1 Area de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, Julián Clavería s/n, 33006 Oviedo, Spain
2 Laboratorio de Proteómica, Centro Nacional de Biotecnología, Cantoblanco, 28049 Madrid, Spain
Correspondence
Jesús Sánchez
jsm{at}fq.uniovi.es
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ABSTRACT |
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INTRODUCTION |
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Bacterial death phenomena in communities appear to be active processes related to a multicellularity trait likewise subject to environmental factors and developmental processes (Rice & Bayles, 2003). Lytic phenomena associated with development were reported early on in Streptomyces coelicolor A3(2) by Wildermuth (1970)
, who described two central developmental fates for the Streptomyces mycelium: the surface layer, leading to spore formation, and the underlying, non-sporulating hyphae, leading to lysis. This phenomenon was subsequently interpreted by assuming that the lysis of the substrate mycelium could serve the purpose of providing nutrients for the developing aerial structures (Chater, 1984
; revised by Hodgson, 1992
). Analysis of the mobilization of radioactively labelled amino acids during the development of S. antibioticus ATCC 11891 on surface cultures supports the reutilization hypothesis (Braña et al., 1986
). The existence of an orderly process of internal cell dismantling was further addressed in S. antibioticus ATCC 11891 by electron microscopy analysis of the mycelium growing in confluent lawns (Miguelez et al., 1999
).
In this paper, we present a complete morphological analysis of the main developmental stages that accompany the growth of confluent surface cultures of S. antibioticus ATCC 11891. To obtain a reliable picture of the cell-death phenomenon, which constitutes the Streptomyces developmental hallmark, we used previously assayed viability staining with propidium iodide (PI) and SYTO 9 (Fernandez & Sanchez, 2001, 2002
). This technique has been widely used for determining membrane integrity in bacteria (Miller & Quarles, 1990
; Lloyd & Hayes, 1995
; Bunthof et al., 1999
, 2001
) and involves staining the nucleic acids of the damaged (leaky) cells with PI. The study was complemented by other fluorescence and electron microscopy analyses. A number of interesting features were uncovered by our work, such as the existence of very early cell death in a young compartmentalized mycelium. The availability of a reliable model for the development-associated death processes may now enable the genetic analysis of this interesting phenomenon, which is central to the biology of the bacterium.
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METHODS |
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Obtaining protoplasts from surface cultures.
Protoplasts were obtained from the S. antibioticus compartmentalized mycelium as described elsewhere for submerged cell cultures (Okanishi et al., 1974; Kieser et al., 2000
). Young mycelium grown on a cellophane disc was scraped off, resuspended in 10·3 % sucrose solution (about 0·6 g sucrose per 15 ml), and washed twice by centrifugation at 10 000 r.p.m. The pellet was resuspended in 4 ml buffer P plus lysozyme (2 mg ml1, Sigma) and incubated at 37 °C for about 30 min. The formation of protoplasts was monitored under a phase-contrast microscope. When appropriate, the protoplasts were visualized after staining under the confocal laser-scanning fluorescence microscope, as described below.
Viability staining.
Culture samples were obtained and processed for microscopy at different times of incubation, as described previously (Fernandez & Sanchez, 2001, 2002
). GAE plates were prepared with Difco agar, inoculated as described above and used to obtain solid blocks of the agar cultures with a scalpel. These were further trimmed to squares of about 10 mm in size and introduced into a hand microtome (11 mm hole diameter) previously cooled to 4 °C, with the growth surface oriented towards the side. Sections of about 0·3 mm were obtained. The limit of the agar and the limit of Streptomyces mycelium were defined at the lateral edge of the sample by fluorescence and/or phase-contrast observation. To improve visualization of the individual hyphae in some of the developmental phases, the stained samples were squashed in the slide by applying a gentle pressure on the coverslip. The mycelium becomes disorganized and loose, allowing the observation of specific details of the hyphae. The permeability assay described previously for Streptomyces was used to stain samples (Fernandez & Sanchez, 2002
). This involves staining the cells with a cell-impermeant nucleic acid stain (PI) in order to detect the dead cell population of S. antibioticus, and with SYTO 9 green fluorescent nucleic acid stain (LIVE/DEAD Bac-Light Bacterial Viability Kit, Molecular Probes, L-13152) to detect the viable cells. The SYTO 9 green fluorescent stain labels all the cells, i.e. both those with intact and those with damaged membranes. In contrast, PI only penetrates bacteria with altered membrane permeability and presents substantial fluorescence enhancement upon binding nucleic acids. This causes a reduction in the SYTO 9 stain fluorescence when both dyes are present. Thus, in the presence of both stains, bacteria with intact cell membranes appear fluorescent green, whereas membrane-compromised bacteria appear red (Haugland, 2002
). The stain mix was prepared as recommended by the manufacturer and was added directly over the samples on the slide. For the viability in liquid cultures assay, a drop of the above mixture was added to a drop of the liquid mycelial sample (Fernandez & Sanchez, 2001
). The coverslip was placed over the surface and submerged samples, staining taking place for at least 10 min in the dark. The samples were observed under a Leica TCS-SP2-AOBS confocal laser-scanning microscope at wavelengths of 488 nm and 568 nm excitation and 530 nm (green) or 630 nm (red) emission. Images were mixed using the Leica Confocal Software. In some cases, the samples were also observed in differential interference contrast mode, available with the same equipment.
