Fig. 1 Interaction pattern of the Streptomyces spore wall synthesizing complex SSSC.
Interactions were identified by bacterial two-hybrid analyses (Kleinschnitz et al., 2011). Interactions with the serine/threonine kinase PkaI (SCO4778), suggesting control by protein phosphorylation, are given in red. Interactions of SSSC proteins with putative CWG synthesis enzymes (grey) are indicated by blue color. Penicillin binding proteins are drawn in green.
Fig. 2 Structural analysis of S. coelicolor A3(2) cell wall glycopolymers.
Vegetative cell walls were hydrolysed for 15 minutes under mild acidic conditions. The fragments were separated according to their molecular weight using high resolution PAGE. A minimum of 19 bands with regular distances could be detected indicating that the corresponding polymer consists of at least 19 uniform subunits. Colorimetric assays confirmed the presence of PDP in the bands recovered from PAGE.
Fig. 3 Effect of pdtA inactivation on spore viability.
Representative pictures showing the measurement of spore viability of S. coelicolor M145 (A) and mutant ΔpdtA (B) by SYTO®9/propidium iodide double staining. SYTO®9 is a membrane permeable DNA stain, indicating viable spores (green). Propidium iodide marks dead spores (red), since it can only enter cells after membrane damage, displacing SYTO®9, due to its use in higher concentration. Cultures were grown on cover slips inserted into MS agar at 30°C. Bar = 2µm. For statistical analysis (C) at least 3000 spores of randomly selected images from at least three independent plates were counted. Compared to the wildtype, the SSΔpdtA mutant showed a strong decrease (Mann-Whitney test, ** = p ≤ 0.01) in average spore viability. Data are presented as median with interquartile range.
Fig. 4 Aberrant growth and miss-localization of PG-synthesis under salt stress in S. coelicolor ΔpdtA.
Strains were grown for 48 hours on cover slips inserted into MS agar supplemented with 6% NaCl (A). Following staining with a fluorescent vancomycin derivative (Van-Fl) to visualize sites of PG synthesis, the mycelium was observed by phase contrast (pc) and fluorescence microscopy (Van-Fl). The wild type M145 incorporates new PG (arrows) only at septal cross walls and at the tips of growing hyphae (A). In contrast, many of the tips of the aberrant branches of SSΔ2578 did not bind Van-Fl and PG incorporation occurred at many places at the lateral walls (B).
Effect of overbalancing phosphorylation activity on proper sporulation (A) of S. coelicolor and the resistance of germinating spores to vancomycin (B).
Live-dead staining of spore chains of the eSTPK mutants NLΔPkaI and NLΔ4775-4779 revealed the presence of dead spores (red) or spores without DNA (black). Expression of a second copy of any eSTPK gene of cluster SCO4775-4779 caused a similar sporulation defect in S. coelicolor M145, NLΔPkaI, or NLΔ4775-4779. Spores of different strains were plated onto LB agar and filter discs containing 5 µg vancomycin were applied. Whereas, M145 and the pkaI mutant NLΔPkaI were resistant, NLΔ4775-4779 spores showed vancomycin sensitivity, suggesting an impaired spore wall. Vancomycin sensitivity of M145 or NLΔPkaI was also caused by expressing a second copy of each kinase gene, with the exception of pkaI. Supplementation of the agar plates with 3 mM MgCl2, known to rescue mutants impaired in cell wall synthesis restored vancomycin resistance to all strains. A, no plasmid integrated; B, ::pSET152-pkaH; C, ::pSET152-SCO4776; D, ::pSET152-pkaD; E, ::pSET152-pkaI; F, ::pSET152-pkaJ.