coelicolor membrane; therefore, incorrect localization is not the reason for lack of complementation of the Δpmt mutation in IB25. Even though both genes were expressed from the strong and inducible PtipA promoter, hemagglutinin-tagged PmtMtu appeared to be less abundant than hemagglutinin-tagged PmtSco, when expressed in S. coelicolor under full induction Talazoparib chemical structure (to ensure that this fainter band was not due to a difference in the amount of protein loaded, the membrane was stained with Coomassie brilliant blue, Fig. S3). In addition, there appeared to be limited degradation of this protein, presumably related to the fact the S. coelicolor has an abundance of extracellular proteases
(Jayapal et al., 2007). It is unlikely that this slightly lower abundance
is the reason for lack of complementation, because hemagglutinin-tagged PmtSco was able to complement the Δpmt mutation for φC31 plaque formation even in the absence of inducer when expression relied on background PtipA transcription levels, revealing that even low levels of functional Pmt are sufficient for complementation (Fig. S4). The previous result prompted us to look for differences between PmtSco and PmtMtu to search for clues to the nonfunctionality of PmtMtu in S. coelicolor. Protein mannosylation by PmtMtu requires Sec translocation, and it has been proposed that physical interactions between the Sec complex and Pmt explain this requirement (VanderVen et al., 2005); therefore, GSK 3 inhibitor the nonfunctionality of PmtMtu in S. coelicolor could result from its inability to interact with the S. coelicolor Sec translocon. Upon alignment of the Pmt protein sequences from
mycobacteria and Streptomyces species, it was clear that the main difference is the presence in the Streptomyces Pmt sequences, including that of S. coelicolor, of an N-terminal extension. According to the prediction for topology of mycobacterial Pmt, this N-terminal extension should be located on the intracellular side of the membrane (Lommel & Strahl, ID-8 2009; Fig. S5). Because this extension could prove important for Pmt function in S. coelicolor (if, for example, it is required specifically for interaction with the S. coelicolor Sec translocon), we constructed two modified versions of the Rv1002c gene to encode chimeric Pmt proteins and cloned them in pIJ6902; in the first construct (pBL20, Table 1), 55 amino acids of PmtSco were affixed to the N-terminus of PmtMtu, giving PmtMtu + 55, whereas in the second construct (pBL21, Table 1), 178 amino acids of PmtSco, which include the first extracellular loop where acidic residues essential for activity are localized (VanderVen et al., 2005), were substituted for the equivalent N-terminal region of PmtMtu (Fig. S5). When pBL20 was introduced into the Δpmt mutant IB25, no complementation was observed, either for φC31 plaque formation (Fig. 4a, plate 5) or for Apa glycosylation (Fig. 4b and c, lane 5).