Natural transformation is a major mechanism of horizontal gene transfer, allowing many bacteria to acquire new functions, including antibiotic resistance. Although several proteins involved in this process have been identified, the precise organization of the machinery responsible for transporting single-stranded DNA across the membrane has until now remained only partially understood.
In this study, the authors used the structural modeling capabilities of AlphaFold3 to reconstruct the complete architecture of the single-stranded DNA transport complex in Streptococcus pneumoniae. The generated model reveals the association of three proteins - ComEC, ComFA, and ComFC-forming a tripartite complex. ComEC constitutes a transmembrane channel through which single-stranded DNA crosses the membrane, while ComFA and ComFC, located in the cytoplasm, ensure the handling of transforming DNA as it exits the channel.
 @DRCM, CEA
| (A) Machinery and steps involved in DNA capture during bacterial transformation in Streptococcus pneumoniae (Sp). The proteins studied are shown in color: ComEC, a transmembrane protein containing a conserved channel, and the ComFA-ComFC pair, known to interact with each other and whose involvement in this process we investigated. (B) AlphaFold structural model of the three-protein complex ComEC, ComFC, and ComFA in interaction with single-stranded DNA.
|
To test the robustness of the model, the researchers then targeted amino acids predicted to be essential for protein-protein or protein-DNA interactions. The resulting mutations caused substantial decreases in transformation efficiency, by factors exceeding 1,000 in some combinations, confirming the critical role of these residues in DNA transport. These experiments support a model in which a single ComEC monomer forms a narrow channel through which single-stranded DNA passes and show that this mechanism is conserved beyond the Firmicutes, in the Gram-negative bacterium Helicobacter pylori, suggesting an ancient shared mechanism. The structuring interaction between ComEC and ComFC, organized around a particularly conserved “hook–pocket” motif, appears to be a central element for the stability and function of the complex. Moreover, the study demonstrates that the overall organization of the ComEC-ComFA-ComFC triplet is conserved in Firmicutes.
Beyond the functional validation of a complete structural–molecular model, this work illustrates the value of integrated approaches that combine AI-based prediction with experimental genetics. It provides new insight into the mechanisms enabling DNA transport in bacteria and contributes to a deeper understanding of the processes underlying microbial adaptation and evolution.
Contact : Pablo Radicella
Référence : Article PNAS