Quantitative Proteome and Phosphoproteome Analyses of Streptomyces coelicolor Reveal Proteins and Phosphoproteins Modulating Differentiation and Secondary Metabolism.

Streptomycetes are multicellular bacteria with complex developmental cycles. They are of biotechnological importance as they produce most bioactive compounds used in biomedicine, e.g. antibiotic, antitumoral and immunosupressor compounds. Streptomyces 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 Streptomyces coelicolor We identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor 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.

The SCO4117 ECF Sigma Factor Pleiotropically Controls Secondary Metabolism and Morphogenesis in Streptomyces coelicolor.

Extracytoplasmic function (ECF) sigma factors are a major type of bacterial signal-transducers whose biological functions remain poorly characterized in streptomycetes. In this work we studied SCO4117, a conserved ECF sigma factor from the ECF52 family overexpressed during substrate and aerial mycelium stages. The ECF52 sigma factors harbor, in addition to the ECF sigma factor domain, a zinc finger domain, a transmembrane region, a proline-rich C-terminal extension, and a carbohydrate-binding domain. This class of ECF sigma factors is exclusive to Actinobacteria. We demonstrate that SCO4117 is an activator of secondary metabolism, aerial mycelium differentiation, and sporulation, in all the culture media (sucrose-free R5A, GYM, MM, and SFM) analyzed. Aerial mycelium formation and sporulation are delayed in a SCO4117 knockout strain. Actinorhodin production is delayed and calcium-dependent antibiotic production is diminished, in the ΔSCO4117 mutant. By contast, undecylprodigiosin production do not show significant variations. The expression of genes encoding secondary metabolism pathways (deoxysugar synthases, actinorhodin biosynthetic genes) and genes involved in differentiation (rdl, chp, nepA, ssgB) was dramatically reduced (up to 300-fold) in the SCO4117 knockout. A putative motif bound, with the consensus "CSGYN-17bps-SRHA" sequence, was identified in the promoter region of 29 genes showing affected transcription in the SCO4117 mutant, including one of the SCO4117 promoters. SCO4117 is a conserved gene with complex regulation at the transcriptional and post-translational levels and the first member of the ECF52 family characterized.

Subcompartmentalization by cross-membranes during early growth of Streptomyces hyphae.

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.

Transcriptomic analysis of Streptomyces coelicolor differentiation in solid sporulating cultures: first compartmentalized and second multinucleated mycelia have different and distinctive transcriptomes.

Streptomycetes are very important industrial bacteria, which produce two thirds of all clinically relevant secondary metabolites. They have a complex developmental-cycle in which an early compartmentalized mycelium (MI) differentiates to a multinucleated mycelium (MII) that grows inside the culture medium (substrate mycelium) until it starts to growth into the air (aerial mycelium) and ends up forming spores. Streptomyces developmental studies have focused mainly on the later stages of MII differentiation (aerial mycelium and sporulation), with regulation of pre-sporulation stages (MI/MII transition) essentially unknown. This work represents the first study of the Streptomyces MI transcriptome, analyzing how it differs from the MII transcriptome. We have used a very conservative experimental approach to fractionate MI from MII and quantify gene expressions. The expression of well characterized key developmental/metabolic genes involved in bioactive compound production (actinorhodin, undecylprodigiosin, calcium-dependent antibiotic, cpk, geosmin) or hydrophobic cover formation-sporulation (bld, whi, wbl, rdl, chp, ram) was correlated with MII differentiation. Additionally, 122 genes conserved in the Streptomyces genus, whose biological function had not been previously characterized, were found to be differentially expressed (more than 4-fold) in MI or MII. These genes encoded for putative regulatory proteins (transcriptional regulators, kinases), as well as hypothetical proteins. Knowledge about differences between the MI (vegetative) and MII (reproductive) transcriptomes represents a huge advance in Streptomyces biology that will make future experiments possible aimed at characterizing the biochemical pathways controlling pre-sporulation developmental stages and activation of secondary metabolism in Streptomyces.

Pre-sporulation stages of Streptomyces differentiation: state-of-the-art and future perspectives.

Streptomycetes comprise very important industrial bacteria, producing two-thirds of all clinically relevant secondary metabolites. They are mycelial microorganisms with complex developmental cycles that include programmed cell death (PCD) and sporulation. Industrial fermentations are usually performed in liquid cultures (large bioreactors), conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that there was no differentiation. In this work, we review the current knowledge on Streptomyces pre-sporulation stages of Streptomyces differentiation.