PMID:23913323

Citation	Arends, K, Celik, EK, Probst, I, Goessweiner-Mohr, N, Fercher, C, Grumet, L, Soellue, C, Abajy, MY, Sakinc, T, Broszat, M, Schiwon, K, Koraimann, G, Keller, W and Grohmann, E (2013) TraG encoded by the pIP501 type IV secretion system is a two-domain peptidoglycan-degrading enzyme essential for conjugative transfer. J. Bacteriol. 195:4436-44
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.
Links	PubMed PMC3807477 Online version:10.1128/JB.02263-12
Keywords	Acetylglucosamine/analogs & derivatives; Acetylglucosamine/pharmacology; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Conjugation, Genetic; Enterococcus faecalis/enzymology; Enterococcus faecalis/genetics; Enterococcus faecium/enzymology; Enterococcus faecium/genetics; Gene Deletion; Gene Expression Regulation, Bacterial/drug effects; Gene Expression Regulation, Bacterial/physiology; Gene Expression Regulation, Enzymologic/drug effects; Gene Expression Regulation, Enzymologic/physiology; Oligosaccharides/pharmacology; Peptidoglycan/metabolism; Plasmids; Proline/analogs & derivatives; Proline/pharmacology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ENTFL:E3WD67	Contributes to	GO:0043684: type IV secretion system complex	ECO:0000315:		C		Mating assays with a traG deletion mutant resulted in no mating as compared to wild-type.	complete CACAO 10760
ENTFL:E3WD67		GO:0009253: peptidoglycan catabolic process	ECO:0000314:		P		Fig. 4 shows Cy3 PG degradation by TraGdTMH.	complete CACAO 10761
ENTFL:E3WD67		GO:0030313: cell envelope	ECO:0000314:		C		Fig. 3 shows that TraG is found in the cell wall and membrane fraction.	complete CACAO 10762
ENTFL:Q8L1D1		GO:0009291: unidirectional conjugation	ECO:0000315:		P		4th paragraph of the initial section of the results: "Transfer rates of the traG deletion mutant were below the detection limit of the assay (<3.5 × 10−9 ± 2.4 × 10−9, mean value of three independent assays), whereas transfer rates with the isogenic strain E. faecalis OG1RF(pIP501) were at least 20,000 times higher and in the expected range for pIP501 of 7.6 × 10−5 ± 5.7 × 10−5 transconjugants per recipient cell (mean value of three independent assays)."	complete CACAO 12326

References

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