María Teresa López-García

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.

Mycelium differentiation and development of Streptomyces coelicolor in lab-scale bioreactors: Programmed cell death, differentiation, and lysis are closely linked to undecylprodigiosin and actinorhodin production

Streptomycetes are mycelium-forming bacteria that produce two thirds of clinically relevant secondary metabolites. Secondary metabolite production is activated at specific developmental stages of Streptomyces life cycle. Despite this, Streptomyces differentiation in industrial bioreactors tends to be underestimated and the most important parameters managed are only indirectly related to differentiation: modifications to the culture media, optimization of productive strains by random or directed mutagenesis, analysis of biophysical parameters, etc. In this work the relationship between differentiation and antibiotic production in lab-scale bioreactors was defined. Streptomyces coelicolor was used as a model strain. Morphological differentiation was comparable to that occurring during pre-sporulation stages in solid cultures: an initial compartmentalized mycelium suffers a programmed cell death, and remaining viable segments then differentiate to a second multinucleated antibiotic-producing mycelium. Differentiation was demonstrated to be one of the keys to interpreting biophysical fermentation parameters and to rationalizing the optimization of secondary metabolite production in bioreactors.

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.

Transcriptomic Analysis of Liquid Non-Sporulating Streptomyces coelicolor Cultures Demonstrates the Existence of a Complex Differentiation Comparable to That Occurring in Solid Sporulating Cultures

Streptomyces species produce many clinically relevant secondary metabolites and exhibit a complex development that includes hyphal differentiation and sporulation in solid cultures. Industrial fermentations are usually performed in liquid cultures, conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that no differentiation took place. The aim of this work was to compare the transcriptomes of S. coelicolor growing in liquid and solid cultures, deepening the knowledge of Streptomyces differentiation. Microarrays demonstrated that gene expression in liquid and solid cultures were comparable and data indicated that physiological differentiation was similar for both conditions. Eighty-six percent of all transcripts showed similar abundances in liquid and solid cultures, such as those involved in the biosynthesis of actinorhodin (actVA, actII-4) and undecylprodigiosin (redF); activation of secondary metabolism (absR1, ndsA); genes regulating hydrophobic cover formation (aerial mycelium) (bldB, bldC, bldM, bldN, sapA, chpC, chpD, chpE, chpH, ramA, ramC, ramS); and even some genes regulating early stages of sporulation (wblA, whiG, whiH, whiJ). The two most important differences between transcriptomes from liquid and solid cultures were: first, genes related to secondary metabolite biosynthesis (CDA, CPK, coelichelin, desferrioxamine clusters) were highly up-regulated in liquid but not in solid cultures; and second, genes involved in the final stages of hydrophobic cover/spore maturation (chpF, rdlA, whiE, sfr) were up-regulated in solid but not in liquid cultures. New information was also provided for several non-characterized genes differentially expressed in liquid and solid cultures which might be regulating, at least in part, the metabolic and developmental differences observed between liquid and solid cultures.

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.

Characterization of SCO4439, a D-alanyl-D-alanine carboxypeptidase involved in spore cell wall maturation, resistance, and germination in Streptomyces coelicolor

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.

New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces.

Integrative plasmids are one of the best options to introduce genes in low copy and in a stable form into bacteria. The ΦC31-derived plasmids constitute the most common integrative vectors used in Streptomyces. They integrate at different positions (attB and pseudo-attB sites) generating different mutations. The less common ΦBT1-derived vectors integrate at the unique attB site localized in the SCO4848 gene (S. coelicolor genome) or their orthologues in other streptomycetes. This work demonstrates that disruption of SCO4848 generates a delay in spore germination. SCO4848 is co-transcribed with SCO4849, and the spore germination phenotype is complemented by SCO4849. Plasmids pNG1–4 were created by modifying the ΦBT1 integrative vector pMS82 by introducing a copy of SCO4849 under the control of the promoter region of SCO4848. pNG2 and pNG4 also included a copy of the PermE* in order to facilitate gene overexpression. pNG3 and pNG4 harboured a copy of the bla gene (ampicillin resistance) to facilitate selection in E. coli. pNG1–4 are the only integrative vectors designed to produce a neutral phenotype when they are integrated into the Streptomyces genome. The experimental approach developed in this work can be applied to create phenotypically neutral integrative plasmids in other bacteria.

Cell immobilization of Streptomyces coelicolor : effect on differentiation and actinorhodin production

Streptomycetes are mycelium-forming bacteria that produce two thirds of the clinically relevant secondary metabolites. Despite the fact that secondary metabolite production is activated at specific developmental stages of the Streptomyces spp. life cycle, different streptomycetes show different behaviors, and fermentation conditions need to be optimized for each specific strain and secondary metabolite. Cell-encapsulation constitutes an interesting alternative to classical fermentations, which was demonstrated to be useful in Streptomyces, but development under these conditions remained unexplored. In this work, the influence of cell-encapsulation in hyphae differentiation and actinorhodin production was explored in the model Streptomyces coelicolor strain. Encapsulation led to a delay in growth and to a reduction of mycelium density and cell death. The high proportion of viable hyphae duplicated extracellular actinorhodin production in the encapsulated cultures with respect to the non-encapsulated ones.

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.

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.