Journal of Bacteriology, February 2003, p . 1455-1458, Vol . 185, No . 4

Differentiation and Anaerobiosis in Standing Liquid Cultures of Streptomyces coelicolor

Geertje van Keulen,¹ Henk M . Jonkers,² Dennis Claessen,¹ Lubbert Dijkhuizen,^1* and Han A . B . Wösten^1,

Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands,¹ Microsensor Research Group, Max Planck Institute for Marine Microbiology, Bremen, Germany²

Received 19 July 2002/ Accepted 15 October 2002

S . coelicolor mutants have been isolated that are impaired in formation of aerial hyphae (bld mutants) or in formation of spores (whi mutants) . bld mutants grown on complex medium are affected in the production of the surface-active peptide SapB (16) and thus cannot escape into the air by their inability to lower the water surface tension (15) . whi mutants are not impaired in SapB production . The aerial hyphae of these mutants, however, are affected in septation, cell wall thickening, and/or spore pigmentation . The phenotypes of a number of bld mutants are known to be suppressed by growth on minimal medium supplemented with mannitol, arabinose, or other sugars (8, 16) .

In contrast to agar media, differentiation does not occur in liquid shaken cultures . So far, it has not been reported whether formation of aerial hyphae and spores occurs in standing liquid cultures of S . coelicolor . To investigate this, S . coelicolor M145 (9) was grown at 30°C in polystyrene microtiter plates (Costar 3370; Corning Inc.) or in petri dishes . Minimal NMMP medium (9) was used in the absence of polyethylene glycol 6000 and supplemented with 25 mM glucose and 0.25% Casamino Acids (gNMMP) or 25 mM mannitol (mNMMP) as the carbon source . Alternatively, S . coelicolor was grown in the complete medium tryptic soy broth, R2YE, or YEME (9) . Spores or mycelium from a liquid shaken culture were used as inoculum . A submerged mycelium was produced in all media . In contrast, aerial hyphae were only formed in minimal medium (gNMMP and mNMMP) . After 3 days of submerged growth in these media, colonies formed at the water-air interface from which aerial hyphae developed (Fig . 1A) . These colonies were fixed at this interface by a rigid light-reflecting film that surrounded the colony . Scanning electron microscopy revealed that the film had no clear ultrastructure and was therefore not an extension of the rodlet layer that coats the aerial hyphae (Fig . 1F and G) . The rigid film was rich in protein but did not contain SapB or rodlins as determined by immunodetections with antibodies raised against these polypeptides (data not shown) . The film could function as a fungal hydrophobin membrane by lowering the water surface tension (18) . Colonies grown on gNMMP formed few aerial hyphae often restricted to radial zones or rings (Fig . 1B) . In contrast, colonies grown on mNMMP formed a confluent layer of aerial hyphae (Fig . 1C) . The aerial hyphae formed on both media metamorphosed into grey-pigmented spores (Fig . 1DE) . Viability of these spores was similar to that of spores isolated from agar cultures before and after freeze drying (results not shown) . bld and whi mutants (bldJ261, bldD53 [17], bldH109 [3], bldA39 [12], whiA [J2401], whiB [J2402], whiG [J2400] [7], whiH [J2403 [K . Flärdh, John Innes Center, Norwich, United Kingdom], whiI [J2450] [1], whiJ [J2452] [J . A . Aínsa, John Innes Center]) did not form floating air-interface colonies on gNMMP and mNMMP, and consequently aerial hyphae could not be formed . However, submerged growth was unaffected, as was shown by total protein determinations using the DC protein determination kit (Bio-Rad) .

 
The standing cultures studied may resemble flooded soil, a condition S . coelicolor may escape from by forming floating sporulating colonies . How the hyphae move from the lower zone in the liquid to the air interface to form a sporulating colony is intriguing since S . coelicolor has no means of motility by, e.g., flagella . Within the genome sequence of S . coelicolor two gene clusters, SCO0649-SCO0658 and SCO6499-SCO6508, are present with homology to gas vesicle gene clusters from halophilic archaea and cyanobacteria (2) . It is therefore not unlikely that S . coelicolor forms gas vesicles to become buoyant to reach the air interface .

In minimal medium, not only were aerial hyphae formed but submerged hyphae also attached to the hydrophobic surface of the microtiter plates . In contrast, hyphae did not adhere in the nutrient-rich media YEME, tryptic soy broth, and R2YE . Attachment of hyphae grown in gNMMP and mNMMP was quantified using a staining assay with crystal violet, which was measured at an optical density at 590 nm (11, 13) . The number of attaching hyphae increased with culture age (Fig . 2) and correlated in a linear way with the increase in total biomass as measured by total protein determination (data not shown) . Cultures grown on glucose formed more biomass than those grown on mannitol, and more hyphae attached . The stationary phase was reached after 7 and 10 days of growth in gNMMP and mNMMP, respectively, after which the number of attaching hyphae no longer increased . Recently, it was shown that hyphae in contact with air or a hydrophobic solid produce rodlins . These proteins are involved in formation of the rodlet layer of aerial hyphae and mediate attachment to a hydrophobic surface (4) . The latter may be instrumental in the establishment of symbiotic or pathogenic interactions . For instance, streptomycetes have been shown to associate in a beneficial way to the cuticle of leaf-cutting ants, which grow fungi as their main source of food (5, 6) .

 
Growing standing liquid cultures are expected to develop oxygen gradients . Indeed, using a sensitive Clark-type oxygen microsensor (10) it was shown that 7-day-old YEME and gNMMP cultures were anoxic at a depth of 0.7 and 2 mm, respectively (Fig . 3) . Yet, biomass still increased as measured by total protein determinations (data not shown), indicating an active metabolism . Standing mNMMP cultures also developed oxygen gradients, but they were less steep and cultures did not become anoxic (Fig . 3) . Apparently, diffusion matches the requirement for oxygen, as can be explained by the reduced growth rate in this medium (Fig . 2) . Because steep oxygen gradients are present in soil (14), S . coelicolor should be able to overcome or even grow under conditions with no or low amounts of oxygen . The genome sequence of S . coelicolor revealed gene clusters, e.g., genes SCO0216-SCO0219, SCO4947-SCO4950, and SCO6532-SCO6535, that putatively encode a typical four-subunit respiratory nitrate reductase (2) . This protein is involved in anaerobic metabolism, indicating that S . coelicolor possesses enzymes to accommodate metabolism under anoxic or microaerobic conditions . Because S . coelicolor did not form colonies at the air interface in anoxic nutrient-rich standing cultures, oxygen availability may not be the signal for their formation .

 
In conclusion, standing cultures of S . coelicolor are an attractive model system to study a wide range of phenomena not studied thus far, such as attachment of streptomycetes to solids, differentiation at a liquid interface, and anaerobic metabolism .

G . van Keulen and D . Claessen are supported by a grant of the Dutch Program EET (Economy, Ecology and Technology), a joint initiative of the Ministries of Economic Affairs, Education, Culture, and Sciences and of Housing, Spatial Planning, and the Environment (EETK98020) .

Present address: Department of Microbiology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands .

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