Despite the fact that most industrial processes for secondary metabolite production are performed with submerged cultures, a reliable developmental model for Streptomyces under these culture conditions is lacking. With the exception of a few species which sporulate under these conditions, it is assumed that no morphological differentiation processes take place. In this work, we describe new developmental features of Streptomyces coelicolor A3(2) grown in liquid cultures and integrate them into a developmental model analogous to the one previously described for surface cultures. Spores germinate as a compartmentalized mycelium (first mycelium). These young compartmentalized hyphae start to form pellets which grow in a radial pattern. Death processes take place in the center of the pellets, followed by growth arrest. A new multinucleated mycelium with sporadic septa (second mycelium) develops inside the pellets and along the periphery, giving rise to a second growth phase. Undecylprodigiosin and actinorhodin antibiotics are produced by this second mycelium but not by the first one. Cell density dictates how the culture will behave in terms of differentiation processes and antibiotic production. When diluted inocula are used, the growth arrest phase, emergence of a second mycelium, and antibiotic production are delayed. Moreover, pellets are less abundant and have larger diameters than in dense cultures. This work is the first to report on the relationship between differentiation processes and secondary metabolite production in submerged Streptomyces cultures.Streptomyces is a soil bacterium that produces numerous clinically useful antibiotics (1, 54, 66), as well as many molecules that affect eukaryotic systems, such as inducers of eukaryotic cellular differentiation, inducers and inhibitors of apoptosis (59), and protein C kinase inhibitors with antitumor activity (such as staurosporine and others) (48, 57). Moreover, its remarkably complex developmental cycle makes this microorganism an interesting subject for study. The traditional developmental cycle of this bacterium describes two differentiated mycelial structures, a substrate (vegetative) mycelium and an aerial (reproductive) mycelium (10,25,33). In the substrate mycelium, septa are thought to be widely spaced and to define compartments containing several nucleoids (10, 63). After a short growth arrest phase characterized by reduced macromolecular synthesis (25), aerial hyphae develop from simple branching from substrate mycelium (29). Finally, the tips of the aerial mycelium differentiate into hydrophobic spore chains (8). Recently, we have been able to extend what is known about the developmental cycle in surface confluent cultures a great deal. Our main contribution has been to reveal the existence of a very young compartmentalized mycelium that dies following an orderly pattern, leaving alternating live and dead segments in the same hypha (37). Subsequently, the remaining live mycelium grows in successive waves that vary according to the density of the ...

Development-associated cell-death processes were investigated in detail during the growth and differentiation of Streptomyces antibioticus ATCC 11891 on confluent surface cultures, by using fluorescent viability probes, membrane and activity fluorescence indicators, and electron microscopy analysis. A previously unsuspected complexity was revealed, namely the presence of a very young compartmentalized mycelium that dies following an orderly pattern, leaving alternating live and dead segments in the same hypha. This death round is followed by the growth of a second mycelium which develops rapidly from the live segments of the first mycelium and dies massively in a second death round, which extends over the phases of aerial mycelium formation and sporulation. INTRODUCTIONStreptomyces forms two differentiated structures when grown as surface cultures (Waksman, 1967): a substrate (vegetative) mycelium and an aerial (reproductive) mycelium. The substrate mycelium is usually described as a branched structure with hyphae that have a mean diameter of 0?7 mm, bounded by a mucopeptide cell wall 0?01-0?02 mm wide and crossed with relatively scarce septa which delimit compartments usually containing many nucleoids (Waksman, 1967;Hodgson, 1992;Chater, 1993;Chater & Losick, 1997). This mycelium is assumed to be present, though in different stages of cellular degeneration, during all growth phases, which in Streptomyces antibioticus cultures last for about 90 h of cultivation on solid medium (Mendez et al., 1985; Braña et al., 1986;Miguelez et al., 1999). After about 35 h or more of cultivation, a mycelium network develops into the air that grows and produces chains of spores with hydrophobic surfaces (Chater, 1984). In early studies, aerial hyphae were reported as originating from simple branching from substrate hyphae (Hopwood & Glauert, 1961), and they are preceded by a short period of reduced macromolecular synthesis (Granozzi et al., 1990).Bacterial death phenomena in communities appear to be active processes related to a multicellularity trait likewise subject to environmental factors and developmental processes (Rice & Bayles, 2003). Lytic phenomena associated with development were reported early on in Streptomyces coelicolor A3(2) by Wildermuth (1970), who described two central developmental fates for the Streptomyces mycelium: the surface layer, leading to spore formation, and the underlying, non-sporulating hyphae, leading to lysis. This phenomenon was subsequently interpreted by assuming that the lysis of the substrate mycelium could serve the purpose of providing nutrients for the developing aerial structures (Chater, 1984; revised by Hodgson, 1992). Analysis of the mobilization of radioactively labelled amino acids during the development of S. antibioticus ATCC 11891 on surface cultures supports the reutilization hypothesis (Braña et al., 1986). The existence of an orderly process of internal cell dismantling was further addressed in S. antibioticus ATCC 11891 by electron microscopy analysis of the mycelium growin...

Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one of the bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized phosphorylation motif "x(pT)xEx".

Macroscopic growths at geographically separated acid mine drainages (AMDs) exhibit distinct populations. Yet, local heterogeneities are poorly understood. To gain novel mechanistic insights into this, we used OMICs tools to profile microbial populations coexisting in a single pyrite gallery AMD (pH ∼2) in three distinct compartments: two from a stratified streamer (uppermost oxic and lowermost anoxic sediment-attached strata) and one from a submerged anoxic non-stratified mat biofilm. The communities colonising pyrite and those in the mature formations appear to be populated by the greatest diversity of bacteria and archaea (including ‘ARMAN' (archaeal Richmond Mine acidophilic nano-organisms)-related), as compared with the known AMD, with ∼44.9% unclassified sequences. We propose that the thick polymeric matrix may provide a safety shield against the prevailing extreme condition and also a massive carbon source, enabling non-typical acidophiles to develop more easily. Only 1 of 39 species were shared, suggesting a high metabolic heterogeneity in local microenvironments, defined by the O2 concentration, spatial location and biofilm architecture. The suboxic mats, compositionally most similar to each other, are more diverse and active for S, CO2, CH4, fatty acid and lipopolysaccharide metabolism. The oxic stratum of the streamer, displaying a higher diversity of the so-called ‘ARMAN'-related Euryarchaeota, shows a higher expression level of proteins involved in signal transduction, cell growth and N, H2, Fe, aromatic amino acids, sphingolipid and peptidoglycan metabolism. Our study is the first to highlight profound taxonomic and functional shifts in single AMD formations, as well as new microbial species and the importance of H2 in acidic suboxic macroscopic growths.

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.

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Programmed cell death (PCD) is an active cellular suicide that occurs in eukaryotes and bacteria in response to both abiotic and biotic stresses. In contrast to eukaryotic apoptosis, little is known about the molecular machinery that regulates bacterial PCD. In a previous work, we described the existence of PCD phenomena in Streptomyces (Manteca et al., Res. Microbiol. 2006, 157, 143-152). In the present study, we performed a proteomic analysis of PCD in Streptomyces coelicolor, for which we developed a system to obtain dead and live cell-enriched samples. PCD in this filamentous bacterium is accompanied by the appearance of enzymes involved in the degradation of cellular macromolecules, regulatory proteins, and stress-induced proteins. We argue that some of these proteins have specific functions in the PCD pathway and putative roles for the identified proteins have been proposed. The increased amounts of several antioxidant proteins suggest oxidative stress as either the cause or consequence of the cell death.

Streptomyces species produce many clinically important secondary metabolites, including antibiotics and antitumorals. They have a complex developmental cycle, including programmed cell death phenomena, that makes this bacterium a multicellular prokaryotic model. There are two differentiated mycelial stages: an early compartmentalized vegetative mycelium (first mycelium) and a multinucleated reproductive mycelium (second mycelium) arising after programmed cell death processes. In the present study, we made a detailed proteomics analysis of the distinct developmental stages of solid confluent Streptomyces coelicolor cultures using iTRAQ (isobaric tags for relative and absolute quantitation) labeling and LC-MS/MS. A new experimental approach was developed to obtain homogeneous samples at each developmental stage (temporal protein analysis) and also to obtain membrane and cytosolic protein fractions (spatial protein analysis). A total of 345 proteins were quantified in two biological replicates. Comparative bioinformatics analyses revealed the switch from primary to secondary metabolism between the initial compartmentalized mycelium and the multinucleated hyphae. Molecular & Cellular Proteomics 9:1423-1436, 2010.