Research projects

Control and activity of the Streptomyces Spore wall Synthesizing Complex SSSC

Coworkers

Nadja Steblau (PhD student), Bernd Vollmer (PhD student), Annette Latus (technician)

A major determinant of bacterial cell shape is the peptidoglycan (PG), which consists of glycan strands, cross-linked by short peptides. Bacteria control their shape by the specific localization of their cell wall synthesizing machineries. A key component of rod-shaped bacteria is the actin-like MreB, which polymerizes into filaments. MreB was shown to interact with other morphogenic proteins in rod-shaped bacteria and forms a lateral cell wall synthesis complex with MreC, MreD, PBP2, and RodA, directing incorporation of newly synthesized PG along the cylindrical part of the cell in a helical manner (White & Gober Trends Microbiol.2012. 20:74-9.

In contrast to rod-shaped bacteria which depend on MreB proteins to control their cell wall assembly, many Gram-positives of the phylum Actinobacteria grow in a different way by building their cell walls at the poles. Corynebacteria and mycobacteria do not contain mreB genes. They acquire rod shape by polarised growth, depending on the coiled-coil protein DivIVA. Similarly, streptomycetes, which do not divide by binary fission and grow by apical tip extension to form a multiply branching mycelium, also depend on DivIVA to direct this mode of apical growth (http://www.ncbi.nlm.nih.gov/pubmed/21036658). Against this background, it was a surprise that an mreB cluster comprising mreB, mreC, mreD, pbp2 and sfr (rodA) is present in S. coelicolor (http://www.ncbi.nlm.nih.gov/pubmed/10954092). Later it was shown that mreB and its paralogue mbl

were dispensable for apical growth of vegetative hyphae. Instead, spore morphology was affected and the spores had lost their resistance to heat, detergent, high osmolarity, lysozyme, and vancomycin (http://www.ncbi.nlm.nih.gov/pubmed/16677297, indicating a defective spore wall. A very similar phenotype was observed for mutants defective in one of the other mre-genes (https://www.ncbi.nlm.nih.gov/pubmed/21244527). MreB and its paralogue Mbl were localized by fluorescence microscopy to the sporulation septa and the spore envelope www.ncbi.nlm.nih.gov/pubmed/21257777 .

Analysis of protein-protein interaction by a bacterial two-hybrid system indicated that the S. coelicolor Mre proteins form a spore wall synthesizing complex which closely resembles the lateral wall synthesizing complex of rod-shaped bacteria (https://www.ncbi.nlm.nih.gov/pubmed/21244527). Screening of a genomic library identified several novel putative components of this complex, e.g.:

SCO2097, an actinomycetes specific membrane protein encoded by the dcw cluster,
SCO4129 (cmdD), previously shown to be involved in morphological differentiation
the eukaryotic type serine/threonine kinase SCO4778 (pkaI), suggesting control of spore wall synthesis by phosphorylation
putative cell wall glycopolymer (CWG) biosynthesis proteins (SCO2578 (pdtA) and SCO2584), indicating a close interconnection between the synthesis of PG and spore wall glycopolymers

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.

To confirm the proposed role of the Mre-interaction partners in spore wall synthesis, selected genes (SCO2097, SCO2584, SCO2578 (pdtA) SCO1403, SCO6494 or the tagF

-like SCO2997) were inactivated (https://www.ncbi.nlm.nih.gov/pubmed/21244527, www.ncbi.nlm.nih.gov/pubmed/21890702). All these mutants (beside ΔpdtA) showed normal growth as substrate mycelium but were severely affected in proper sporulation. These results suggest that the Mre-proteins co-operate with the newly identified proteins forming the Streptomyces Spore wall Synthesizing Complex SSSC. The SSSC probably orchestrates PG and CWG synthesis during septum formation to build the thickened spore wall required to resist detrimental environmental conditions, like heat, high osmolarity or cell wall damaging agents.

S. coelicolor

A3(2) contains two PG-linked CWGs, teichulosonic acid and a polydiglycosylphosphate, consisting of galactose and N-acetylglucosamine phosphate (Shashkov et al., 2012, Carbohydr. Res., 359:70–75). High resolution PAGE identified a PDP polymer of at least 19 repeating units www.ncbi.nlm.nih.gov/pubmed/27401190.

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.

The LytR-CpsA-Psr (LCP) protein PdtA (SCO2578), a TagV-like CWG transferase, has a dual function in the S. coelicolor A3(2) life cycle. Despite the presence of 10 additional LCP homologs, PdtA is crucial for proper sporulation. A pdtA deletion mutant produced 34% non-viable spores.

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.

The amount of PDP in spore walls was significantly reduced, suggesting that the reduced PDP content affected integrity of the spore envelope. Interestingly, apical tip extension and normal branching of vegetative mycelium were severely impaired on high-salt medium. This growth defect coincided with the miss-localization of peptidoglycan synthesis. This indicates that PdtA itself or the PDP attached to the peptidoglycan by the glycopolymer transferase PdtA also has a crucial function in apical tip extension of vegetative hyphae under stress conditions (https://www.ncbi.nlm.nih.gov/pubmed/27422828).

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).

During morphological differentiation Streptomyces faces the problem, how to build dozens of septal cross walls at the same time. For this process the membrane- and PG-synthesizing machineries have to be provided in sufficient quantities and positioned properly. Moreover, the activities of the complexes have to be controlled to prevent aberrant sporulation by sporadic formation of single cross walls in a non-coordinated manner. Interaction of the Ser/Thr protein kinase eSTPK PkaI (SCO4778) with multiple SSSC proteins suggested that sporulation septation in streptomycetes might be controlled by protein phosphorylation. Following co-expression of pkaI with either mreC or pbp2 in E. coli, specific phosphorylation of MreC and PBP2 by PkaI was demonstrated and the phosphosites were identified by LC-MS/MS (http://www.ncbi.nlm.nih.gov/pubmed/25927987).pkaI is part of a cluster of five independently transcribed eSTPK genes (SCO4775-SCO4779). Deletion of pkaI alone (NLΔPkaI) delayed sporulation and produced some aberrant spores. The five-fold mutant NLΔ4775-4779 was more severely affected and produced aberrant spores with an increased sensitivity to vancomycin indicating an impaired spore wall. Moreover, overbalancing phosphorylation activity by expressing a second copy of any of these kinase genes (under control of its native promoter) also interfered with proper sporulation. Spore chains contained 15-55 % dead spores and the germinating spores were sensitive to vancomycin. Vancomycin sensitivity could be complemented by MgCl2.

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.

Our data suggest that elaborate protein phosphorylation controls activity of the SSSC to ensure proper sporulation by suppressing premature cross-wall synthesis.