The Interaction of the F-Like Plasmid-Encoded TraN Isoforms with Their Cognate Outer Membrane Receptors

Area of Science:

• Bacterial genetics and molecular biology
• Microbial evolution
• Horizontal gene transfer mechanisms

Background:

• Horizontal gene transfer via conjugation is crucial for bacterial evolution and the spread of antimicrobial resistance.
• F-like plasmids utilize mating pair stabilization (MPS) for efficient DNA transfer, mediated by interactions between donor TraN outer membrane (OM) proteins and recipient chromosomal OM proteins.
• Seven TraN sequence types exist, grouped into four structural types (TraNα, TraNβ, TraNγ, TraNδ), each with specific OM protein pairings.

Purpose of the Study:

• To investigate how subtle sequence differences in recipient outer membrane proteins (OMPs) affect TraN-mediated mating pair stabilization (MPS) and conjugation efficiency.
• To identify the specific amino acid determinants responsible for the differential binding of TraNα variants to OmpW.
• To explore the binding capabilities of TraNβ with different recipient OMPs and its impact on conjugation.

Main Methods:

• Comparative analysis of TraNα variants (TraNα1 and TraNα2) binding to OmpW in different bacterial species (E. coli, C. rodentium).
• AlphaFold2 structure prediction to identify potential interaction sites.
• Experimental confirmation of amino acid effects on OmpW binding specificity.
• Site-directed mutagenesis of OmpK36 to assess the impact of insertions on TraNβ-mediated conjugation efficiency.
• Testing TraNβ binding to OmpK35.

Main Results:

• TraNα2 (from Salmonella enterica pSLT) binds OmpW in both E. coli and C. rodentium, while TraNα1 (from R100-1 plasmid) binds OmpW only in E. coli.
• A single amino acid difference in loop 3 of OmpW was identified as the key determinant for this binding specificity, confirmed experimentally.
• Single amino acid insertions in loop 3 of OmpK36 significantly altered TraNβ-mediated conjugation efficiency of the K. pneumoniae plasmid pKpQIL.
• TraNβ was found to mediate MPS by binding to OmpK35, in addition to OmpK36, indicating broader receptor compatibility.

Conclusions:

• Subtle sequence variations in bacterial outer membrane receptors (like OmpW and OmpK36) critically influence the efficiency and specificity of TraN-mediated conjugation.
• These findings highlight selective pressures shaping plasmid-host compatibility and the dynamics of horizontal gene transfer in bacterial populations.
• The discovery that TraNβ can bind multiple OMPs expands our understanding of MPS mechanisms and their role in the dissemination of genetic material, including antibiotic resistance genes.