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© The Rockefeller University Press,
0021-9525/1999//515 $5.00
The Journal of Cell Biology, Volume 145, Number 3,
, 1999 515-525
Regular Articles |
Hyphal Death during Colony Development in Streptomyces antibioticus: Morphological Evidence for the Existence of a Process of Cell Deletion in a Multicellular Prokaryote
During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.
Key Words: Streptomyces antibioticus • programmed cell death • colony development • hyphal death • multicellular development
Abbreviations used in this paper: PCD, programmed cell death.
Address correspondence to Manuel B. Manzanal, Laboratorio de Microbiología, Facultad de Medicina, Universidad de Oviedo, Julián Clavería s/n, 33006 Oviedo, Spain. Tel.: 34-985103559. Fax: 34-985103534.
CELL death has long been known to be a fundamental feature of animal development, but only very recently has it become a fashionable subject of general biological interest. This is because programmed cell death (PCD,1 an active physiological process of cell deletion) has been recognized as playing a relevant role in the turnover of self-renewing tissues, morphogenesis, embryonic development (Ellis et al., 1991; Cohen et al., 1992; Wyllie, 1992; Vaux et al., 1994; Sanders and Wride, 1995; Raff, 1996; Jacobson et al., 1997), and more recently in neurodegenerative diseases and cancer progression (Williams, 1991; Sen and D'Incalci, 1992; Ameisen, 1994; Barr and Tomei, 1994; Kerr et al., 1994; Cohen et al., 1996; Kusiak et al., 1996). Most studies on cell death have been carried out in animal cells. By contrast, little or no attention has been given to the process of cell death in prokaryotes, although they have been widely used as models for the study of many other basic cellular processes.
Members of the genus Streptomyces (mycelial gram-positive soil bacteria) possess two characteristics which make them attractive microorganisms for the study of cell death at the prokaryote level: first, unlike Escherichia coli (the most studied and best known single-cell prokaryote), which continuously divides by binary fission into two functionally and structurally identical daughter cells (which, therefore, are potentially immortal), the streptomycetes grow through the formation of long, multinucleoid hyphae that, with time, undergo senescence and die; second, they execute a complex developmental cycle that represents one of the probably several evolutionary attempts at multicellularity (Champness, 1988; Shapiro, 1988; Chater, 1989a; Chater and Losick, 1997). In fact, colonies of streptomycetes are now viewed as multicellular organisms containing morphologically and biochemically differentiated populations of hyphae organized into separate somatic and germ cell lineages (Chater, 1993; Champness and Chater, 1994; Bruton et al., 1995), the development of which is governed by an intricate system of intercellular communication (Horinouchi and Beppu, 1992; Kaiser and Losick, 1993; Willey et al., 1993). The colony growth cycle of the streptomycetes is initiated when a spore germinates, giving rise to one or more long multinucleoid filaments. These filaments elongate and branch repeatedly, originating a vegetative mycelium (substrate mycelium) that develops over, and into, the culture medium. As the colony ages, specialized branches emerge from the substrate mycelium and grow away from the surface of the colony, originating the reproductive aerial mycelium. Then, the aerial hyphae septate into chains of uninucleoid compartments, which finally metamorphose into thick-walled spores (McVittie, 1974; Hardisson and Manzanal, 1976; Hodgson, 1992; Chater, 1998). Along this cycle, large numbers of hyphae (including the original substrate hyphae and any portion of the aerial mycelium which does not differentiate into spores) degenerate and die. Since this phenomenon was reported for the first time (Wildermuth, 1970), autolysis has been the term most frequently used in the literature to describe the process of hyphal death which accompanies colony development in streptomycetes (Kalakoutskii and Agre, 1976; Ensign, 1978; Locci and Sharples, 1984; Méndez et al., 1985; Braña et al., 1986; Chater, 1989a,b; Hodgson, 1992; Kelemen et al., 1995).
We present studies demonstrating that autolysis (a form of cell death that follows degradation of the bacterial cell wall by the uncontrolled, lytic action of murein hydrolases) is not the predominant mechanism for hyphal death in Streptomyces antibioticus. Instead, the hyphae undergo progressive disorganization of internal cell constituents (including extensive genome digestion), preceding loss of plasma membrane integrity. Cell wall degradation, if it occurs, is just a very late event. In addition, analysis of DNA degradation carried out by gel electrophoresis revealed two rounds of hyphal death during colony development: during emergence of the aerial hyphae and during formation of the spores. We conclude that during the life cycle of S. antibioticus the mycelium does not undergo a random process of autolysis, but a highly regulated process of PCD.
