Two-dimensional gel analysis of proteins from cell wall fractions of S. coelicolor M145 (A) and a ΔtatC mutant derivative (B). Strains were cultured on R5, and cell wall proteins were isolated as described. Proteins were separated in the first dimension by isoelectric focusing (pH gradient 4–7) and in the second dimension by SDS/PAGE. Protein spots that migrated to specific positions in the M145 cell wall fractions but were absent from the corresponding position in cell wall fractions of the ΔtatC strain are circled. The identities of the protein spots were determined by in-gel tryptic digestion, followed by mass spectrometry.

Agarase is a Tat substrate. (A) The agarase signal peptide. The consecutive arginines of the twin-arginine motif are highlighted in bold underline. (B) S. coelicolor strains M145 (WT) and TP1 (ΔtatC::Apra^R) were grown on MM-C minimal medium for 5 days and were stained with Lugol solution. (C) Strains M145 (WT) and TP4 (ΔtatC) harboring pIJ6902-dagA in single copy were grown on minimal media containing 0.5% glucose, apramycin, and thiostrepton for 3 days before staining with Lugol solution.

Export of agarase mediated by S. coelicolor signal peptides. The y axis shows the signal peptides from a range of S. coelicolor cell wall-associated proteins (listed in Tables 2 and 4). The x axis gives a measure of agarase export from each fusion protein compared with agarase bearing its native signal peptide (set at 100%). Each construct carries the native agarase promoter and ribosome-binding site, and, therefore, the activity should be a measure of the efficiency of export directed by each particular signal peptide. The assay was verified by using signal peptides from SCO3053, SCO5660, and SCO6199, which were included as negative controls because they lack twin arginines in the signal peptide and were detected in the cell wall fractions of both M145 and the ΔtatC mutant by MudPIT analysis. None of these signal peptides mediated extracellular agarase activity. The signal peptides from the following proteins also were tested and were found to be negative in this assay: SCO0432, SCO0474, SCO0494, SCO0930, SCO1230, SCO1396, SCO1824, SCO1948, SCO1968, SCO2226, SCO2383, SCO2446, SCO2591, SCO2637, SCO2786, SCO2821, SCO3456, SCO4010, SCO4142, SCO4152, SCO4884, SCO4885, SCO5074, SCO5113, SCO5447, SCO5461, SCO6009, SCO6644, SCO6738, and SCO7399. ∗, two versions of each of these signal peptides (designated long and short), representing two alternative start sites, were tested (see Tables 2 and 4).