title: Conjugative type IV secretion in Gram-positive pathogens: TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM.
abstract: 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.
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria. Electronic address: verena.kohler@uni-graz.at.
authors.forename: Verena
authors.initials: V
authors.lastname: Kohler
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, University Medical Center Freiburg, Freiburg, Germany; Faculty of Biology, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany. Electronic address: ines.probst@uniklinik-freiburg.de.
authors.forename: Ines
authors.initials: I
authors.lastname: Probst
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria. Electronic address: andreas.aufschnaiter@uni-graz.at.
authors.forename: Andreas
authors.initials: A
authors.lastname: Aufschnaiter
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria; Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden. Electronic address: sabrina.buettner@su.se.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Büttner
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria. Electronic address: lisa-marie.schaden@edu.uni-graz.at.
authors.forename: Lisa
authors.initials: L
authors.lastname: Schaden
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria; Omics Center Graz, BioTechMed-Graz, Graz, Austria. Electronic address: gerald.rechberger@uni-graz.at.
authors.forename: Gerald N
authors.initials: GN
authors.lastname: Rechberger
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria. Electronic address: guenther.koraimann@uni-graz.at.
authors.forename: Günther
authors.initials: G
authors.lastname: Koraimann
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, University Medical Center Freiburg, Freiburg, Germany; Faculty of Life Sciences and Technology, Beuth University of Applied Sciences, Berlin, Germany. Electronic address: elisabeth.grohmann@beuth-hochschule.de.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Grohmann
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria. Electronic address: walter.keller@uni-graz.at.
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism / Q000648:ultrastructure; D003227:Conjugation, Genetic; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D013293:Enterococcus faecalis / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D015964:Gene Expression Regulation, Bacterial; D046928:Peptidoglycan Glycosyltransferase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D017384:Sequence Deletion; D000069319:Type IV Secretion Systems / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D000069319:Type IV Secretion Systems; D046928:Peptidoglycan Glycosyltransferase; D010450:Endopeptidases
keywords: Endopeptidase; Enterococcus faecalis; Lytic transglycosylase; Type IV secretion system; VirB1; VirB8