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Microscopy
At different times of incubation, samples of the cultures (exhibiting uniformity of development) were obtained and processed for microscopy as follows. Blocks of agar containing mycelium were cut out from the culture medium and dissected into small pieces (
3–4 mm in width and 8–10 mm in length). The pieces were fixed overnight at room temperature in 1% wt/ vol osmium tetroxide in 0.1 M veronal acetate buffer (pH 6.0), and postfixed with 0.5% wt/vol uranyl acetate in 0.1 M veronal acetate buffer (pH 6.0) for 2 h. After this, pieces were dehydrated through graded acetone solutions over a 2-h period at room temperature, embedded in Epon 812 resin and polymerized at 60°C for 36 h. Before polymerization, pieces were properly positioned to facilitate vertical sectioning of the whole mycelium. For electron microscopic observations, ultrathin sections of silver-gray interference color (thickness, 60–90 nm) were obtained with an LKB Ultramicrotome III equipped with a diamond knife and mounted on Formvar-coated copper grids. To improve contrast, ultrathin sections were poststained in the dark for 10 min on uranyl acetate drops (2% wt/ vol aqueous uranyl acetate), followed by counterstaining with lead citrate (pH 12, 1.5 min). Ultrathin sections were examined in a Philips EM300 electron microscope at an operating voltage of 60 kV, and photographed with Scientia electron microscopy film (AGFA; developed for 4 min in Kodak D19). For light microscopic observations, thin sections (1 µm thick) were mounted on slides, stained in Toluidine blue (0.1% wt/vol Toluidine blue in 0.1% aqueous sodium borate) for 1 min, and examined in a Nikon light microscope.
For high-resolution scanning electron microscopy, agar blocks containing mycelium were fixed with osmium tetroxide (1% wt/vol in 0.1 M veronal acetate buffer, pH 6.0) for 2 h, passed through increasing concentrations of acetone, and dried to critical point with a Balzers CPD-030 apparatus. The dried samples were mounted on aluminum stubs, coated with gold by vacuum evaporation (SCD-004 sputter coat; Balzers) and examined with a Jeol JSM-6100 scanning electron microscope.
Image Analysis
High-contrast photographic negatives were digitized using a Hewlett Packard 4C slide scanner. The digitized images were imported into Scion Image (Beta 2 version for Windows 95; Scion Corp.) for digital analysis. Plots of pixel intensity from zones of interest were obtained by positioning rectangular selections (452 pixels width, 600 pixels height) over such zones, or by tracing linear selections (5 pixels width) perpendicularly to the wall, passing from resin to the middle of the cells. Plots were generated by measuring pixel intensities (intensity range of each pixel 256 gray levels [8 bits]) along the linear selections (Profile Plot function) or in the rectangular selections (Surface Plot function). By using the LUT tool, pseudocolor images were generated in which the different components of the cell displayed an arbitrarily different color. All plots were obtained from hyphae considered to be cut diametrically (i.e., showing a clear-cut cell wall profile consisting of two electron-dense zones separated by a less electron-dense zone). Final images were composed and prepared for printing by using Adobe Photoshop software (4.0 version; Adobe Systems). All the images were printed using a Epson SC-800 printer.
Analytical Procedures
At various times of incubation, the mycelium from three plates (8.5 cm diam) was gently scraped from the cellophane with a plastic spatula, pooled, and suspended in 10 mM potassium phosphate buffer (pH 7.0). The suspensions were then sonicated at full power for 2 min on a MSE Soniprep 150 sonicator. Samples of disrupted mycelial suspensions (1.5 ml, by triplicate) were taken, 1.5 ml of 0.5 N perchloric acid was added, and the samples were maintained for 30 min at 0°C in an ice bath. After centrifugation, the pellets were extracted three times with 0.5 N perchloric acid at 70°C. Supernatants were pooled and assayed for RNA by the orcinol method (Schneider, 1957). Pellets were dissolved in 1.0 N NaOH and assayed for protein (Lowry et al., 1951). For dry cell weight determinations, samples (1.5 ml by triplicate) of disrupted mycelial suspensions were collected in preweighted glass vials and dried at 100°C to constant weight.
