Plasmid-driven strategies for clone success in Escherichia coli

Abstract

Escherichia coli is the most widely studied microbe in history, but the population structure and evolutionary trends of its extrachromosomal elements known as plasmids remain poorly delineated. Here we used long-read technology to high-resolution sequence the entire plasmidome and the corresponding host chromosomes from an unbiased longitudinal survey covering two decades and over 2000 E. coli isolates. We find that some plasmids have persisted in lineages even for centuries, demonstrating strong plasmid-lineage associations. Our analysis provides a detailed map of recent vertical and horizontal evolutionary events involving plasmids with key antibiotic resistance, competition and virulence determinants. We present genomic evidence of both chromosomal and plasmid-driven success strategies adopted by distant lineages by independently inheriting the same genomic elements. Further, we use in vitro experiments to verify the importance of key bacteriocin-producing plasmids for clone success. Our study has general implications for understanding plasmid biology and bacterial evolutionary strategies.

Fig. 1. Plasmid diversity and typing scheme on the set of predicted plasmid sequences obtained from 1999 ExPEC hybrid assemblies.

A Maximum-likelihood phylogeny of the 1999 ExPEC isolates with an associated hybrid assembly. Isolates corresponding to the ten most prevalent sequence types (ST) in the NORM study are indicated with a distinct color and their position marked in the phylogeny. In the case of ST131, the distinct clades present in the clone (A, B, C1, C2) are represented. B The closest mge-cluster solution (perplexity = 62, minimum cluster size = 49) to the network approach was considered to visualize the position of the pTs in the 2D embedding space. For visualization purposes, we computed the mean tsne1D and tsne2D coordinates of all sequences belonging to each pT and considered those coordinates to represent the position and anchor each pT in the embedding. Each pT is labeled with its associated pT consisting of two digits indicating their graph component and associated bin and its size is proportional to the average plasmid length. C Barplot indicating the total number of plasmid sequences assigned to each pT and belonging to the main STs found in the ExPEC collection.

Fig. 2. Synteny analysis and visualization of the main IncF large plasmid types (pT).

For each pT, we selected a plasmid sequence (tagged with its sequence ID) representing the most predominant features present in the cluster (as summarized in Supplementary Data 2). The gene synteny analysis was performed using clinker considering a minimum identity threshold of 0.8 to draw a link between genes. Virulence and antibiotic resistance genes were identified using the EcOH database (indexed in Abricate) and AMRFinderPlus and their names curated according to well-studied ExPEC reference plasmids.

Fig. 3. Dated phylogenies of the four main ExPEC clones and presence/absence pattern of the most prevalent plasmid types (pT) found in each sequence type (ST).

A Dated phylogeny of ST95 with clades indicated based on fimH/O/H typing and presence/absence of the pColV-like plasmids (pTs 10-1 and 9-1) and the pUTI89-like plasmid (pT 2-3). B Dated phylogeny of ST69 with clades indicated based on fimH/O/H typing and presence/absence of the pUTI89-like plasmid (pT 2-2). C Dated phylogeny of ST73 with clades indicated based on fimH/O/H typing and presence/absence of the pT 13-1. D Dated phylogeny of ST131 with clades indicated based on existing nomenclature (A, B, B0, C0, C1, C2), fimH/O/H typing and presence/absence of multiple associated pTs.

Fig. 4. Distribution and classification of colicin genes in the ExPEC population.

A Stacked barplot showing the preferred genomic location of colicin genes: chromosome, plasmid, virus (bacteriophage) or unclassified (unknown). In the case of plasmid location, we have indicated the particular plasmid type (pT) associated. B Distribution of the colicin genes in the ExPEC phylogeny (as shown in Fig. 1A). Each colicin was colored according to whether the gene was encoded in the chromosome, plasmid, virus (bacteriophage) or an unclassified contig.

Fig. 5. Schematic representation of the bacteriocin susceptibility assay.

A Description of bacteriocinogenic plasmids from pT 9-1 (ST131 clade B) and pT 10-1 (ST95). These archetypical pColV-like plasmids encode two bacteriocin gene clusters: microcin V and colicin Ia. Darker and lighter arrows indicate respectively the toxin (cvaC, cia) and immunity genes (cvi, iia) of each cluster. Control isolates either harbor no plasmid (ST131 clade B isolate 27-56) or harbor plasmid versions encoding only a single bacteriocin (colicin Ia for ST131 B isolate 31-16, microcin V for ST95 isolate 31-17). B Susceptibility of E. coli MG1655 to microcin V. Production of microcin V is induced by iron limitation (simulated experimentally with the addition of the chelating agent 2,2’-bipyridyl). Bacteriocinogenic activity is demonstrated by growth inhibition in the spotted area (in a dilution-dependent way and without formation of individual plaques). C Susceptibility of E. coli MG1655 to colicin Ia. Production of colicin Ia is induced through SOS induction (stimulated experimentally with UV irradiation). Bacteriocinogenic activity is demonstrated as in B. Created in BioRender. Gama, J. (2025) https://BioRender.com/e79b562.

Fig. 6. Bacteriocin susceptibility of NORM isolates.

Pruned phylogeny of the 51 E. coli NORM isolates tested experimentally for bacteriocin susceptibility (right). Susceptibility to microcin V and colicin Ia is individually indicated in the heatmap in blue, while yellow indicates bacteriocin insensitivity (no effect detected). Isolates harboring the archetypical pColV-like bacteriocinogenic plasmids are indicated in gray.