Biology subjects

JOHNSTON, C. H. G.

Publications and source records attributed to JOHNSTON, C. H. G..

3 recordsLinked to original sources

bioRxiv2025

A tripartite protein complex promotes DNA transport during natural transformation in firmicutes.

Natural genetic transformation is a conserved mechanism of bacterial horizontal gene transfer, which is directed entirely by the recipient cell and facilitates the acquisition of new genetic traits such as antibiotic resistance. Transformation proceeds via the capture of exogenous DNA, its internalisation in single strand form (ssDNA) and its integration into the recipient chromosome by homologous recombination. While the proteins involved in these steps have mainly been identified, the specific mechanisms at play remain poorly characterised. This study takes advantage of recent advances in structural modelling to explore the uptake of ssDNA during transformation. Using the monoderm human pathogen Streptococcus pneumoniae, we model a tripartite protein complex composed of the transmembrane channel ComEC, and two cytoplasmic ssDNA-binding proteins ComFA and ComFC. Using targeted mutation and transformation assays, we propose that pneumococcal ComEC features a narrow channel for ssDNA passage, and we show this channel is conserved in the diderm Helicobacter pylori. We identify key residues involved in protein-protein and protein-ssDNA interactions in the pneumococcal tripartite complex model and we show them to be crucial for transformation efficiency. Structural modelling reveals that this tripartite protein complex and its interaction with ssDNA are conserved in firmicutes. Overall, this study validates a tripartite complex required for the internalisation of ssDNA during transformation in firmicutes, providing new insights into the molecular mechanisms involved in this horizontal gene transfer mechanism central to bacterial adaptation. It also demonstrates the power of recent structural modelling techniques such as AlphaFold3 as hypothesis generators and guides for designing experiments. Significance statementNatural genetic transformation is a key mechanism of horizontal gene transfer, conserved in bacteria. The investigation of transmembrane channel ComEC and its interaction partners was previously hindered by the difficulty of manipulating these proteins experimentally. Thanks to state-of-the-art structural modelling with AlphaFold3 and subsequent thorough experimental validation of this model using targeted mutation and transformation assays, we demonstrate the importance of the ComEC/ComFA/ComFC complex for single strand DNA (ssDNA) uptake during natural transformation in the human pathogen Streptococcus pneumoniae. Similar models in several other species suggest a widely conserved organization of this complex in firmicutes. In addition, we demonstrate that the ComEC transmembrane channel is also crucial for ssDNA uptake during natural transformation in Helicobacter pylori.

microbiology↗

bioRxiv2023

Pneumococcal competence, a populational health sensor driving multilevel heterogeneity in response to antibiotics.

Competence for natural transformation is a central driver of genetic diversity in bacteria. In the human pathogen Streptococcus pneumoniae, competence exhibits a populational character mediated by the stress-induced ComABCDE quorum-sensing (QS) system. Here, we explore how this cell-to-cell communication mechanism proceeds and the functional properties acquired by competent cells grown under lethal stress. We show that populational competence development depends on self-induced cells stochastically emerging in response to stresses, including antibiotics. Competence is demonstrated to propagate through the population from a low threshold density of self-induced cells, defining a biphasic Self-Induction and Propagation (SI&P) QS mechanism. We also reveal that a competent population displays either increased sensitivity or improved tolerance to lethal doses of antibiotics, dependent in the latter case on the competence-induced ComM division inhibitor. Remarkably, these surviving competent cells also display an altered transformation potential. Thus, the unveiled SI&P QS mechanism shapes pneumococcal competence as a health sensor of the clonal population, promoting a bet-hedging strategy that both responds to and drives cells towards heterogeneity.

microbiology↗

bioRxiv2022

The RecA-directed recombination pathway of natural transformation initiates at chromosomal replication forks in Streptococcus pneumoniae.

Homologous recombination (HR) is a crucial mechanism of DNA strand exchange that promotes genetic repair and diversity in all kingdoms of life. Bacterial HR is driven by the universal recombinase RecA, assisted by dedicated mediators that promote its polymerization on single-stranded DNA (ssDNA). In bacteria, natural transformation is a prominent HR-driven mechanism of horizontal gene transfer specifically dependent on the conserved DprA recombination mediator. Transformation involves internalisation of exogenous DNA as ssDNA, followed by its integration into the chromosome by RecA-directed HR. How DprA-mediated RecA filamentation on transforming ssDNA is spatiotemporally coordinated with other cellular processes remains unknown. Here, we tracked the localisation of functional fluorescent fusions to DprA and RecA in Streptococcus pneumoniae and revealed that both accumulate in an interdependent manner with internalised ssDNA at replication forks. In addition, dynamic RecA filaments were observed emanating from replication forks, even with heterologous transforming DNA, which probably represent chromosomal homology search. In conclusion, this unveiled interaction between HR transformation and replication machineries highlights an unprecedented role for replisomes in anchoring transforming ssDNA to the chromosome, which would define a pivotal early HR step for its chromosomal integration.

microbiology↗