Journal of Microbiological Methods 91 (2012) 318–320
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A sandwich-culture technique for controlling antibiotic production and morphological development in Streptomyces coelicolor Inez P. de Jong, Dennis Claessen ⁎ Molecular Biotechnology, Institute Biology Leiden (IBL), Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands
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Article history: Received 17 July 2012 Received in revised form 12 September 2012 Accepted 12 September 2012 Available online 18 September 2012
a b s t r a c t Antibiotic-producing streptomycetes form complex colonies consisting of vegetative and aerial hyphae. Here, we describe a sandwich-culture technique in which the mycelium grows in between two membranes, thereby preventing morphogenesis and antibiotic production. Both processes are restored by removal of the top membrane, thus providing a switch to coordinate their timing. © 2012 Elsevier B.V. All rights reserved.
Keywords: Antibiotics Development Growth Heterogeneity Streptomyces
Streptomycetes are ﬁlamentous bacteria with a complex life cycle. After spore germination a network of interconnected hyphae, called a mycelium, is established. After a period of vegetative growth hyphae start to grow away from the substrate into the air. These aerial hyphae give rise to chains of spores that after their dispersal can initiate growth elsewhere. Aerial growth coincides with the production of many secondary metabolites, including many clinically relevant antibiotics. In addition, these bacteria produce numerous antifungals and immunosuppressive agents, making these organisms of outstanding interest for pharmaceutical industries (Hopwood, 2007). The formation of aerial hyphae and spores introduces a level of complexity to the colony that makes the study of the vegetative mycelium underneath the aerial structures complicated. For instance, it is nearly impossible to physically dissect the distinct tissues due to the complex three-dimensional nature of the colony. We therefore adapted a method to grow streptomycetes between two membranes, based on a method described for ﬁlamentous fungi (Wösten et al., 1991; Ásgeirsdóttir et al., 1999; Fig. 1). First, a cellophane disk (Focus Packaging & Design Ltd, Louth (UK); sterilized in water) was placed onto the surface of an R5 agar plate (Kieser et al., 2000). Then, 2 ml of melted 1% agarose was poured over the cellophane surface. After the agarose had solidiﬁed, 0.5 μl of a spore suspension (2 × 10 3 spores ml −1 to obtain colonies derived from a single spore) was inoculated on to the agarose. These cultures were incubated for 12 h at 30 C before a second cellophane disk was placed on top of the growing colony (Fig. 1). The sandwiched colonies were further ⁎ Corresponding author at: Institute Biology Leiden, Molecular Biotechnology, Leiden University, Sylviusweg 72, 2300 RA, Leiden, The Netherlands. Tel.: +31 71 527 5052. E-mail address: [email protected]
(D. Claessen). 0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2012.09.012
grown at 30 C for up to 14 days. Under these conditions, colonies of the model streptomycete Streptomyces coelicolor M145 (Kieser et al., 2000) grew relatively ﬂat in between the two membranes and had a smooth appearance (Fig. 2A). Microscopic analysis revealed that individual hyphae were easily discriminated at the periphery of sandwiched colonies, unlike those grown on top of a cellophane disk (data not shown). Notably, we noticed that growth under these conditions proceeded up to at least 7 days as indicated by the enlargement of the diameter of the trapped colony (data not shown). However, the size of sandwiched colonies was smaller than those that had grown without a membrane on top. After 14 days, the diameter of colonies that had grown in between the membranes was 8 ± 2 mm. In contrast, the diameter of colonies grown without a cellophane disk on top was 12±3 mm. While the colonies grown without a membrane were sporulating abundantly, no signs of aerial growth (and sporulation) were detected in the sandwiched cultures, even after 14 days of growth (Fig. 2A). Aerial growth was also abolished in sandwiched cultures of all other streptomycetes we tested, including Streptomyces lividans, Streptomyces avermitilis, Streptomyces griseus and Streptomyces venezuelae (data not shown). Interestingly, we noticed that the production of the pigmented antibiotics undecylprodigiosin and actinorhodin was abolished in S. coelicolor (Fig. 2A). This is consistent with the notion that morphological development and the production of antibiotics are intimately related (van Wezel and McDowall, 2011). To see if the failure to produce antibiotics was resulting from the absence of aerial hyphae or the presence of the membrane, we analyzed sandwiched colonies of a bldF mutant strain, which is unable to form aerial hyphae but produces a red-pigmented compound (Puglia and Cappelletti, 1984). Interestingly, growth of the bldF mutant strain in between cellophane disks abolished the production of the red mycelial-associated pigment
