APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2009, p. 2920–2924 Vol. 75, No. 90099-2240/09/$08.00�0 doi:10.1128/AEM.02288-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Streptomyces Development in Colonies and Soils�

Angel Manteca1,2* and Jesus Sanchez1

Area de Microbiologia, Departamento de Biologia Funcional e IUBA, Facultad de Medicina, Universidad de Oviedo,33006 Oviedo, Spain,1 and Department of Biochemistry and Molecular Biology, University of Southern Denmark,

Campusvej 55, Odense M, DK-5230 Odense, Denmark2

Received 6 October 2008/Accepted 20 February 2009

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth ratewas much lower than that in standard laboratory cultures, and the life span of the previously named firstcompartmentalized mycelium was remarkably increased.

Streptomycetes are gram-positive, mycelium-forming, soilbacteria that play an important role in mineralization pro-cesses in nature and are abundant producers of secondarymetabolites. Since the discovery of the ability of these micro-organisms to produce clinically useful antibiotics (2, 15), theyhave received tremendous scientific attention (12). Further-more, its remarkably complex developmental features makeStreptomyces an interesting subject to study. Our researchgroup has extended our knowledge about the developmentalcycle of streptomycetes, describing new aspects, such as theexistence of young, fully compartmentalized mycelia (5–7).Laboratory culture conditions (dense inocula, rich culture me-dia, and relatively elevated temperatures [28 to 30°C]) result inhigh growth rates and an orderly-death process affecting thesemycelia (first death round), which is observed at early timepoints (5, 7).

In this work, we analyzed Streptomyces development underconditions resembling those found in nature. Single coloniesand soil cultures of Streptomyces antibioticus ATCC 11891 andStreptomyces coelicolor M145 were used for this analysis. Forsingle-colony studies, suitable dilutions of spores of these spe-cies were prepared before inoculation of plates containingGYM medium (glucose, yeast extract, malt extract) (11) orGAE medium (glucose, asparagine, yeast extract) (10). Ap-proximately 20 colonies per plate were obtained. Soil cultureswere grown in petri dishes with autoclaved oak forest soil (11.5g per plate). Plates were inoculated directly with 5 ml of aspore suspension (1.5 � 107 viable spores ml�1; two indepen-dent cultures for each species). Coverslips were inserted intothe soil at an angle, and the plates were incubated at 30°C. Tomaintain a humid environment and facilitate spore germina-tion, the cultures were irrigated with 3 ml of sterile liquid GAEmedium each week.

The development of S. coelicolor M145 single colonies grow-ing on GYM medium is shown in Fig. 1. Samples were col-lected and examined by confocal microscopy after differentincubation times, as previously described (5, 6). After sporegermination, a viable mycelium develops, forming clumps

which progressively extend along the horizontal (Fig. 1a and b)and vertical (Fig. 1c and d) axes of a plate. This mycelium isfully compartmentalized and corresponds to the first compart-mentalized hyphae previously described for confluent surfacecultures (Fig. 1e, f, and j) (see below) (5); 36 h later, deathoccurs, affecting the compartmentalized hyphae (Fig. 1e and f)in the center of the colony (Fig. 1g) and in the mycelial layersbelow the mycelial surface (Fig. 1d and k). This death causesthe characteristic appearance of the variegated first mycelium,in which alternating live and dead segments are observed (Fig.1f and j) (5). The live segments show a decrease in fluores-cence, like the decrease in fluorescence that occurs in solidconfluent cultures (Fig. 1 h and i) (5, 9). As the cycle proceeds,the intensity of the fluorescence in these segments returns, andthe segments begin to enlarge asynchronously to form a new,multinucleated mycelium, consisting of islands or sectors onthe colony surfaces (Fig. 1m to o). Finally, death of the deeperlayers of the colony (Fig. 1q) and sporulation (Fig. 1r) takeplace. Interestingly, some of the spores formed germinate (Fig.1s), giving rise to a new round of mycelial growth, cell death,and sporulation. This process is repeated several times, andtypical, morphologically heterogeneous Streptomyces coloniesgrow (not shown). The same process was observed for S.antibioticus ATCC 11891, with minor differences mainly in thedevelopmental time (not shown).

