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  • TraM is one of two transfer proteins

    2020-01-15

    TraM is one of two transfer proteins from pIP501 T4SS that have structural similarity to the A. tumefaciens VirB8 protein (Fercher et al., 2016, Goessweiner-Mohr et al., 2013), a central member of the inner membrane complex (Bailey et al., 2006, Guglielmini et al., 2014, Trokter et al., 2014). VirB8 homologs are crucial for their respective systems and are proposed to act as scaffolding factor for the assembly of the conjugative core complex (Porter et al., 2012, Sivanesan et al., 2010). Further, crystal structures in combination with mutational analyses suggested that their oligomerisation is physiologically relevant (Das and Xie, 2000, Den Hartigh et al., 2008). TraG is a VirB1-like protein, essential for conjugation (Arends et al., 2013), with a modular architecture, comprising of a transmembrane helix (TMH) at the N-terminus, followed by a putative soluble lytic transglycosylase (SLT) domain (Engel et al., 1991). At its C-terminus TraG contains a cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain (Bateman and Rawlings, 2003, Rigden et al., 2003). VirB1 is required for efficient T4SS function, since virB1 gpr40 agonist in A. tumefaciens led to 1–10% residual virulence and attenuated tumor formation (Berger and Christie, 1994, Höppner et al., 2004, Mushegian et al., 1996). Lytic transglycosylases show the same substrate specificity as lysozyme, but generate, with a terminal 1-6-anhydro-N-acetylmuramic moiety, a different reaction product (Nocadello et al., 2016, Thunnissen et al., 1994, Vollmer et al., 2008). These enzymes are often associated with protein complexes in secretion systems (Höppner et al., 2005, Koraimann, 2003) generating localized lesions early in the biogenesis of the complex (Bayer et al., 1995, Berger and Christie, 1994, Zahrl et al., 2005). In this study, we show that impaired transfer frequency observed in an E. faecalis pIP501 in-frame traG deletion mutant (Arends et al., 2013) is fully recovered by providing full-length wild type traG in trans, but not when its C-terminal CHAP domain is missing. We can assign lytic transglycosylase as well as endopeptidase activity to the enzymatic domains of TraG by analysis of peptidoglycan degradation products with tandem mass spectrometry. Further, we detect a novel interaction between the VirB1-like protein TraG and the translocation channel member TraM with a bacterial-2-hybrid assay and show via fluorescence microscopy that both proteins localize at the E. faecalis membrane. With a combined approach of fluorescence microscopy and flow cytometry we demonstrate that the correct localization of TraM at the cell membrane depends on TraG, suggesting that TraG represents an essential protein in translocation complex assembly.
    Material and methods
    Results
    Discussion We have previously shown that the VirB1-like protein TraG is an essential T4SS component. However, complementation experiments with in trans expression of TraGΔTMH did not restore transfer efficiency (Arends et al., 2013). Now, we were able to completely recover conjugative transfer in our traG in-frame knockout mutant upon in trans expression of full-length TraG. Therefore, we conclude that the transmembrane helix of TraG could play an essential role in the function of the protein. Interestingly, a truncated variant lacking the CHAP domain could not restore transfer efficiency either, which might be due to impaired protein folding or a significant decrease in peptidoglycan degrading activity of the protein. CHAP domains are often associated with other domains cleaving peptidoglycan (Kausmally et al., 2005, Pritchard et al., 2004) and most peptidoglycan muramidases from T4SSs also possess a CHAP domain at their C-terminus (Bhatty et al., 2013). This suggests that peptidoglycan degrading two-domain enzymes only deploy their full activity when their respective partner domain is present. Most peptidoglycan metabolizing enzymes associated with T4SSs from G+ origin are essential for conjugation, and mutants lacking these enzymes exhibit impaired transfer efficiencies (Bantwal et al., 2012, Laverde Gomez et al., 2014). The hydrolase domain of CwlT from Bacillus subtilis ICEBs1 was shown to be essential, whereas its peptidase domain is partially dispensable for conjugative transfer of ICEBs1 (DeWitt and Grossman, 2014). PrgK from E. faecalis pCF10 comprises three enzymatic domains, namely LytM, a “goose-type” SLT and a CHAP domain and was shown to be essential for conjugative transfer. Transfer was completely abolished when prgK was knocked out, but two domains were sufficient for near-wild type plasmid transfer levels. Intriguingly, in trans expression of muramidases from other systems, such as TraG from pIP501, partially complemented prgK (Laverde Gomez et al., 2014). In contrast, complete deletion of genes encoding for G- peptidoglycan degrading enzymes only causes a reduction in transfer efficiency of 10–100 fold (Bayer et al., 1995, Berger and Christie, 1994, Winans and Walker, 1985).