Control of Morphological Differentiation of Streptomyces coelicolor A3(2) by Phosphorylation of MreC and PBP2

Abstract

During morphological differentiation of Streptomyces coelicolor A3(2), the sporogenic aerial hyphae are transformed into a chain of more than fifty spores in a highly coordinated manner. Synthesis of the thickened spore envelope is directed by the Streptomyces spore wall synthesizing complex SSSC which resembles the elongasome of rod-shaped bacteria. The SSSC includes the eukaryotic type serine/threonine protein kinase (eSTPK) PkaI, encoded within a cluster of five independently transcribed eSTPK genes (SCO4775-4779). To understand the role of PkaI in spore wall synthesis, we screened a S. coelicolor genomic library for PkaI interaction partners by bacterial two-hybrid analyses and identified several proteins with a documented role in sporulation. We inactivated pkaI and deleted the complete SCO4775-4779 cluster. Deletion of pkaI alone delayed sporulation and produced some aberrant spores. The five-fold mutant NLΔ4775-4779 had a more severe defect and produced 18% aberrant spores affected in the integrity of the spore envelope. Moreover, overbalancing phosphorylation activity by expressing a second copy of any of these kinases caused a similar defect. Following co-expression of pkaI with either mreC or pbp2 in E. coli, phosphorylation of MreC and PBP2 was demonstrated and multiple phosphosites were identified by LC-MS/MS. Our data suggest that elaborate protein phosphorylation controls activity of the SSSC to ensure proper sporulation by suppressing premature cross-wall synthesis.

Polydiglycosylphosphate Transferase PdtA (SCO2578) of Streptomyces coelicolor A3(2) Is Crucial for Proper Sporulation and Apical Tip Extension under Stress Conditions

“…Moreover, more than one-third of the produced spores were nonviable, and the remaining spores (or the developing mycelium) showed an increased sensitivity to lysozyme and osmotic stress. A similar phenotype was also reported for SSSC mutants defective in the mre genes mreB, mreC, mreD, pbp2, sfr, and mbl (23,42) or the Ser/Thr kinase gene pkaI, which directs the phosphorylation of MreC and PBP 2 (25). Surprisingly, the ⌬pdtA mutant was highly sensitive to rifampin, which targets the RNA polymerase and does not interfere with envelope synthesis.…”

“…Proper sporulation and synthesis of the thickened spore wall were shown to depend on the Streptomyces spore wall-synthesizing complex (SSSC), which highly resembles the elongasome of rod-shaped bacteria (23,24). The activity of the SSSC is probably controlled by protein phosphorylation involving multiple eukaryote-like serine/threonine protein kinases (eSTPK) (25). Besides MreBCD, RodZ, and various penicillin-binding proteins, the SSSC also contains proteins possibly involved in anionic glycopolymer synthesis, suggesting that the SSSC not only directs PG synthesis but also determines the glycopolymer content of spore envelopes (24).…”

Elucidation of the genetic mechanisms contributing to moenomycin resistance in actinobacteria

“…We also overexpressed PGT genes in S. coelicolor in hope that increased quantities of MmA target proteins might lead to increases in MmA susceptibility; however no changes in antibiotic resistance have been detected (data not shown). Finally, we examined following S. coelicolor strains disrupted in cell wall biosynthesis (pbp2, sco2997 (tagF-like), sco2578 (tagV-like)) and cytokinesis (mreB, mreC) genes described by us in previous works [13]. As compared to M145, mreB, mreC and sco2997 mutants were more susceptible to MmA after 48 h of growth in presence of antibiotic disc, however later on (72 h) halo of growth inhibition disappeared (Fig.…”

Recent advances in understanding Streptomyces

“…After septation, SepG relocalises to the prespore periphery, where it may interact with the cell wall synthesising complex during spore wall remodelling. Five protein kinases, encoded by a cluster of adjacent genes, phosphorylate key proteins of this complex, keeping them inactive until sporulation septation begins: it is assumed that activation involves one or more of the more than 50 phosphatases encoded by the S. coelicolor genome 94 .…”

“…FtsZ then condenses into Z-rings, which guide septation, and SepG relocates to the periphery of the prespore compartments 93 . The spore wall remodelling complex, held in an inactive phosphorylated state, is activated by one or more of many phosphorylases 94 and, apparently with the involvement of SepG, causes prespores to become rounder and thicker walled. I tentatively suggest that the increased amounts of SepG at this stage may depend on the Wbl-type regulator WhiD (possibly influenced by nitric oxide [NO] – see Figure 3), since sepG and whiD null mutants have very similar phenotypes 93, 95 .…”