Figure 2 shows the different types of mycelia present in S.coelicolor cultures under the conditions described above, de-pending on the compartmentalization status. Hyphae weretreated with different fluorescent stains (SYTO 9 plus pro-pidium iodide for nucleic acids, CellMask plus FM4-64 for cellmembranes, and wheat germ agglutinin [WGA] for cell walls).Samples were processed as previously described (5). Theyoung initial mycelia are fully compartmentalized and havemembranous septa (Fig. 2b to c) with little associated cell wallmaterial that is barely visible with WGA (Fig. 2d). In contrast,the second mycelium is a multinucleated structure with fewermembrane-cell wall septa (Fig. 2e to h). At the end of thedevelopmental cycle, multinucleated hyphae begin to undergothe segmentation which precedes the formation of spore chains(Fig. 2i to m). Similar results were obtained for S. antibioticus(not shown), but there were some differences in the numbers ofspores formed. Samples of young and late mycelia were freeze-substituted using the methodology described by Porta andLopez-Iglesias (13) and were examined with a transmission

* Corresponding author. Mailing address: Area de Microbiologia,Departamento de Biologia Funcional e IUBA, Facultad de Medicina,Universidad de Oviedo, 33006 Oviedo, Spain. Phone: (34) 985103555.Fax: (34) 985103148. E-mail: mantecaangel@uniovi.es.

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electron microscope (Fig. 2n and o). The septal structure ofthe first mycelium (Fig. 2n) lacks the complexity of the septalstructure in the second mycelium, in which a membrane with athick cell wall is clearly visible (Fig. 2o). These data coincidewith those previously described for solid confluent cultures (4).

The main features of S. coelicolor growing in soils are shownin Fig. 3. Under these conditions, spore germination is a very

slow, nonsynchronous process that commences at about 7 days(Fig. 3c and d) and lasts for at least 21 days (Fig. 3i to l),peaking at around 14 days (Fig. 3e to h). Mycelium does notclump to form dense pellets, as it does in colonies; instead, itremains in the first-compartmentalized-mycelium phase duringthe time analyzed. Like the membrane septa in single colonies,the membrane septa of the hyphae are stained with FM4-64

FIG. 1. Confocal laser scanning fluorescence microscopy analysis of the development-related cell death of S. coelicolor M145 in surface culturescontaining single colonies. Developmental culture times (in hours) are indicated. The images in panels l and n were obtained in differentialinterference contrast mode and correspond to the same fields as in panels k and m, respectively. The others are culture sections stained with SYTO9 and propidium iodide. Panels c, d, k, l, p, and q are cross sections; the other images are longitudinal sections (see the methods). Panels h andi are images of the same field taken with different laser intensities, showing low-fluorescence viable hyphae in the center of the colonies that developinto a multinucleated mycelium. The arrows in panels e and s indicate septa (e) and germinated spores (s). See the text for details.

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(Fig. 3j and k), although only some of them are associated withthick cell walls (WGA staining) (Fig. 3l). Similar results wereobtained for S. antibioticus cultures (not shown).

In previous work (8), we have shown that the myceliumcurrently called the substrate mycelium corresponds to theearly second multinucleated mycelium, according to our no-menclature, which still lacks the hydrophobic layers character-istic of the aerial mycelium. The aerial mycelium thereforecorresponds to the late second mycelium which has acquiredhydrophobic covers. This multinucleated mycelium as a wholeshould be considered the reproductive structure, since it isdestined to sporulate (Fig. 4) (8). The time course of lysine6-aminotransferase activity during cephamycin C biosynthesishas been analyzed by other workers using isolated colonies ofStreptomyces clavuligerus and confocal microscopy with greenfluorescent protein as a reporter (4). A complex medium anda temperature of 29°C were used, conditions which can beconsidered similar to the conditions used in our work. In-

terestingly, expression did not occur during the develop-ment of the early mycelium and was observed in the myce-lium only after 80 h of growth. This suggests that the secondmycelium is the antibiotic-producing mycelium, a hypothesispreviously confirmed using submerged-growth cultures of S.coelicolor (9).

