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 internalization 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 characterized. This study takes advantage of recent advances in structural modeling 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 modeling 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 internalization of ssDNA during transformation in Firmicutes, providing insights into the molecular mechanisms involved in this horizontal gene transfer mechanism central to bacterial adaptation. It also demonstrates the power of recent structural modeling techniques such as AlphaFold3 as hypothesis generators and guides for designing experiments.