Membrane staining.
Lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64) (Molecular Probes, T-3166), was added directly to the culture medium before pouring into the plates (Vida & Emr, 1995) at a final concentration of 1 µg ml1 that did not affect growth. At the appropriate times, culture samples were obtained and processed for microscopy, as described above for viability staining, and observed under the confocal laser-scanning microscope at wavelengths of 550 nm excitation and 700 nm emission.
Activity cell staining.
S. antibioticus was grown on the surface of cellophane discs and scraped off with a plain spatula at different times. The harvested mycelium was incubated at 30 °C in a carboxyfluorescein diacetate (CFDA) solution (Molecular Probes, C-1157) at a final concentration of 30 µM for 15 min, washed twice with distilled water and observed directly under the confocal laser-scanning microscope at wavelengths of 488 nm excitation and 530 nm emission. The samples were sometimes additionally stained with PI, after CFDA staining.
Analysis of cell membrane potential with rhodamine-123.
Rhodamine-123 is a vital stain that selectively accumulates in cells with an intact membrane potential (Haugland, 2002) and has been widely employed in both eukaryotic (Darzynkiewicz et al., 1992
) and prokaryotic (Lopez-Amoros et al., 1995
) cell studies. Mycelium harvested from cellophane surface cultures of S. antibioticus was incubated at 30 °C for 15 min in a solution of rhodamine-123 (0·2 mg ml1 final concentration, Sigma), washed twice with distilled water and observed under the confocal microscope at wavelengths of 514 nm excitation and 563 nm emission. Some samples were additionally tested for viability with PI and SYTO 9 staining. In this case, the rhodamine emission was recorded at 580 nm in order to prevent interference with the SYTO emission spectrum.
Cell wall staining.
Wheat germ agglutinin (WGA) conjugated with Texas red (Molecular Probes W-21405), which binds selectively to N-acetylglucosamine and N-acetylneuraminic acid, was extensively used to stain cell walls. Solid cultures of Streptomyces were scraped from cellophane with a plain spatula at various times. Harvested mycelium was processed as described elsewhere (Schwedock et al., 1997). Cells were fixed in 2·8 % paraformaldehyde, 0·0045 % glutaraldehyde in PBS (0·14 M NaCl, 2·6 mM KCl, 1·8 mM KH2PO4 and 10 mM Na2HPO4) for 15 min at room temperature, washed twice with PBS and incubated for 1 min in 2 mg lysozyme ml1 in glucose/Tris/EDTA (GTE: 50 mM glucose, 20 mM Tris/HCl, pH 8, 10 mM EDTA). The samples were washed again with PBS and blocked in 2 % BSA in PBS for 5 min. WGA was added at a concentration of 100 µg ml1 in 2 % BSA in PBS, and incubated at room temperature for 3 h. Finally, the samples were washed eight times with PBS and observed under the confocal microscope at wavelengths of 595 nm excitation and 615 nm emission.
Electron microscopy.
