Solution characterization of the dynamic conjugative entry exclusion protein TraG

• Published in: Structural dynamics (Melville, N.Y.) N.Y.), 2022-11, Vol.9 (6), p.064702-064702-16
• Main Authors: Bragagnolo, Nicholas, Audette, Gerald F.
• Format: Article
• Language: English
• Subjects: Antibiotics; Bacteria; Bacterial infections; Conjugation; Dichroism; Domains; Drug resistance; Genes; Mass spectrometry; Membranes; Pathogens; Plasmids; Proteins; Size exclusion chromatography; Virulence; X-ray scattering
• ISSN: 2329-7778; 2329-7778
• DOI: 10.1063/4.0000171

The R100 plasmid and the secretion system it encodes are representative of F-like conjugative type IV secretion systems for the transmission of mobile DNA elements in gram-negative bacteria, serving as a major contributor to the spread of antibiotic resistance in bacterial pathogens. The TraG protein of F-like systems consists of a membrane-bound N-terminal domain and a periplasmic C-terminal domain, denoted TraG*. TraG* is essential in preventing redundant DNA transfer through a process termed entry exclusion. In the donor cell, it interacts with TraN to facilitate mating pair stabilization; however, if a mating pore forms between bacteria with identical plasmids, TraG* interacts with its cognate TraS in the inner membrane of the recipient bacterium to prevent redundant donor–donor conjugation. Structural studies of TraG* from the R100 plasmid have revealed the presence of a dynamic region between the N- and C-terminal domains of TraG. Thermofluor, circular dichroism, collision-induced unfolding–mass spectrometry, and size exclusion chromatography linked to multiangle light scattering and small angle x-ray scattering experiments indicated an N-terminal truncation mutant displayed higher stability and less disordered content relative to full-length TraG*. The 45 N-terminal residues of TraG* are hypothesized to serve as part of a flexible linker between the two independently functioning domains.