Solution characterization of the dynamic conjugative entry exclusion protein TraG

Abstract

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.

Genomic Insights into Moderately Thermophilic Methanotrophs of the Genus Methylocaldum

“…Several predicted functions were only found in the two moderate thermophilic M. szegediense strains, including K12056, corresponding to the conjugal transfer mating pair stabilization protein TraG, which is one of the pilus assembly proteins in bacterial type IV secretion systems (T4SS) [ 32 ]. While M. szegediense Norfolk has only two conjugal transfer proteins, TraG and TraN, the M. szegediense O-12 genome contains two TraG, as well as TraW, TraF, TraE, and TraL, which participate in pilus assembly; TraA (pilin); TraN and TraU, which are responsible for mating pair stabilization; TraV, TraK, and TraB, which form the core complex; TraD and TraC, which are ATPase proteins reported to be complexed in the cytosol and inner membrane; and also TrbL, which is a subset of proteins described as F-like T4SS [ 32 , 33 ].…”