BIOTECHNOLOGY TECHNIQUES Volume 10 No.2 (February 1996) pp.7932 Received as revised 1st December.

INFLUENCE OF MEDIUM COMPOSITION ON SPORULATION BY STREPTOMYCES COELICOLOR A3(2) GROWN ON DEFINED SOLID MEDIA. Atul Karandikar *, George P. Sharples and Glyn Hobbs. Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF.

Summary. Reproducible sporulation using solid-plate cultures of Streptomyces coelicolor A3(2) was obtained on a defined medium with glucose as the primary source of carbon and sodium nitrate as the sole source of nitrogen. The type of agar usedas a solidifying agent, and the inclusion of trace salts to the medium, significantly affected spomlation. An interrelationship between the medium carbon to nitrogen ratio and sporulation was observed, with clear promotion of spore formation under nitrogen-limited conditions, whilst carbon-limited conditions suppressed spomlation. The influence of medium phosphate upon sporulation is also reported, with high concentrationsexerting an inhibitory effect.

Introduction. The isolation and subsequentmanipulation of streptomycetesoften dependsupon inducing the organisms concernedto form spores. The process of sporulation in this group of bacteria has been studied largely in Streptomycescoelicolor and efforts have chiefly been focused at the ultrastructural and genetic levels (Hodgson, 1992). In contrast, little is known of the cultural conditions that promote the formation of spores. The present study reports upon aspectsof the culture medium that affect sporulation in S.coelicolor. Furthermore, a method usedto optimise the carbon:nitrogen ratio of the medium to achieve high spore productivity is described.

Materials and Methods. Organism. Streptomycescoelicolor A3(2) strain 1147 (Hopwood, 1959) was used throughout this study. Media and growth conditions. The media used were based upon that of Hopwood (1960), which were supplemented with OSml of trace salts (Hobbs et al., 1989). In all cases, the primary carbon source was sterilised separatelyand the pH of the media adjusted to 7.2. The minimal media were solidified by the addition of 1.5% (w/v) Agar No.3 (Oxoid) unless otherwise stated. The surfaces of agar plates were overlaid with cellophane membrane discs (325P, Courtaulds Cannings, Bristol, UK) prior to inoculation and incubation as previously describedby Shahabet al. (1994). Extraction of solutes from agar and estimation of dry weights and low molecular weight compounds. In all cases, procedureswere followed according to Shahab.et al ( 1994). Qualitative and quantitative estimation of sporulation. Impression mounts were preparedfrom the centre of the plates as described by Chater (1972). Glass coverslips (1.5 x 1.5cm) were washedsequentially in chloroform, alcohol, water, alcohol, and chloroform in a fume cupboard

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and then left to dry in a petri dish. Preparationswere mounted face down onto a drop of TritonXl00 (25ml) on a glass slide and viewed by phase-contrastmicroscopy. Sporulation was assessedby determining the percentageof random fields of view, that contained phase-dark spores,when viewed at 400x magnification.

Results. Preliminary medium studies. Several preliminary experiments were conducted to establish a suitable medium formulation to promote sport&ion in Scoelicolor. The defined medium describedby Hobbs et al (1989) resulted in poor sporulation and an alternative medium based

upon Hopwood (1960) was adopted. In an attempt to develop an understanding of those componentsof the medium that affected sport&ion we modified the original formulation. An initial examination of the solidifying agents revealed clear variability in the extent of sporulation when different commercial agars were used in the medium. Spore formation was achieved on Agar No.3 (Oxoid), Ionagar No.2 (Oxoid), Bactoagar (Difco) and Plant Extract Agar (Lab M). However, Agar No. 1 (Oxoid) did not support sporulation yet marked pigment production did occur, a phenomenon not observed with the other agars. In addition to the influence of agar type on sporulation, the inclusion of trace salts also significantly affected sporeformation. The omission of trace salts in the original medium did not support production of spores. However, addition of trace salts (Hobbs et al., 1989) resulted in prolific sporulation. The effect of the carbon and nitrogen source on sporulation. A limited number of carbon and nitrogen sources were studied for their ability to support sporulation. In all cases, the concentration of carbon source was 222mM and the concentration of nitrogen source was 30mM with respectto nitrogen. Of those nitrogen sourcestested, sodium nitrate and aspamgine promoted sporulation while ammonium chloride, ammonium nitrate and alanine grown cultures showed no evidence of spore formation, even after three weeks incubation. The nature of the carbon sourcesthat were examined had little effect upon sporulation when incorporated in a medium containing a permissive nitrogen source (sodium nitrate) and a suitable agar (Agar No.~), glucose, glycerol, xylose, fructose,galactoseand lactoseall supportedspore formation.

On the basis of these results, the minimal medium of Hopwood (1960) supplementedwith trace salts (Hobbs et al., 1989) and containing sodium nitrate as the sole source of nitrogen (30mM), glucose as the primary carbon source (222mM) and agar No.3 (1.5% w/v) as the solidifying agent, was adopted. The e#kct of the medium carbonrnitrogen (C:N) ratio upon sporulation. The C:N ratio of a growth medium has long been known to influence the extent of sporulation in certain fungi (Madelin, 1956). It was therefore of interest to examine this relationship in the bacterium S.coelicolor.