DNA Extraction and Agarose Gel Electrophoresis
Standard methods were used for DNA extraction (Hopwood et al., 1985). In brief, samples of mycelium collected from cellophane membranes were suspended in a lysing solution consisting of 5 mM Tris-HCl (pH 8.0), 25% sucrose, and lysozyme (1 mg/ml). After 1 h at 37°C, the solution was successively treated with Pronase (1 mg/ml) and 1% SDS. After phenol/chloroform extraction, the DNA solution was incubated for 1 h at 37°C in the presence of RNase (40 µg/ml) and then precipitated by adding an equal volume of 100% ethanol. The precipitated DNA was washed with 70% ethanol and suspended in TE buffer. The final DNA solution was checked spectrophotometrically for purity and concentration. Equal amounts of DNA from each sample were electrophoresed in 1% agarose gels in TBE buffer at 60–80 mA for 1–2 h. The gels were stained with ethidium bromide and photographed under UV light.
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After 48 h of incubation, the colonies contained a well-developed aerial mycelium (Fig. 6, left). In semithin sections, the aerial mycelium appeared as a loose network of hyphae that develop upwards into the air (Fig. 6 a). Ultrathin sections through representative zones of these colonies, illustrating the vertical distribution of hyphae undergoing cell degeneration, are shown in Fig. 6, b–d. No hyphae with symptoms of cellular degeneration were encountered in the aerial mycelium at this stage of development, where a majority of the hyphae showed a dense, heavily stained cytoplasm (Fig. 6 b). Below this zone and near the boundary with the culture medium (not only above, but also within it), the substrate mycelium appeared as an intricate network of hyphae in different stages of cellular degeneration, ranging from hyphae with electron-transparent areas in their cytoplasm to hyphae in which the cytoplasmic components had completely disappeared (Fig. 6, c and d). At the bottom of the colony (130 µm below the surface of the culture medium), a minority of the hyphae displayed a quite different morphological form of cell death, which was identifiable in 5–10% of the hyphae present in that region (Fig. 6, right). These hyphae did not collapse nor did they undergo progressive disorganization of the nucleoid and cytoplasm with maintenance of cell wall integrity. Instead, there was an early rupture of the wall and plasma membrane followed by rapid release of cellular contents into the surrounding medium, as suggested by the almost total absence of hyphae exhibiting stages of cellular degeneration later than those shown in Fig. 6, b and c. Only very few hyphae showing the initial stages of cell wall degradation could be examined and all these hyphae showed lightly stained cytoplasm with nucleoids of various sizes and shapes irregularly distributed through it.
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Hyphal Death and Associated Biochemical Changes
S. antibioticus was cultured on sterile cellophane membranes overlaid on solid GAE medium. At different times during growth, samples of mycelium were carefully removed from the cellophane membranes and used either to examine the state of DNA by agarose gel electrophoresis or to estimate changes in the macromolecular content of the hyphae.
Changes in DNA Integrity.
As Fig. 9 a shows, DNA extracted from samples collected during the first 24 h of incubation (lanes 2 and 3) did not shows signs of degradation. However, after 36 h of incubation and continuing through later time points (Fig. 9 a, lanes 4–9), DNA degradation was apparent as a smear beneath a band of high molecular weight DNA. As can be seen, DNA degradation coincided in time with the presence in the colony of hyphae undergoing nuclear degeneration (see Figs. 5–7). On the other hand, there were two time points at which DNA degradation reached a maximum (Fig. 9 b, 48 and 72 h of incubation). These corresponded to the same time points at which the colonies were found to form aerial hyphae and to differentiate into spores, respectively (see Figs. 6 and 7).
Changes in Dry Cell Weight and Macromolecular Content.
As Fig. 9 c shows, over the period 12–32 h after inoculation the cultures entered a phase of rapid growth during which all the growth parameters increased steadily. This was followed by a long period of slow biomass accumulation (which began with the emergence of the aerial mycelium and extended throughout its development), during which the total contents of RNA and protein in the mycelium varied significantly: there was a marked decrease during emergence of the aerial hyphae (between 32 and 44 h of incubation), followed by a slight increase between 44 and 64 h of incubation and by a moderate but persistent decrease during the period of spore formation.
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Therefore, the main objectives of this study were to characterize the sequence of ultrastructural changes leading to hyphal death in S. antibioticus, to identify the zones of the colony where they occur, and to determine the developmental time at which they appear. Our electron microscopy study has revealed some previously unrecognized aspects of hyphal death in streptomycetes. We have discerned that nucleoid degradation is a relatively early event in the hyphal death process and that this degradation clearly precedes the rupture of the plasma membrane. Moreover, we also observed that nucleoid degradation is accompanied by progressive digestion of cytoplasmic contents and distortion of the hyphal shape, and that all this occurs with maintenance of cell wall integrity and without disturbing the general organization of the colony.
