1 Scientific RepoRts | 6:27045 | DOI: 10.1038/srep27045 www.nature.com/scientificreports Aggregation of germlings is a major contributing factor towards mycelial heterogeneity of Streptomyces Boris Zacchetti 1 , Joost Willemse 1 , Brand Recter 2 , Dino van Dissel 1 , Gilles p. van Wezel 1 , H. A. B. Wösten 2 & Dennis Claessen 1 Streptomycetes are flamentous bacteria that produce numerous valuable compounds, including the majority of clinically used antibiotics. At an industrial scale, most of these compounds are produced in bioreactors. Growth of streptomycetes under these conditions is characterized by the formation of complex mycelial particles, whose sizes follow a bimodal distribution. Given the correlation between specifc productivity and morphology, this size heterogeneity poses a potential drawback in industry. Recent work indicates that mycelial morphology is controlled by a number of genes that encode proteins required for the synthesis of cell surface-associated glycans. Using a quantifable system based on fuorescent markers, we here show that these glycans mediate aggregation between germlings and young mycelia, yielding mycelial particles that originate from many diferent individuals. We also demonstrate that at later time points aggregation between distinct particles is no longer detectable. Notably, the absence of the corresponding glycan synthases yields mycelia that are homogeneous in size, identifying mycelial aggregation as a driving factor towards size heterogeneity. Given that aggregation is widespread within streptomycetes and can also occur between diferent Streptomyces strains, our work paves the way to improve Streptomyces as a cell factory for the production of known metabolites, but possibly also to discover new ones. Streptomycetes are flamentous bacteria that are renowned for their ability to produce several types of bioactive compounds. Standing out among these are a plethora of antibiotics, but also numerous antiviral compounds, anticancer agents and molecules that suppress the immune system 1,2 . Due to their saprophytic lifestyle, strepto- mycetes also show a remarkable capacity to produce and efciently secrete various hydrolytic enzymes that allow them to degrade almost any naturally occurring polymer 3 . Te extensive industrial use of streptomycetes does not come as a surprise when these factors are taken into account. In contrast to most other bacteria, streptomycetes grow by forming thread-like cells called hyphae 4 . Tese hyphae extend at their tip, while new hyphae emerge via subapical branching of pre-existing ones. Tis mode-of-growth leads to the construction of complex networks of interconnected hyphae called mycelia. When grown in liquid, these mycelia display markedly diferent morphologies 5 . Some strains, including the industrial workhorse Streptomyces lividans, predominantly form dense mycelial particles called pellets. In contrast, other strains form so-called mats, which are loosely entangled and scarcely dense mycelial networks 5 . Notably, a strong correlation exists between morphology and productivity in streptomycetes 5–7 . While growth as pellets is preferred for the production of antibiotics, it is suboptimal for the production of enzymes. For efcient enzyme production mycelial mats are more suitable, which relates to the fact that nutrients are more easily accessible to all hyphae. In fact, hyphae in the central part of pellets ofen sufer from nutrient stress leading to a phase of controlled cell death 8,9 . To further complicate matters, recent work from our lab demonstrated that liquid-grown Streptomyces cultures are heterogeneous and contain at least two populations of mycelial particles that difer in size 10 . Tis size 1 Microbial Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands. 2 Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Correspondence and requests for materials should be addressed to H.A.B.W. (email: h.a.b.wosten@uu.nl) or D.C. (email: D.Claessen@biology.leidenuniv.nl) received: 29 February 2016 Accepted: 13 May 2016 Published: 31 May 2016 opeN