Conjugative type IV secretion in Gram-positive pathogens: TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM

Plasmid. 2017 May;91:9-18. doi: 10.1016/j.plasmid.2017.02.002. Epub 2017 Feb 17.


Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.

Keywords: Endopeptidase; Enterococcus faecalis; Lytic transglycosylase; Type IV secretion system; VirB1; VirB8.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Base Sequence*
  • Binding Sites
  • Biological Transport
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Cell Wall / metabolism
  • Cell Wall / ultrastructure
  • Conjugation, Genetic
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Enterococcus faecalis / genetics*
  • Enterococcus faecalis / metabolism
  • Enterococcus faecalis / ultrastructure
  • Gene Expression Regulation, Bacterial*
  • Peptidoglycan Glycosyltransferase / genetics
  • Peptidoglycan Glycosyltransferase / metabolism
  • Plasmids / chemistry*
  • Plasmids / metabolism
  • Protein Binding
  • Protein Domains
  • Sequence Deletion*
  • Type IV Secretion Systems / metabolism


  • Bacterial Proteins
  • Carrier Proteins
  • Type IV Secretion Systems
  • Peptidoglycan Glycosyltransferase
  • Endopeptidases