Biology subjects

Seddon, C.

Publications and source records attributed to Seddon, C..

4 recordsLinked to original sources

bioRxiv2024

Cryo-EM structure of the conjugation H-pilus reveals the cyclic nature of the TrhA pilin

Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor-recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF, and describe its cryo electron-microscopic (cryo-EM) structure at 2.2 [A] resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stochiometric ratio of 1:1. While there were previous reports that the T-pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N- and C-termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxylate residues. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination. SignificanceA major medical challenge is the spread of bacteria which are resistant to antibiotics. The resistance genes are spread via mobilized DNA, mainly via a process named conjugation. During conjugation, a resistant bacterium (donor), transfers the resistance DNA to another bacterium (recipient) in a contact-dependent manner. The initial donor-recipient interaction is mediated by a hollow filament expressed by the donor, named the conjugation pilus, that binds the recipient. This pilus is built via polymerization of a small protein subunit, pilin. Here, we report the atomic structure of the H-pilus, whose pilin subunit has an unusual cyclic structure where the N- and C-termini of the protein are covalently linked by a peptide bond.

microbiology↗

bioRxiv2024

Cryo-EM structure and evolutionary history of the conjugation surface exclusion protein TraT

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF revealed that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol modified -helical barrel domain. Unexpectedly, we identified chromosomal TraT homologues from multiple Gram-negative phyla which formed numerous deep-branching lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences largely cluster into two separate lineages that have more recently evolved, incorporating TraT from Enterobacterales IncF and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.

microbiology↗

bioRxiv2023

The interaction of the F-like plasmid-encoded TraN isoforms with their cognate outer membrane receptors

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of seven TraN sequence types, which are grouped into four structural types, we named TraN, TraN{beta}, TraN{gamma}, TraN{delta}. Moreover, we have shown specific pairing between TraN and OmpW, TraN{beta} and OmpK36 of Klebsiella pneumoniae, TraN{gamma} and OmpA and TraN{delta} and OmpF. In this study we found that although structurally similar, TraN encoded by the pSLT plasmid (TraN2) binds OmpW in both Escherichia coli and Citrobacter rodentium while TraN encoded by the R100-1 plasmid (TraN1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraN{beta}-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraN{beta} can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. ImportanceConjugation plays a central role in the spread of antimicrobial resistance genes amongst bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilisation (MPS), mediated by tight interactions between the plasmid encoded outer membrane protein (OMP) TraN in the donor (of which there are seven sequence types grouped into the four structural isoforms , {beta}, {gamma}, {delta}) and an OMP receptor in the recipient (OmpW, OmpK36, OmpA and OmpF, respectively). In this study we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

microbiology↗

bioRxiv2020

OmpK36 and TraN facilitate conjugal transfer of the Klebsiella pneumoniae carbapenem resistance plasmid pKpQIL

We investigated the mechanism of conjugal transfer of the endemic Klebsiella pneumoniae carbapenem resistance plasmid, pKpQIL. Transfer efficiency of this plasmid was found to be dependent on the expression of the major outer membrane porin, OmpK36, in recipient cells. We also found that conjugal uptake is reduced in recipients expressing an OmpK36 isoform associated with the globally pervasive K. pneumoniae ST258 clade (OmpK36ST258). This reduction was attributed to a glycine-aspartate insertion in loop 3 of OmpK36ST258, which constricts the pore by 26%. Deletion of finO, which encodes an RNA-binding protein, derepressed transfer of pKpQIL and enabled visualisation of the conjugation pilus and OmpK36-dependent conjugation in real time. While deletion of traN abolished pKpQIL conjugation, substituting traN in pKpQIL with its homologue from R100-1 circumvented OmpK36 dependency. These results suggest that OmpK36 in recipient K. pneumoniae and the pKpQIL-encoded TraN in donor bacteria cooperate to facilitate plasmid transfer. This is the first report since 1998 to suggest a novel recipient cell receptor for IncF plasmid transfer and supports the idea that TraN mediates receptor specificity for plasmids belonging to this incompatibility group.

microbiology↗