TraG encoded by the pIP501 type IV secretion system is a two-domain peptidoglycan-degrading enzyme essential for conjugative transfer

Abstract

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.

Fig 1

Domain composition of TraG. TraG shows a modular structure. The VirB1 ortholog TraG_pIP501 protein (GenBank sequence accession no. CAD44387.1) contains 369 amino acids. A transmembrane helix (TMH) is predicted at the N terminus (positions 20 to 36, HMMTOP), as well as a signal peptide with a putative cleavage site at positions 47 to 48 (SignalP 3.0). A specific lytic transglycosylase (SLT) domain is predicted at positions 80 to 165 (gray box); a cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain is putatively located at the C terminus (positions 243 to 369, white box). A peptidoglycan binding motif is predicted for the SLT domain (conserved domain search).

Fig 2

Expression of traG downstream genes in the pIP501 tra operon is not affected by traG deletion. Immunodetection of proteins TraH (A), TraK (B), and TraM (C) using polyclonal antibodies against the respective proteins showed comparable expression levels for all three proteins in lysates from E. faecalis OG1RF(pIP501 wt) and E. faecalis OG1RF(pIP501ΔtraG). The plasmid-free E. faecalis OG1RF strain was used as a negative control. Lanes 1, E. faecalis OG1RF lysate; lanes 2, E. faecalis OG1RF(pIP501 wt) lysate; lanes 3, E. faecalis OG1RF(pIP501ΔtraG) lysate.

Fig 3

(A) TraG subcellular localization. TraG localizes to the cell envelope of E. faecalis JH2-2 harboring pIP501. The localization of TraG in the cell fractions was detected by immunoblotting with polyclonal anti-TraG antibodies. Plasmid-free E. faecalis JH2-2 was applied as a negative control. Lane 1, cell wall fraction; lane 2, membrane fraction; lane 3, cytoplasmic fraction; lane 4, empty well; lane 5, cell wall fraction; lane 6, membrane fraction; lane 7, cytoplasmic fraction; lanes 1 to 3 represent samples from E. faecalis JH2-2 cells without pIP501; lanes 5 to 7 contain samples from pIP501-harboring cells. (B) TraN subcellular localization. TraN localizes to the cytoplasmic fraction and thus serves as a negative control for the localization of TraG to the cell envelope. Lane 1, cell wall fraction; lane 2, membrane fraction; lane 3, cytoplasmic fraction.

Fig 4

Cy3 PG degradation by TraGΔTMH. Cy3-labeled PG was measured before (green lines) and after (red lines) digestion. Line scans are shown for a window of 50 pixels (2 mm). rfu, relative fluorescence units. Values above each box indicate the reduction of the fluorescence in each Cy3 PG spot and therefore reflect enzymatic activity (for details, see Materials and Methods). (A) Cy3 PG was digested with the indicated proteins or incubated with buffer alone. (B and C) Inhibition of TraGΔTMH with indicated concentrations of hexa-N-acetylchitohexaose and bulgecin A, respectively.

Fig 5

TraGΔTMH and mutant protein muramidase activity on l-[³H]lysine-labeled PG from E. faecalis JH2-2. Activities (in cpm) are given as average values of three independent measurements with standard deviations. MBP and lysozyme (from hen egg white) were applied as negative and positive controls, respectively.