coelicolor is more proteolytic than

S lividans (Kieser e

coelicolor is more proteolytic than

S. lividans (Kieser et al., 2000; Jayapal et al., 2007). When supernatants of the Δppm mutant IB31 carrying the cloned apa gene (in pBL1) were analyzed, the GSK2126458 in vitro Apa protein was still expressed and secreted, as evidenced by the presence of a clear band detected by the 6A3 monoclonal antibodies (Fig. 1b, lane 2), but it was not glycosylated, as indicated by the slightly lower mass observed and by the lack of reaction with ConA (Fig. 1c, lane 2). This result indicates that PpmSco is essential for glycosylation of M. tuberculosis Apa by S. coelicolor. To determine whether the S. coelicolor Δppm mutant IB31 could be complemented by M. tuberculosis Ppm (PpmMtu), the Rv2051c gene was amplified from M. tuberculosis H37Rv DNA and cloned under the control of the strong PtipA promoter (plasmid pBL10, Table 1); the S. coelicolor ppm gene (sco1423) and upstream flanking region were cloned this website in pSET152 as a control (plasmid pBL13, Table 1). Phage φC31 was able to form plaques in the S. coelicolor Δppm mutant IB31 carrying either pBL10 or pBL13, encoding PpmMtu and PpmSco, respectively (Fig. 1a, plates 3 and 4; Table S2). In addition, introduction of these same plasmids into the S. coelicolor Δppm mutant

expressing Apa [IB31(pBL1)] restored glycosylation of this protein, as indicated by the presence of bands in Western blots detected with monoclonal antibodies (Fig. 1b, lanes 3 and 4), which showed restoration of ConA reactivity (Fig. 1c, lanes 3 and 4). To demonstrate activity of PpmMtu in S. coelicolor, an in vitro assay was carried out to detect labeling of the membrane polyprenyl phosphate by GDP-[14C]mannose in purified membrane fractions.

Streptomyces coelicolor harbors Orotidine 5′-phosphate decarboxylase a single C45 membrane polyprenol (Wehmeier et al., 2009; Fig. S1), and clear labeling of this molecule was observed in membranes of wild-type S. coelicolor (J1928) as indicated by a single-labeled band (Fig. 2, lane 1), but not in membranes of the Δppm mutant (IB31; Fig. 2, lane 2). Complementation was confirmed by this in vitro assay, because labeling of the membrane polyprenyl phosphate was restored when either pBL13 (PpmSco) or pBL10 (PpmMtu) was introduced into the Δppm mutant (Fig. 2, lanes 3 and 4, respectively), confirming that PpmMtu is functional when expressed in S. coelicolor. PpmMtu is a protein composed of two distinct domains. The N-terminal hydrophobic domain D1 (Met1-Tyr593) is responsible for lipoprotein N-acyltransferase (Lnt) activity, whereas the C-terminal domain D2 (Met594-Glu874) is the Ppm catalytic domain (Gurcha et al., 2002; Tschumi et al., 2009). We therefore decided to analyze whether the isolated D2 domain of PpmMtu was functional in S. coelicolor in the absence of the D1 domain. To do this, the portion of the Rv2051c gene encoding the D2 domain was cloned in pIJ6902 under control of the PtipA promoter (pBL11) and introduced into the S. coelicolor Δppm mutant IB31.

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