On the other hand, when pBL21 was introduced into the Δpmt mutant IB25, mycelium could only be grown under noninducing conditions (i.e. in the absence of thiostrepton) where no complementation of the Δpmt mutation was observed (Fig. S4); adding thiostrepton to the medium resulted in a lack of growth, either in liquid medium or on solid medium, meaning that expression of this chimeric Pmt protein was lethal (Fig. S6). Therefore, replacement of the N-terminal region containing the first extracellular loop of PmtMtu by that of PmtSco was apparently not innocuous. One possibility is that this construct results

in a misfolded protein that is toxic; we consider this unlikely, given the structural conservation shown by both Pmt proteins (Fig. S5). Another possible explanation is that the lethal phenotype is the result of a nonproductive interaction between selleck the chimeric Pmt and the Sec translocon, perhaps affecting Sec function to such an extent as to make it nonfunctional. We attempted to show specific interactions between components of the S. coelicolor Sec translocon and either PmtSco or PmtMtu using the bacterial two-hybrid system of Buparlisib chemical structure Karimova et al. (1998), but no significant interactions could be observed (data not shown).

It has also been recently suggested that interaction between corynebacterial Pmt and Lnt might be essential for Pmt function in these bacteria, as there was no detectable glycosylation of a lipoprotein that is normally glycosylated in the absence of Lnt (Mohiman et al., 2012). Mycobacteria are closely related to corynebacteria, so it find more is conceivable that PmtMtu is not functional in S. coelicolor because it is unable to interact with S. coelicolor Lnt1. Because our results show that Lnt1 is not required for PmtSco function, this might reveal a fundamental difference between these two bacterial groups. Because PmtMtu failed to complement the Δpmt deletion of S. coelicolor in vivo, we wondered

whether Pmt activity could be detected in vitro, using the assay previously described for glycosylation of the synthetic A3 peptide derived from the Apa protein with a purified membrane fraction (Cooper et al., 2002). As can be seen in Fig. S7, membranes of wild-type S. coelicolor J1928 were able to mannosylate the A3 peptide, whereas those of the Δpmt mutant IB25 were not. When plasmid pBL12 encoding PmtSco was introduced into the Δpmt strain IB25 in vitro activity was restored, but no Pmt activity was detected when pBL9 (encoding PmtMtu) was introduced into this strain, meaning that this enzyme is not functional when expressed in S. coelicolor. This result supports the idea that PmtMtu is not capable of forming a productive interaction with the S.