The significance of the first compartmentalized myceliumhas been obscured by its short life span under typical labora-tory culture conditions (5, 6, 8). In previous work (3, 7), wepostulated that this structure is the vegetative phase of thebacterium, an hypothesis that has been recently corroboratedby proteomic analysis (data not shown). Death in confluentcultures begins shortly after germination (4 h) and continuesasynchronously for 15 h. The second multinucleated myceliumemerges after this early programmed cell death and is thepredominant structure under these conditions. In contrast, asour results here show, the first mycelium lives for a long timein isolated colonies and soil cultures. As suggested in our

FIG. 2. Analysis of S. coelicolor hyphal compartmentalization with several fluorescent indicators (single colonies). Developmental culture times(in hours) are indicated. (a, e, and i) Mycelium stained with SYTO 9 and propidium iodide (viability). (b, f, and j) Hyphae stained with Cell Mask(a membrane stain). (c, g, and l) Hyphae stained with FM 4-64 (a membrane stain). (d, h, and m) Hyphae stained with WGA (cell wall stain). Septain all the images in panels a to j, l, and m are indicated by arrows. (k) Image of the same field as panel j obtained in differential interference contrastmode. (n and o) Transmission electron micrographs of S. coelicolor hyphae at different developmental phases. The first-mycelium septa (n) arecomprised of two membranes separated by a thin cell wall; in contrast, second-mycelium septa have thick cell walls (o). See the text for details.IP, propidium iodide.

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previous work (5, 6, 8), if we assume that the compartmental-ized mycelium is the Streptomyces vegetative growth phase,then this phase is the predominant phase in individual colonies(where it remains for at least 36 h), soils (21 days), and sub-merged cultures (around 20 h) (9). The differences in the lifespan of the vegetative phase could be attributable to the ex-tremely high cell densities attained under ordinary laboratoryculture conditions, which provoke massive differentiation andsporulation (5–7, 8).

But just exactly what are “natural conditions”? Some au-thors have developed soil cultures of Streptomyces to studysurvival (16, 17), genetic transfer (14, 17–19), phage-bacteriuminteractions (3), and antibiotic production (1). Most of thesestudies were carried out using amended soils (supplementedwith chitin and starch), conditions under which growth andsporulation were observed during the first few days (1, 17).

These conditions, in fact, might resemble environments thatare particularly rich in organic matter where Streptomycescould conceivably develop. However, natural growth condi-tions imply discontinuous growth and limited colony develop-ment (20, 21). To mimic such conditions, we chose relativelypoor but more balanced carbon-nitrogen soil cultures (GAEmedium-amended soil) and less dense spore inocula, condi-tions that allow longer mycelium growth times. Other condi-tions assayed, such as those obtained by irrigating the soil withwater alone, did not result in spore germination and mycelialgrowth (not shown). We were unable to detect death, thesecond multinucleated mycelium described above, or sporula-tion, even after 1 month of incubation at 30°C. It is clear thatin nature, cell death and sporulation must take place at the endof the long vegetative phase (1, 17) when the imbalance ofnutrients results in bacterial differentiation.

FIG. 3. Confocal laser scanning fluorescence microscopy analysis of the development-related cell death and hyphal compartmentalization of S.coelicolor M145 growing in soil. Developmental culture times (in days) are indicated. The images in panels b, f, and h were obtained in differentialinterference contrast mode and correspond to the same fields as the images in panels a, e, and g, respectively. The dark zone in panel h correspondsto a particle of soil containing hyphae. (a, c, d, e, g, i, j, and k) Hyphae stained with SYTO 9, propidium iodide (viability stain), and FM4-64(membrane stain) simultaneously. (i) SYTO 9 and propidium iodide staining. (j) FM4-64 staining. The image in panel k is an overlay of the imagesin panels i and j and illustrates that first-mycelium membranous septa are not always apparent when they are stained with nucleic acid stains (SYTO9 and propidium iodide). (l) Hyphae stained with WGA (cell wall stain), showing the few septa with thick cell walls present in the cells. Septa areindicated by arrows. IP, propidium iodide.

FIG. 4. Cell cycle features of Streptomyces growing under natural conditions. Mycelial structures (MI, first mycelium; MII, second mycelium)and cell death are indicated. The postulated vegetative and reproductive phases are also indicated (see text).

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In summary, the developmental kinetics of Streptomyces un-der conditions resembling conditions in nature differs substan-tially from the developmental kinetics observed in ordinarylaboratory cultures, a fact that should be born in mind whenthe significance of development-associated phenomena isanalyzed.

This research was funded by a grant from the DGI, MEC Subdirec-cion General de Proyectos de Investigacion, Spain (grant BIO2007-66313). A.M. was supported by a postdoctoral grant from the Minis-terio Ciencia e Innovacion, Spain.

We thank Priscilla A. Chase for revising the text.

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