New Method for Monitoring Programmed Cell Death and Differentiation in Submerged Streptomyces Cultures

Abstract

Vital stains were used in combination with fluorimetry for the elaboration of a new method to quantify Streptomyces programmed cell death, one of the key events in Streptomyces differentiation. The experimental approach described opens the possibility of designing online protocols for automatic monitoring of industrial fermentations.Streptomyces is an extremely important bacterium for industry, since approximately two-thirds of all antibiotics are synthesized by members of this genus (4). Furthermore, streptomycetes produce large numbers of eukaryotic cell differentiation inducers and apoptosis inhibitors and inducers (19,24,25). Moreover, some authors consider that bacteria with complex life cycles (streptomycetes, cyanobacteria, etc.) are the evolutionary origin of some of the protein domains involved in programmed cell death (PCD) processes, including eukaryotic apoptosis: APATPases (apoptotic ATPases), kinases, caspases, nucleases, etc. As such, these bacteria would constitute a simple and convenient model by which to study this important phenomenon (1,3,9,12,21,26).The classical Streptomyces developmental model in confluent solid cultures assumed that differentiation processes took place along the transverse axis of the cultures (bottom up): completely viable vegetative mycelia (substrate) grew on the surface and inside agar until they underwent a PCD process, after which they differentiated into a reproductive (aerial) mycelium that grew into the air (reviewed in reference 8). Although most industrial processes for secondary metabolite production are performed in liquid cultures, Streptomyces strains generally do not sporulate under these conditions (6,18,22), and most authors assumed that differentiation did not take place. Recently, a detailed analysis of Streptomyces differentiation in surface and submerged cultures has been performed, describing novel aspects of the differentiation processes of this bacterium (10-17). A previously unidentified compartmentalized mycelium (MI) initiates the developmental cycle and then dies following a highly ordered sequence (PCD) (10,11,14). Subsequently, the remaining viable segments enlarge, yielding a multinucleated mycelium (MII) that grows in successive waves that determine the characteristic complex growth curves of this microorganism. In surface cultures, two types of second mycelium were defined, based on the absence (in early development) or presence (in late development) of the hydrophobic layers characteristic of aerial hyphae (5). The traditionally denominated substrate (vegetative) mycelium corresponds, in fact, to the early second multinucleated mycelium that still lacks the hydrophobic layers coating the aerial mycelium (15). We proposed that the first compartmentalized mycelium fulfills the true vegetative role in Streptomyces development in soil (17). According to this scheme, the second early and late multinucleated mycelia should be considered jointly as part of the reproductive phase, since they are destined to sporulate (17). The second multinuclea...

Streptomyces Differentiation in Liquid Cultures as a Trigger of Secondary Metabolism

“…A simple methodology based on fluorometric measures of cultures stained with SYTO9 and propidium iodide was designed to quantify PCD in liquid cultures [ 69 ]. This method allows the efficiency of antibiotic production to be predicted based on the level of PCD [ 69 ].…”

Quantitative Proteome Analysis of Streptomyces coelicolor Nonsporulating Liquid Cultures Demonstrates a Complex Differentiation Process Comparable to That Occurring in Sporulating Solid Cultures

“…18,19 The traditionally denominated substrate mycelium corresponds to MII lacking hydrophobic layers, and the aerial mycelium to MII coated with these hydrophobic layers (Figure 1). 15 The only mycelial phases present in liquid cultures were MI and MII without hydrophobic layers 8,9 (Figure 1).…”

Streptomyces as a Source of Antimicrobials: Novel Approaches to Activate Cryptic Secondary Metabolite Pathways

“…The lack of a reliable developmental model in streptomycetes liquid cultures has hindered the precise identification of reliable phenotypes for use in the analysis and optimization of industrial fermentations. It has been demonstrated that antibiotics are produced by the substrate mycelium at the end of the proliferation phase, i.e., second mycelium morphology (after PCD process) [27,30,37]. However, despite the fact that there is a distinctive mycelium producing antibiotics (MII) [27, 30] (hence there is not secondary metabolite production until the differentiation of MII), antibiotic production has additional regulations, and each Streptomyces strain does not display its entire potential secondary metabolism at a specific developmental condition.…”

“…The N-acetylglucosamine resulting from peptidoglycan dismantling accelerates the development and antibiotic production [108,109] and might be one of the signals released during PCD. A simple methodology based on fluorimetric measures was designed to quantify PCD in liquid cultures [37], allowing prediction of the efficiency of antibiotic production based on the level of PCD.…”