0

50

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150

C/N Ratio

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Fig 1. The effect of C:N ratio sporulation. In all cases, glucose was constant at 280mM.

The relationship between C:N ratio (molar ratio) and sporulation (Fig 1) illustrates that spore formation was restricted to C:N ratios between the range of 40-100, whilst lower C:N ratios (~40) did not permit spore formation. It was also evident that sporulation was reduced at C:N ratios above 90. The determination of conditions supporting carbon or nitrogen limited growth. Possible inferences from the aforementioned C:N ratio data were that sporulation may be triggered in response to a specific nutrient limitation. By varying the concentration of one nutrient whilst maintaining the other constant, it was possible to determine the C:N ratios that resulted in carbon-limited and nitrogen-limited growth (Fig 2a and Fig 2b). 200

200

l ‘i 0

i p)

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H $ r

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H z B

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Z .cn :

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EoEoo,0

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Fig 2a. The effect of increasing gtucose levels upon maximum biomass production by plate cultures. In all cases, nitrate was constant at 50mM.

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Fig 2b. The effect of increasing nitrate levels upon maximum biomass production by plate cultures. In all cases, glucose was constant at 140mM.

Carbon-limited growth was achieved at C:N ratios below 20. Furthermore, these levels were non-permissive for sporulation and therefore, suggest that carbon limitation is not the trigger for sporulation. Nitrogen limitation was clearly defined at C:N ratios above 42. These ratios were permissive for spore formation suggesting that nitrogen limitation is a prerequisite for sporulation.

The effect of phosphate concentration on sporulation. Phosphorus has been reported to have a profound effect upon production of secondary metabolites in this organism (Doull & Vining, 1990; Hobbs et al., 1991, 1992). Thus, it was decided to investigate the role of this essential nutrient upon sporulation. A series of media containing different concentrations of phosphate were compared for their ability to support sporulation. Sporulation was most abundant at 5.7mM of phosphate. At high concentrations of phosphate (>57mM), sporulation was clearly inhibited whilst lower phosphate concentrations (<0.29mM) led to both poor growth and spore production. It was noted that blue pigment was exclusively produced at concentrations of phosphate between 0.57mM and 1.44mM. This suggests that sporulation and pigment production are both sensitive to the concentration of phosphate in the medium although the threshold of sensitivity appears to be different.

Discussion. Clearly sporulation is profoundly affected by both quality and quantity of nutrients in the growth medium. Our study suggests that the interrelationship between the carbon and the nitrogen concentrations prevailing in the medium influence the ability of the organism to sporulate. Furthermore, there is the suggestion that nitrogen-limitation is a prerequisite for

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stimulation of spornlation in this organism. This contradicts recent reports concerning S. by Lee and Rho (1993), that nitrogen-limitation is not responsible for stimulation of spore formation. It was also observedthat significant spore formation could only be realised provided that a sufficient supply of exogenous carbon sources was available to complete the process. Excess exogenousglucose however produced an inhibitory effect upon spomlation, possibly as a result of catabolite repressionupon the utilisation of other carbon sourcesrequired for sport&ion, such as glycogen (Hodgson and Chater, 1981; Dawes, 1992). aIbidoj7uvus

Results indicated that high levels of phosphate inhibited both spomlation and production of blue pigment. The influence of phosphateupon control of secondarymetabolism has long been established (Martin, 1977). In this organism, high levels of phosphate have previously been reported to inhibit secondary metabolism in submerged cultures with optimal production of secondarymetabolites favoured at lower concentrations(Doull and Vining, 1990; Hobbs et al., 1991, 1992). This relationship is analogous to the effect of phosphateupon sport&ion by solid plate cultures. Futhermore, the depletion of phosphate in submergedcultures has been reported to stimulate formation of spores in several species of streptomycetes(Daza et al., 1989).

References. Chater, K. F. (1972). J.Gen. Microbial., 72,9-28. Dawes, E. A. (1992). Sot. Gen. Microbial. Symp. 47, 81-122. Daza, A et al. (1989). J. Gen. Microbial., 135,2483-2491. Doull, J. L. and Vining, L. C. (1991). Appl. Microbial. Biotech., 32,449454. Hobbs, G et al. (1989). Appl. Microbial. Biotechnol., 31,272-277. Hobbs, G. et al.. (1991). J. Gen. Microbial. 136,2291-2296. Hobbs, G. et al. (1992). J. Bacterial., 174, 1487-1494. Hodgson, D. A. (1992). Sot. Gen. Microbial. Symp. 47,407440. Hodgson, D. A. and Chater, K. F. (1981). J. Gen. Microbial., 124, 339-348. Hopwood, D. A. (1959). Ann. N. Y. Acad. Sci., 81, 887-898. Hopwood, D. A. (1960). J. Gen. Microbial., 22, 295-302. Lee, K. Y. and Rho, Y. T. (1993). J. Gen. Microbial., 139, 3 131-3137. Madelin, M. F. (1956). Arm. Bot, 20, 307-330. Martin, J. F. (1977). Adv. Biochem. Eng., 6, 105-127. Shahab,N et al. (1994). Biotech. Letts., 16, 1015-1020.

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