Samples of the cultures grown on the surface of cellophane discs were scraped off at different times of incubation with a plain spatula and incorporated in 3 % agar blocks. These were cut into small pieces and fixed at 4 °C in 2·5 % glutaraldehyde. After washing in 0·1 M phosphate buffer, pH 7·3, the cells were postfixed overnight at 4 °C in a solution of 1 % (w/v) osmium tetroxide in 50 mM phosphate buffer pH 7·3. Pieces were dehydrated through graded acetone solutions over a 2 h period at room temperature. In the 70 % dehydration step, 2 % (w/v) uranyl acetate was added and the samples were left for 1 h to contrast the cells. After completion of the dehydration treatment, the blocks were embedded in London Resin White (LR-White) and polymerized at 60 °C for 40 h. Ultrathin sections of silver-grey interference colour (thickness 6090 nm) were obtained for electron microscopy observations using a diamond knife and mounted on Formvar-coated copper grids. Samples were examined under a Philips EM300 electron microscope at an operating voltage of 60 kV and photographed with Scientia electron microscopy film (AGFA).
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RESULTS |
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Fig. 1(k) shows how the second mycelium grows massively until reaching the phase in which aerial mycelium is visible on the plate surface, i.e. at about 25 h. This coincides with a marked increase in the thickness of the mycelium layer [compare the scales of Fig. 1(i)
with those of Fig. 1(k)
]. Simultaneously, a second massive death round affecting this second mycelium takes place in the deepest zone of the section (Fig. 1k
). The growth of the remaining live mycelium continues and the hyphae start to segment their DNA in nucleoids (Fig. 1l
). The death waves of the mycelium gradually reach the upper zones of the section and, as a consequence, an important proportion of the DNA-segmented hyphae also appear dead at the culmination of this phase (Fig. 1l
). At these times (36 h), spores are not yet being formed.
Finally, the sporulation phase takes place at 4896 h. The spores form a thin layer on the surface of the medium and the mycelium layer presents its maximum thickness. However, as occurred in the first death round (Fig. 1i, j), no fluorescence is observed below the surface spore layer, conceivably due to the disintegration of the dead hyphae and the degradation of nucleic acids (Fig. 1m
, see below). The form of the hyphae cannot be observed under the phase-contrast microscope (Fig. 1n
), only a thick cellular mass that is opaque to light.
Details of the most relevant events occurring during the first 15 h are shown in Fig. 2. Fig. 2(a)
represents a young compartmentalized hypha with dead and live regions marked by a defined boundary. The majority of the live segments of the variegated mycelium show a drop in the intensity of their green fluorescence (Fig. 2a, b
), which is recovered in a later phase (Fig. 2d
f; see below). Fig. 2(b, c, d and f)
shows details of the completion of the first death round (1018 h), which at the end of the process uniformly affects this initial substrate mycelium. As shown also in Fig. 1(f)
, some spores have still not germinated in this time (Fig. 2b, c
). The mycelium appears fragmented due to the disintegration of the dead segments, which can be seen covered by a diffuse red layer likely formed by the intracellular material (Fig. 2e
). Some of the live segments of the variegated mycelium start to enlarge asynchronously (Fig. 2f
; visible also in Fig. 1g
) to form what we call a second mycelium, in order to distinguish it from the aforementioned first mycelium.
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DISCUSSION |
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The second death round described in this work is equivalent to the death process proposed to occur in non-sporulating aerial hyphae of S. coelicolor by Wildermuth (1970), as well as in S. antibioticus ETH7451 surface cultures, a strain which grows faster and also sporulates in submerged conditions (Novella et al., 1992
; Fernandez & Sanchez, 2002
). This is also likely to be the same death process reported by other authors in the strain used by us (Miguelez et al., 1999
). In contrast, the early first death round affecting the compartmentalized young mycelium and the evidence for the presence of such compartmentalization have not been previously described, probably due to their ephemeral existence. It is noteworthy that this compartmentalization was detected with different fluorescence indicators and by electron microscopy. The intrinsic properties of the SYTO 9/PI fluorescent colourants used (Haugland, 2002
) imply that the corresponding segments are separated by at least a membrane (see below). Intriguingly, the red-fluorescent compound FM 4-64, a membrane-specific dye previously used in Streptomyces (Grantcharova et al., 2003
), stained some zones of the S. antibioticus compartmentalized hyphae more intensely than others (Fig. 3a, f
), a phenomenon also reported in Escherichia coli (Fishov & Woldringh, 1999
). As in the latter bacterium, the intense red-fluorescent regions in Streptomyces may be explained by the presence of membrane domains with greater affinity for the dye (Fishov & Woldringh, 1999
). WGA staining and electronic microscopy analysis revealed that these compartments lack any cellular walls detectable by these approaches.