I.P. de Jong, D. Claessen / Journal of Microbiological Methods 91 (2012) 318–320
Cellophane disk Agar medium
(Fig. S2). Strikingly, similar results were obtained when the membrane was removed after 7 or 14 days. This indicates that morphological development and antibiotic production under these conditions are initiated upon removal of the top membrane, irrespective of colony age. The sandwich-culture technique employed here provides a simple and highly reproducible tool to coordinate the timing of morphological development and antibiotic production in streptomycetes, which is useful to study the control of these processes. Moreover, we anticipate that this method facilitates the analysis of vegetative mycelium, which is otherwise largely obscured by the aerial mycelium. For instance, this approach has proven to be useful for the localization of protein secretion by vegetative hyphae in ﬁlamentous fungi (Ásgeirsdóttir et al., 1999; Levin et al., 2007). Therefore, this method is not only an attractive tool to coordinate and study morphogenesis, but also to explore
12 h Cellophane disk
B Agar medium
Fig. 1. Schematic overview of a sandwich-culture technique for streptomycetes.
(Fig. 2B). This red pigment was the antibiotic undecylprodigiosin, as deletion of redD, encoding the pathway-speciﬁc activator required for the production of the antibiotic undecylprodigiosin (Takano et al., 1992), abolished pigmentation in bldF (Fig. S1). To exclude that the failure to produce antibiotics was due to oxygen limitation, we punctured the top membrane in close proximity to and in the center of the growing colonies. However, this did not restore antibiotic production (data not shown). These data show that the physical barrier imposed by the cellophane disk not only prevents aerial growth but also antibiotic production. To see if aerial growth and antibiotic production could be restored by removal of the membrane, sandwiched wild-type cultures were grown for 4 days after which the top membrane was removed (Fig. 2C). Remarkably, after removal of the membrane the morphology of the colony changed rapidly. Already after 3 h we noticed a form of differentiation in the vegetative mycelium manifested by the formation of rings in the center and at the periphery of the colony (Fig. 2C). Strikingly, 6 h after removal of the membrane we detected that colonies started to produce the red-pigmented antibiotic undecylprodigiosin, coinciding with the appearance of aerial hyphae. These aerial hyphae started to form in the center of the colony, and increased in number until a conﬂuent lawn had covered the entire surface of the colony after 24 h (Fig. 2C). These aerial hyphae then further differentiated into gray-pigmented spores, which were visible after 48 h after removal of the membrane. At that time, the bluepigmented antibiotic actinorhodin was also visible. The latter is consistent with the detected induction of gfp expression (under control of the actII-ORF4 promoter) after removing the top membrane
Fig. 2. Effects on growth, development and antibiotic production in S. coelicolor using the sandwich-culture technique. A. Formation of aerial hyphae and pigmented antibiotics in a 14-day-old colony of the S. coelicolor wild-type strain (left) is abolished in colonies grown in between cellophane disks (right). B. Undecylprodigiosin production in a bldF mutant (left) is abolished by growth of the strain in between cellophane disks (right). C. Restoration of aerial growth, and antibiotic production in a 4-day-old wild-type colony after removal of the cellophane disk. The numbers indicate the time in hours after removal of the membrane. Red and Act are the pigmented antibiotics undecylprodigiosin and actinorhodin, respectively. Aerial growth is visible as the white color appearing on top of the colony.
I.P. de Jong, D. Claessen / Journal of Microbiological Methods 91 (2012) 318–320
heterogeneity in growth and secretion in vegetative mycelium on solid substrates. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.mimet.2012.09.012. References Ásgeirsdóttir, S.A., Scholtmeijer, K., Wessels, J.G.H., 1999. A sandwiched-culture technique for evaluation of heterologous protein production in a ﬁlamentous fungus. Appl. Environ. Microbiol. 65, 2250–2252. Hopwood, D.A., 2007. Streptomyces in Nature and Medicine: The Antibiotic Makers. Oxford University Press, USA. Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., Hopwood, D.A., 2000. Practical Streptomyces Genetics. Norwich, UK, The John Innes Foundation.
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