Streptomycetes are multicellular bacteria with complex developmental cycles. They are of biotechnological importance as they produce most bioactive compounds used in biomedicine, antibiotic, antitumoral and immunosupressor compounds. genomes encode many Ser/Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of We identified and quantified 3461 proteins corresponding to 44.3% of the proteome across three developmental stages: vegetative hypha (first mycelium); secondary metabolite producing hyphae (second mycelium); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signaling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism. Phosphoproteomics revealed 85 unique protein phosphorylation sites, 58 of them differentially phosphorylated during differentiation. Computational analysis suggested that these regulated protein phosphorylation events are implicated in important cellular processes, including cell division, differentiation, regulation of secondary metabolism, transcription, protein synthesis, protein folding and stress responses. We discovered a novel regulated phosphorylation site in the key bacterial cell division protein FtsZ (pSer319) that modulates sporulation and regulates actinorhodin antibiotic production. We conclude that manipulation of distinct protein phosphorylation events may improve secondary metabolite production in industrial streptomycetes, including the activation of cryptic pathways during the screening for new secondary metabolites from streptomycetes.

Bacteria of the genus Streptomyces are a model system for bacterial multicellularity. Their mycelial life style involves the formation of long multinucleated hyphae during vegetative growth, with occasional cross-walls separating long compartments. Reproduction occurs by specialized aerial hyphae, which differentiate into chains of uninucleoid spores. While the tubulin-like FtsZ protein is required for the formation of all peptidoglycan-based septa in Streptomyces, canonical divisome-dependent cell division only occurs during sporulation. Here we report extensive subcompartmentalization in young vegetative hyphae of Streptomyces coelicolor, whereby 1 μm compartments are formed by nucleic acid stain-impermeable barriers. These barriers possess the permeability properties of membranes and at least some of them are cross-membranes without detectable peptidoglycan. Z-ladders form during the early growth, but cross-membrane formation does not depend on FtsZ. Thus, a new level of hyphal organization is presented involving unprecedented high-frequency compartmentalization, which changes the old dogma that Streptomyces vegetative hyphae have scarce compartmentalization.

Reversible protein phosphorylation at serine, threonine and tyrosine is a well-known dynamic post-translational modification with stunning regulatory and signalling functions in eukaryotes. Shotgun phosphoproteomic analyses revealed that this post-translational modification is dramatically lower in bacteria than in eukaryotes. However, Ser/Thr/Tyr phosphorylation is present in all analysed bacteria (24 eubacteria and 1 archaea). It affects central processes, such as primary and secondary metabolism development, sporulation, pathogenicity, virulence or antibiotic resistance. Twenty-nine phosphoprotein orthologues were systematically identified in bacteria: ribosomal proteins, enzymes from glycolysis and gluconeogenesis, elongation factors, cell division proteins, RNA polymerases, ATP synthases and enzymes from the citrate cycle. While Ser/Thr/Tyr phosphorylation exists in bacteria, there is a consensus that histidine phosphorylation is the most abundant protein phosphorylation in prokaryotes. Unfortunately, histidine shotgun phosphorproteomics is not possible due to the reduced phosphohistidine half-life under the acidic pH conditions used in standard LC-MS/MS analysis. However, considering the fast and continuous advances in LC-MS/MS-based phosphoproteomic methodologies, it is expected that further innovations will allow for the study of His phosphoproteomes and a better coverage of bacterial phosphoproteomes. The characterisation of the biological role of bacterial Ser/Thr/Tyr and His phosphorylations might revolutionise our understanding of prokaryotic physiology.

Streptomycetes are important biotechnological bacteria with complex differentiation. Copper is a well-known positive regulator of differentiation and antibiotic production. However, the specific mechanisms buffering cytosolic copper and the biochemical pathways modulated by copper remain poorly understood. Here, we developed a new methodology to quantify cytosolic copper in single spores which allowed us to propose that cytosolic copper modulates asynchrony of germination. We also characterised the SCO2730/2731 copper chaperone/P-type ATPase export system. A Streptomyces coelicolor strain mutated in SCO2730/2731 shows an important delay in germination, growth and sporulation. Secondary metabolism is heavily enhanced in the mutant which is activating the production of some specific secondary metabolites during its whole developmental cycle, including germination, the exponential growth phase and the stationary stage. Forty per cent of the S. coelicolor secondary metabolite pathways, are activated in the mutant, including several predicted pathways never observed in the lab (cryptic pathways). Cytosolic copper is precisely regulated and has a pleiotropic effect in gene expression. The only way that we know to achieve the optimal concentration for secondary metabolism activation, is the mutagenesis of SCO2730/2731. The SCO2730/2731 genes are highly conserved. Their inactivation in industrial streptomycetes may contribute to enhance bioactive compound discovery and production.

This work contributes to the understanding of cell wall modifications during sporulation and germination in Streptomyces by assessing the biological function and biochemical properties of SCO4439, a D-alanyl-D-alanine carboxypeptidase (DD-CPase) constitutively expressed during development. SCO4439 harbors a DD-CPase domain and a putative transcriptional regulator domain, separated by a putative transmembrane region. The recombinant protein shows that DD-CPase activity is inhibited by penicillin G. The spores of the SCO4439::Tn5062 mutant are affected in their resistance to heat and acid and showed a dramatic increase in swelling during germination. The mycelium of the SCO4439::Tn5062 mutant is more sensitive to glycopeptide antibiotics (vancomycin and teicoplanin). The DD-CPase domain and the hydrophobic transmembrane region are highly conserved in Streptomyces, and both are essential for complementing the wild type phenotypes in the mutant. A model for the biological mechanism behind the observed phenotypes is proposed, in which SCO4439 DD-CPase releases D-Ala from peptidoglycan (PG) precursors, thereby reducing the substrate pool for PG crosslinking (transpeptidation). PG crosslinking regulates spore physical resistance and germination, and modulates mycelium resistance to glycopeptides. This study is the first demonstration of the role of a DD-CPase in the maturation of the spore cell wall.