In addition, we have observed that the hyphae die in specific zones and at specific times during the colony life cycle. Studies on the presence and distribution of degenerating hyphae within the mycelium carried out by electron microscopy and analysis of DNA degradation carried out by gel electrophoresis revealed two rounds of hyphal death during colony development in S. antibioticus. The first round coincided with development of the aerial mycelium. It caused massive death in the substrate mycelium but had no apparent effect on the emergent aerial hyphae. The second round was not triggered until sporulation had been initiated and was more selective, since it only affected the basal, nonsporulating parts of the aerial hyphae. These observations are also interesting because they suggest that hyphal death is somehow included into the developmental program of the colony.
All together, the results obtained in this study demonstrate that the hyphae of S. antibioticus do not die via autolysis (see Introduction). Instead, they undergo an orderly process of internal cell dismantling (including extensive genome digestion), followed by shrinkage and distortion of the hyphal shape that resembles PCD in animal development. However, there are some aspects of the cytology and functions of hyphal death in S. antibioticus that distinguish it from PCD in higher organisms. First, dying hyphae do not display features such as reduction in nuclear size, condensation of chromatin, and internucleosomal cleavage of DNA (which gives a characteristic ladder when analyzed by electrophoresis), as is characteristically seen in eukaryotic cells undergoing PCD via apoptosis. This is probably a consequence of the quite different ultrastructural organization of the prokaryotic nucleoid, which lacks a nuclear membrane and contains an extensively folded DNA molecule not arranged into nucleosomes (Kellenberger and Arnold-Schulz-Gahmen, 1992; Robinow and Kellenberger, 1994). Second, dead hyphae do not completely disappear, but form part of the colony structure where they still could potentially perform two, nonmutually exclusive roles: they could provide a mechanical support for aerial hyphae to develop far from the surface of the culture medium, and they could serve as a conducting system for passage of water and solutes within the colony (a process of considerable importance for the aerial hyphae which develop in the absence of a surrounding liquid medium). Moreover, as dying hyphae provide nutrient support for development of the aerial mycelium in S. antibioticus (Méndez et al., 1985), maintenance of cell wall integrity would allow the cytoplasmic contents of these hyphae to be degraded and reused for growth without disturbing the general architecture of the colony.
In conclusion, our study has provided morphological evidence for the existence of a process of PCD in a prokaryotic organism. This adds new support to the hypothesis that the basic structure of the cell death processes has been preserved and extended throughout evolution (Vaux et al., 1994; Vaux and Strasser, 1996; Hochman, 1997). Establishing the mechanisms and signals that regulate such a process will be a major challenge for the future. In this respect, it is important to note that several bacterial plasmids carrying genes capable of killing their host have been reported recently to be responsible for the death of specific subpopulations of bacterial cells (Bugge and Gerdes, 1995; Naito et al., 1995; Yarmolinsky, 1995; Chaloupka and Vinter, 1996; Franch and Gerdes, 1996; Holcík and Lyer, 1997). Interestingly, two such plasmid-encoded killer systems seem to be present in Streptomyces spp. (Holcík and Lyer, 1997, and references therein). Finally, the colony growth cycle of the streptomycetes provides a useful prokaryotic system for the study of the mechanism and role of cell death in development. Such studies may provide insights into the role of cell death in more complex eukaryotic systems and may also provide insights into the evolution of this important phenomenon.
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Acknowledgments |
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This work was supported by grants BIO94-1025 and PM97-0197 from the CICYT, Spain.
Submitted: 4 January 1999
Revised: 25 March 1999
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phage DNA as a molecular size marker. (b) Pixel intensity traces corresponding to lanes 2–9. A digitized image of the gel was transferred to Scion Image, an image analysis program. Lanes were analyzed by tracing linear selections (5 pixels, width) and measuring pixel intensities along the trajectory of each lane with the help of the Profile Plot function of the software. Y-axis: pixel intensity (0–250, arbitrary values). X-axis: lane trajectory. (c) Changes in dry cell weight and macromolecular content during colony development in S. antibioticus. The microorganism was cultured on cellophane-covered GAE medium. At different times of incubation, the mycelium was removed from the cellophane and the dry cell weight (open circles) and the total RNA (filled circles) and protein contents (open boxes) were determined. The plotted values are the mean of three determinations. Arrows at the top of the figure indicate the developmental stage of the cultures. AM, period of aerial mycelium development; SP, period of spore formation.
