Therefore, lyophilized spores were mixed with a matrix solution containing either α-cyano-4-hydroxycinnamic acid [10 mg mL−1 in 50% acetonitrile/0.1% trifluoroacetic acid (TFA)] or sinapinic acid (20 mg mL−1 in 40% acetonitrile/0.1% TFA). These mixtures were spotted for analysis with a Shimadzu Biotech MALDI-TOF Mass Spectrometer (Axima Performance). Spectra of spores isolated from complex R5 medium (Kieser et al., 2000) or complex MS medium (Kieser

et al., 2000) showed peaks ranging from 1 to 12 kDa (Fig. 1a). Relative high intensities were observed for peaks with masses of 5070, 5121, 5182, and 5274 Da (Fig. 1b), which fit the predicted masses of ChpD, ChpH, ChpF, and ChpE, respectively (Claessen et al., 2003; Elliot p38 MAPK assay et al., 2003). Analysis of spores of the S. coelicolor chpABCDH (Claessen et al., 2003), chpABCDEH (Claessen et al., 2003), and chpABCDEFGH (Claessen et al., 2004) strains (obtained after prolonged incubation on MS medium) confirmed

the identity of these peaks, as they were absent in the respective mutants (Supporting Information, Fig. S1a). The rodlin proteins RdlA and RdlB (Claessen et al., 2002, 2004) were also identified at the spore surface as proteins with masses of 10 517 and 10 708 Da, respectively (Fig. S1b). NepA, whose presence on the spore surface has been demonstrated by immuno labeling (de Jong et al., 2009), could not be identified according to its predicted mass of 7725 Da. In contrast, SapB was found on the spore surface represented by a peak at 2027 Da. Interestingly, SapB was not only found on spores obtained from R5 medium (Fig. 1c) but Transferase inhibitor also on those obtained from MS medium (Fig. 1d), a condition in which Diflunisal SapB was formerly shown not to be secreted by the wild-type strain (Capstick et al., 2007). The intensity of the SapB peak on MS medium was about fourfold lower compared to that found with spores from R5 medium. SapB was also identified on spores on defined minimal medium with mannitol as a carbon source (Fig. 1e). Also in this medium, SapB is not secreted into the medium (Willey et al., 1991).

As expected, SapB was absent on spores of the ramR (Fig. S1c) and ramS mutants (Fig. 1c–e) that had been collected from cultures grown on R5 or minimal mannitol medium. Similar results were obtained when TFA extracts of spores were analyzed by MALDI-TOF MS (data not shown). The fact that SapB is not secreted in certain media (Willey et al., 1991; Capstick et al., 2007) suggested a difference between SapB secretion by vegetative hyphae and aerial hyphae and spores. To confirm this, culture media were analyzed for the presence of SapB by MALDI-TOF MS. Agar plates overlaid with cellophane disks were inoculated with spores of the wild-type strain or the ramR or ramS mutant strains. After 5 days of growth at 30 °C, the agar medium underlying the cellophane membrane was collected and melted.