ) and mobilizable plasmids (pMV158). Only conjugative genes are represented. The blue and orange arrows represent MPF genes and the red arrows represent relaxases. T4SS, type 4 secretion system.

Fig. 2.

Plasmid abundance (A and E), size (B and C), and IS density (D) according to mobility type and relaxase class. (C) The median plasmid size for relaxase classes with a significant number of elements for each mobility type. The width of the circles indicates the frequency of the plasmids (see inset legend). Tests of differences (Wilcoxon paired-tests): ***P < 0.001, **P < 0.01.

Fig. 3.

Overview of plasmids and their mobility across the tree of life. The plasmids were grouped according to their NCBI taxonomic classification of the host bacteria. This sketch tree was drawn from the compilation of different published phylogenetic analyses (Denise et al. 2019). The Proteobacteria, Bacteroidetes, and Firmicutes phyla are highlight in gold, turquoise, and purple, respectively.

Fig. 4.

Associations between relaxases. (A) Cooccurrence (edges) of relaxases within the same plasmid. The numbers in blue, red, and orange represent the number of cooccurrences found in pCONJ, pMOB, and pdCONJ, respectively. The thickness of the edges is proportional to the number of cooccurrences of the two relaxases in the same plasmids. (B) Representation of the results of profile–profile alignments between classes of relaxases. Edges represent bidirectional alignments between HMM profiles, and their length represents the probability of the alignment (short links represent a higher probability [up to 96%] and long links represent lower probability [down to 76%]). Are only represented alignments having a probability superior to 70% and an e-value inferior to 10⁻⁴.

Fig. 5.

Phylogenetic trees of MOB_F relaxases and MOB_P1 relaxases. Ultrafast bootstrap values superior to 75 are shown with a light gray circle and values superior to 90 with a black circle. Percentage of Ultrafast bootstrap values superior to 75 and 90 for each the tree are reported in the table. The trees were rooted using the midpoint root. (A) The phylogenetic tree was built using 735 MOB_F proteins with maximum likelihood with IQTree (model WAG + F + R10 and 1000 ultrafast bootstraps, Nguyen et al. 2015). *: clade containing MOB_F12 plasmids used in fig. 7. (B) The phylogenetic tree was built using 850 MOB_P proteins (all retrieved with the MOBP1 HMM profile) with maximum likelihood with IQTree (model VT + F + R10 and 1000 ultrafast bootstraps).

Fig. 6.

(A) Relationship between relaxase protein sequence divergence and wGRR in pairs of plasmids classed in terms of mobility for MOB_P (left) and MOB_F (right). Each curve represents the smoothing spline for comparisons between: pdCONJ (yellow), conjugative (blue), mobilizable (red), conjugative and pdCONJ (dotted black), and comparisons between mobilizable and conjugative plasmids (black). (B) Comparison between selected conjugative and mobilizable plasmids. Plotted using GenoPlotR v.0.8.10 (Guy et al. 2010), based on Blastn analysis of the plasmid nucleotide sequences (e-value < 10⁻⁴). Comparison between two pCONJs (blue) and a pMOB (red). For clarity, we only represent regions of homology larger than 1 kb with more than 50% identity. Genes involved in the plasmid mobility (T4SS and relaxase[s]) and insertion sequences are highlighted.

Fig. 7.

(A) Graphical representation of the algorithm used for the ANI calculation: each plasmid nucleotide sequence was divided into overlapped 1 kb windows and for each plasmid pair, all stretches were compared. ANI scores were obtained by averaging the percentage of identity of all considered windows with identity and coverage >70%, whenever the sum of windows covered at least the 50% of the smallest plasmid in the pair. ANI scores different from 0 were represented as edges connecting the members of the plasmid pair (nodes) in the ANI network. ANI similarity network of the MOB_F12 plasmid family from E. coli and Shigella spp: The genomic relatedness of 353 transmissible MOB_F12 plasmids hosted in E. coli and Shigella spp., and 140 MOBless plasmids belonging to the same PTUs was estimated by pairwise ANI calculations. At the left, nodes are colored by their mobility type, and at the right, by their PTU. “no PTU” means nonassigned PTU. In the right panel, PTUs that are separated from the main group (PTU-F_E) are surrounded by a circle. (B) Graphical representation of the method used for the plasmid proteome network analysis: the protein set of all plasmids to be compared is clustered at 95% amino acid identity and 80% alignment coverage. The homologous protein clusters and their corresponding plasmids are the two kinds of nodes represented in the network and edges connect both types whenever a plasmid contains a member in a given protein cluster. Proteome network of the PTU-F_E plasmids. The proteins of 250 PTU-F_E plasmids were clustered at 95% identity and 80% coverage. Whenever a plasmid has a member in a protein cluster, an edge is linking them. Homologous protein clusters present in a single plasmid were removed from the figure. Plasmids are colored by their mobility type. (C) Comparison between selected PTU-F_E, PTU-E₄₁, and PTU-E₅ plasmids. Plotted using GenoPlotR v.0.8.10 (Guy et al. 2010), based on Blastn analysis of the plasmid nucleotide sequences (e-value < 10⁻⁴ and alignment length > 1000 bp). Comparison between a pCONJ (blue), a pMOB (red), and a pMOBless plasmid (gray). For clarity, we only represent best bidirectional regions of homology larger than 1 kb with more than 50% identity. Genes involved in the plasmid mobility (T4SS and relaxase[s]), antimicrobial resistance (AMR), plasmid partitioning, pathogenicity, and insertion sequences are highlighted.

