Thioredoxin-like proteins in F and other plasmid systems

Abstract

Bacterial conjugation is the process by which a conjugative plasmid transfers from donor to recipient bacterium. During this process, single-stranded plasmid DNA is actively and specifically transported from the cytoplasm of the donor, through a large membrane-spanning assembly known as the pore complex, and into the cytoplasm of the recipient. In Gram negative bacteria, construction of the pore requires localization of a subset of structural and catalytically active proteins to the bacterial periplasm. Unlike the cytoplasm, the periplasm contains proteins that promote disulfide bond formation within or between cysteine-containing proteins. To ensure proper protein folding and assembly, bacteria employ periplasmic redox systems for thiol oxidation, disulfide bond/sulfenic acid reduction, and disulfide bond isomerization. Recent data suggest that plasmid-based proteins belonging to the disulfide bond formation family play an integral role in the conjugative process by serving as mediators in folding and/or assembly of pore complex proteins. Here we report the identification of 165 thioredoxin-like family members across 89 different plasmid systems. Using phylogenetic analysis, all but nine family members were categorized into thioredoxin-like subfamilies. In addition, we discuss the diversity, conservation, and putative roles of thioredoxin-like proteins in plasmid systems, which include homologs of DsbA, DsbB, DsbC, DsbD, DsbG, and CcmG from Escherichia coli, TlpA from Bradyrhizobium japonicum, Com1 from Coxiella burnetii, as well as TrbB and TraF from plasmid F, and the absolute conservation of a disulfide isomerase in plasmids containing homologs of the transfer proteins TraH, TraN, and TraU.

The Multidrug Resistance IncA/C Transferable Plasmid Encodes a Novel Domain-swapped Dimeric Protein-disulfide Isomerase

“…Its core functional components comprise: (i) cell surface pili that establish contact with recipient cells; (ii) a multiprotein secretion channel that facilitates transfer of the plasmid across the bacterial cell envelope; and (iii) a type IV coupling protein receptor that recognizes proteincoated conjugative plasmids at the cytoplasmic face and delivers them into the secretion channel (9). The large extrachromosomal conjugative plasmids or ICEs contain a subset of genes located in the transfer (tra) region that encode protein components (Tra proteins) required for building and assembling the complete conjugative machinery (10). In addition to these essential genes and several genes encoding regulatory proteins, the F plasmids and many other drug-resistant conjugative plas-mids also contain a gene that encodes a thioredoxin (TRX)-like disulfide oxidoreductase (10 -13).…”

Chemical shift assignments of a reduced N-terminal truncation mutant of the disulfide bond isomerase TrbB from plasmid F, TrbBΔ29

“…In the absence of a disulfide bond isomerase, oxidative stress leads to aggregation of periplasmic residents, and, eventually, cell death. 2011, Hemmis 2013. Interestingly, the TrbB redox active site is maintained by the hostencoded redox system rather than through a plasmid-mediated process, indicating a dependence of the plasmid on the redox system of its host (Hemmis 2011).…”

“…1987, Elton 2005, Hemmis 2013). Structural predictions, which here are confirmed by NMR and limited proteolysis experiments, reveal that the N-terminal 29 residues of the mature TrbB protein are largely unstructured.…”

“…Structural predictions, which here are confirmed by NMR and limited proteolysis experiments, reveal that the N-terminal 29 residues of the mature TrbB protein are largely unstructured. Further biochemical analysis revealed that TrbB functions as a disulfide bond isomerase and likely functions to mediate the proper folding and maintenance of disulfide networks in a subset of plasmid F encoded proteins (Hemmis 2011, Hemmis 2013). Interestingly, the TrbB redox active site is maintained by the hostencoded redox system rather than through a plasmid-mediated process, indicating a dependence of the plasmid on the redox system of its host (Hemmis 2011).…”

“…Disulfide bond isomerase activity is completely abrogated in dimerizationdeficient DsbC mutants (Bader 2001), making the lack of an N-terminal dimerization domain in TrbB quite striking. Reflecting these deviations, recent phylogenetic analyses place TrbB in a unique family of plasmid-based disulfide isomerases that are likely responsible for the correct folding and maintenance of a subset of F plasmid-encoded proteins including TraH, TraN, and TraU (Hemmis 2011, Hemmis 2013). …”