If the density of hyphae is high at a particular point on the plate, the consumption of an essential nutrient(s) will overcome the flow of these substances through the agar. This could motivate the establishment of local starvation conditions in the microenvironment close to the hypha, which may lead to stress responses (Kelemen et al., 2001). Gradients of oxygen and free radical concentration (Hahn et al., 2002
; van Keulen et al., 2003
) could have a similar effect. The metabolic activity of an isolated hypha or of a less concentrated group of hyphae would provoke a lesser disturbance of the surrounding medium. The compartmentalization of the mycelium and the survival capacity of some of the cellular segments jointly allow the bacteria to face up to these critical situations and would constitute a good adaptive response in their natural soil habitat. The phenomenon evokes that of persistence within bacterial populations, in which the suicide programme is disabled in a fraction of cells when confronted with potentially lethal damage (Lewis, 2000
). As yet, we do not know the mechanisms underlying the outstanding capacity of the first mycelium to partially inactivate a fraction of cells. An interesting possibility is the presence in Streptomyces of a kind of determining mechanism, such as that reported in Bacillus subtilis, in which the cells that have entered the pathway to sporulate produce and export a killing factor that causes sister cells to lyse (Gonzalez-Pastor et al., 2003
). Nutrient replenishment/depletion effects in the agar medium may analogously motivate the second death round. In this case, however, death affects the much longer segments of the unseptated hyphae that form the aerial mycelium.
All studies to date describing the differentiation and developmental cycle of Streptomyces describe a fully viable substrate mycelium grown in culture medium, from which a reproductive (aerial) mycelium emerges (Wildermuth, 1970; Kalakoutskii & Agre, 1976
; Mendez et al., 1985
; Chater, 1989
; Hodgson, 1992
). There is also consensus that the substrate mycelium and pre-sporulating aerial mycelium are syncytia with sporadic septa (Hodgson, 1992
; Chater, 1993
; Chater & Losick, 1997
). The substrate mycelium in S. antibioticus ATCC 11891 would be the mycelium formed from spores and developed until around 35 h cultivation at 30 °C. The formation of the aerial mycelium would commence from this point on, and at around 60 h cultivation the sporulation process would begin (Mendez et al., 1985
; Miguelez et al., 1999
). In our work, it seems clear that a compartmentalized mycelium is formed after the germination of spores. This mycelium should not be considered as substrate mycelium or aerial mycelium, as it is non-syncytial in nature. The viable mycelium developed from viable segments of the so-called variegated hyphae is already a syncytium (not shown), and in consequence it would correspond to the substrate mycelium described by other authors, lasting until about 35 h, whereas at later times it would correspond to the aerial mycelium. Thus, the former designations substrate and aerial are not strictly applicable to the variegated mycelium or to the second mycelium described in our work.
The features described in this study are not a striking peculiarity of the species and strain used, as they have been detected in all Streptomyces analysed so far, including the genetically well-characterized species S. coelicolor (A. Manteca and J. Sánchez, unpublished results). It has been proposed that micro-organisms, and especially those that present a complex developmental life, such as Streptomyces, Bacillus, Anabaena, Caulobacter, Rhizobium and the myxobacteria, have programmed cell-death mechanisms and/or genes which could be considered to be the phylogenetic precursors of eukaryotic programmed cell death (Yarmolinsky, 1995; Hochman, 1997
; Aravind et al., 1999
; Koonin & Aravind, 2002
; this study). Kinase domains, similar to those from eukaryotic signal domains, are present in the gene products of the Streptomyces genome (Zhang, 1996
; Aravind et al., 1999
; Elizarov & Danilenko, 2001
; Petrickova & Petricek, 2003
), some of which are membrane associated and calcium dependent (Elizarov & Danilenko, 2001
). Moreover, Streptomyces harbours ATPases of the apoptotic type (Aravind et al., 1999
; Koonin & Aravind, 2002
), which are typical components of eukaryotic signalling systems and, in particular, of the apoptotic system. The study of the function of these hypothetical signals and effectors in the Streptomyces cell death processes described here will certainly constitute an important field of investigation in the future.
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ACKNOWLEDGEMENTS |
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Received 17 March 2005;
revised 20 June 2005;
accepted 26 July 2005.
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