Fig. 8.

Characterization of mobility transitions. Graphical representation of the method: starting from the phylogenetic tree of the relaxases and the reconstruction of the ancestral states, we inferred the direction of the changes in terms of mobility and paired recently transited plasmids with the closest plasmid of another mobility. (A) Transitions inferred in terminal branches of the relaxase phylogenetic trees. Arrows and numbers represent the direction and number of transitions, respectively. The size of circle indicates the abundance of each plasmids type. (B) Boxplots representing the size of plasmids that recently changed in terms of mobility (in comparison to the sister-taxa plasmid in the relaxase tree having the ancestral state). Boxplots 1, 2, and 3 represent the size distribution associated with transitions from pCONJ to pMOB, from pMOB to pCONJ, and from pCONJ to pdCONJ respectively. (C) Bubble plot of the median size between plasmid that recently transited mobility and others. The red bubbles represent pMOB, the blue bubbles pCONJ, and the yellow bubbles pdCONJ. The gray dotted line represents the median size of pMOBless. (D) Boxplot of the wGRR between pairs of plasmids where one recently changed from pCONJ to pMOB. The gray (black) boxplot represents the wGRR for plasmids of the same (different) replicon type. (E) Comparison between selected plasmids, plotted using GenoPlotR v.0.8.10 (Guy et al. 2010), based on Blastn analysis of the plasmid nucleotide sequences (e-value < 10⁻⁴ and alignment length > 1000 bp). Top: Comparison between a pdCONJ (yellow) and a conjugative plasmid (blue). Bottom: Comparison between a pMOB (red) and a pCONJ (blue). For clarity, we only represent regions of homology larger than 1 kb with more than 50% identity. Genes involved in the plasmid mobility (T4SS and relaxase[s]) are highlighted. Tests of differences (Wilcoxon paired-tests): ***P < 0.001, **P < 0.01, *P < 0.05, NS (nonsignificant).

Fig. 9.

CDF of the minimal patristic distances in the relaxase phylogenetic trees from a relaxase to another relaxase of a different mobility type. The upper panel depicts the method used to compute the CDF. The patristic distance between each relaxase and its closest closest homolog associated with a different mobility type was retrieved for all trees. When the CDF approaches 1 for low patristic distances, this means that all relaxases are close to a relaxase of a plasmid with a different mobility type in the tree. Here, are indicated the CDF of relaxases identified with protein profiles for MOBP1, MOBF, MOBV, and MOBH.

Fig. 10.

Relationship between the size of mobilizable or conjugative plasmid and the closest related pMOBless. Graphical representation of the method: mobile plasmids were paired with the pMOBless with the highest wGRR. Then the difference in size of both plasmids was calculated and compared with linear model. (A) Each data point in the scatter plot represents the size of pairs of plasmids that are very similar (wGRR > 0.75) and one is pMOBless whereas the other is not. Blue, red, and yellow dots represent pairs between MOBless and pCONJ, pMOB, and pdCONJ, respectively. The histograms on the right represent the distribution of the length difference compared with a linear model (distance to the identity line in gray). All three distributions have an average significantly lower than zero (all Wilcoxon tests, P < 0.0001). (B) Comparison between selected plasmids, plotted using GenoPlotR v.0.8.10, based on Blastn analysis of the plasmid nucleotide sequences (e-value < 10⁻⁴ and alignment length > 1000 bp). Comparison between a pCONJ (blue) and a MOBless plasmid (gray). For clarity, we only represent regions of homology larger than 1 kb with more than 50% identity. Genes involved in the plasmid mobility (T4SS and relaxase[s]) and insertion sequences are highlighted. The pMOBless pSCV50 also encode a relaxase pseudogene not highlighted.

Fig. 11.

Snapshots of the aftermath of the mobility transition events. Possible scenarios for plasmid transitions in terms of mobility type. The similarities in genes repertoires and size are given for the moment right after the transition occurred. MPF represents mating pair formation genes. MOB represents the relaxase.