Mating pair stabilization mediates bacterial conjugation species specificity

Abstract

Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure

“…We investigated the effect of the 24c > t and 25c > t ompK36 mutations on meropenem resistance in a background resembling that of KPC-producing K. pneumoniae strains of the ST258/512 lineage, using derivatives of the laboratory K. pneumoniae strain ICC8001 ( 5 ). We refer to the genes in this strain as wild type (WT), in line with previous work ( 5 , 24 ). To that end, we deleted the gene encoding ompK35 (as this is truncated in 99.8% of the ST258/512 genome collection) and introduced a pKpQIL-like plasmid encoding the KPC-2 carbapenemase ( 24 ) ( Tables 1 and 2 ).…”

“…We refer to the genes in this strain as wild type (WT), in line with previous work ( 5 , 24 ). To that end, we deleted the gene encoding ompK35 (as this is truncated in 99.8% of the ST258/512 genome collection) and introduced a pKpQIL-like plasmid encoding the KPC-2 carbapenemase ( 24 ) ( Tables 1 and 2 ). We then superimposed 24c > t and 25c > t mutations into the chromosomal ompK36 locus ( Fig.…”

Real time visualisation of conjugation reveals the molecular strategy evolved by the conjugative F plasmid to ensure the sequential production of plasmid factors during establishment in the new host cell

“…Our current knowledge of conjugation mainly emerges from experimental genetic, biochemical and structural studies that provided a well-documented understanding of the molecular reactions and factors involved in DNA transfer, while genomic and computational studies uncovered the diversity of conjugative plasmids and their importance in the epidemiology of antibiotics resistance dissemination. It is only recently that the application of optical microscopy has started to provide insights into the organisation of conjugation at the cellular scale (Aguilar et al, 2011; Babic et al, 2011; Babić et al, 2008; Carranza et al, 2021; Clarke et al, 2008; Goldlust et al, 2022; Lawley et al, 2002; Low et al, 2022; Nolivos et al, 2019). In this study, live-cell microscopy combined with specifically developed fluorescent reporters offers a unique view of the cellular dynamics of conjugation while providing insights into the timing and localisation of each key step.…”

“…This could suggest that the pole of the recipients' surface is the preferred location for the donor's F-pilus attachment or the stabilisation of the mating pair. The latter possibility is reinforced by the fact that mating pair stabilisation during F conjugation involves interaction between the plasmid protein TraN exposed at the surface of the donor cells and the host outer membrane protein OmpA of the recipient cells (Klimke and Frost, 1998;Low et al, 2022). OmpA was shown to be enriched and less mobile in the polar regions of E. coli cells (Verhoeven et al, 2013), possibly favouring the stabilisation of the mating pair and the conjugation pore at this location.…”

Spatial controls on eco-evolutionary processes in microbial communities

“…Via an entirely different mechanism, spatial intermixing can also modulate the extent to which conjugative plasmids are transferred through bacterial communities. Conjugation typically requires direct cell to cell contact between a plasmid donor and a potential recipient (Cabezón et al 2015;Low et al 2022). Because higher spatial intermixing describes a system with more interspecific cell to cell contacts (i.e., Figure 1c compared to Figure 1b), such systems should also exhibit higher extents of successful plasmid conjugation, and hence greater opportunities for evolutionary changes among bacterial populations.…”