Vol. 127, No. 3 Printed in U.S.A.
JOURNAL
OF BACTERIOLOGY, Sept. 1976, p. 1443-1454 Copyright C 1976 American Society for Microbiology
Ultrastructural Studies of Sporulation in Streptomyces CARLOS HARDISSON* ArD MANUEL B. MANZANAL
Departamento de Microbiologia, Universidad de Oviedo, Oviedo, Spain Received for publication 29 April 1976
This is the first study of sporogenesis in Streptomyces carried out on a relatively high number of species (seven) which allows us, using also previously published results, to establish a general picture of this process. In the sporogenesis of Streptomyces two basic stages can be considered: the sporulation septum synthesis and the arthrospore maturation. Our ultrastructural study of the sporulation septum formation suggests the existence within this genus of three basic types. Type I is distinguished because the septum is formed from the beginning by two separate cross walls. Within this type we include Streptomyces erythraeus, Streptomyces albus, and Streptomyces aureofaciens and also include Streptomyces venezuelae, Streptomyces griseus, and Streptomyces osteogriseus. Type II is distinguished because there is a deposit of material previous to the synthesis of the double annulus which completes the septum. This type can be divided into two subtypes. In the first the deposits are wedge-shaped and the double annulus is clearly visible, and to this group belong Streptomyces flaveolus, Streptomyces ambofaciens, and Streptomyces coelicolor. In the second the deposits, which have a different shape and are very well developed, constitute almost entirely the sporulation septum in which the double annulus is barely visible; Streptomyces antibioticus and also Streptomyces viridochromogenes belong to this group. Type III, represented by Streptomyces cinnamonensis, is distinguished because the septum is formed by a single cross wall.
The genus Streptomyces belonging to the order Actinomycetales shows a comparatively complex development cycle in which from a branched vegetative mycelium submerged in the substrate is formed an aerial mycelium with hyphae of greater diameter. Through the sporulation process these aerial hyphae give rise to chains of arthrospores, which placed under favorable conditions germinate and repeat the cycle. The term arthrospores has been proposed by Cross (5) to refer to the asexual spores produced by Streptomyces that are formed by the annular ingrowth of specialized, regularly spaced cross walls in existing hyphae enclosed in a fibrQus sheath. The arthrospores are differentiated from bacterial endospores both by the sporogenesis process and by their properties. In recent years great interest has been shown in the study of sporulation processes in bacteria, because they constitute a simple, and therefore an attractive, model for the study of a process of cellular differentiation. Although most of these studies are performed on species of two genera Bacillus and Clostridium, several works have been published on the different processes of sporulation within the Actinomycetes, particularly in the genera Streptomyces and Thermoactinomyces (6).
In this paper we present the results of the ultrastructural study of the sporulation in seven species of Streptomyces. We compare these results with those obtained previously by other authors and make a tentative classification of the genus Streptomyces according to the patterns of the sporulation process.
MATERIALS AND METHODS Microorganisms. The following strains of the Streptomyces genus were used: Streptomyces albus, CMI 52766; Streptomyces ambofaciens, ATCC 23877; Streptomyces antibioticus ATCC 11891; Streptomyces aureofaciens, ATCC 13304; Streptomyces cinnamonensis, ATCC 12308; Streptomyces erythraeus, ATCC 11635; Streptomyces flaveolus, ATCC 3319. Media and culture conditions. The microorganisms were inoculated by streaking on GAE medium (glucose, 1%; asparagine, 0.1%; yeast extract, 0.05%; K2HPO4, 0.05%; MgSO4 - 7H20, 0.05%; FeSO4, 0.001%; and agar, 2%) contained in petri plates. After incubation at 28°C well-isolated colonies whose stage of development was checked by direct observation in the electron microscope were selected for the ultrastructural study of the sporulation. Electron microscopy. Selected whole colonies at different incubation times were cut out of the medium on small blocks of agar and fixed by the RyterKellemberger method (13). The dehydration was carried out according to Burdett and Rogers (2). Samples were embedded in Araldite. The sectioning 1443
1444
J. BACTERIOL.
HARDISSON AND MANZANAL
was carried out in a LKB III ultramicrotome and stained with 2% uranyl acetate. The Philips EM300 was operated at 40 and 80 kV with instrumental magnifications from 3,000 to 30,000. For direct observation, the surface of the colonies was touched gently with Formvar-coated copper grids. Aerial mycelium and arthrospores adhere to the surface of the grids and can be observed directly in the electron
microscope. Usually colonies with abundant aerial mycelium and only a few arthrospores give good samples for the study of sporulation.
RESULTS In the sporulation process of Streptomyces two basic stages may be considered. The first
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FIG. 1-11. Sporulation ofS. erythraeus, S. aureofaciens, and S. albus. Abbreviations: AA, Double edge of ingrowing annulus; FS, fibrous sheath; SS, sporulation septum. Bar equals 200 nm. FIG. 1. Longitudinal section of a sporulating hypha ofS. erythraeus showing a partially complete sporulation septum. Note the constriction of the dividing nuclear material and the invagination of the plasma membrane. FIG. 2. Longitudinal section of a sporulating hypha of S. erythraeus showing two complete sporulation septa and, at the center, another in the process of being formed (arrowhead). FIG. 3. Longitudinal section of a sporulating hypha of S. aureofaciens showing a partially complete sporulation septum (arrowhead). Note the two separate cross walls and the plasma membrane invagination. FIG. 4. Longitudinal section of an aerial hypha of S. albus at the onset of sporulation showing three forming sporulation septa (arrows). FIG. 5. Enlarged detail of the above micrograph. Note the invagination of the plasma membrane and that the sporulation septum is formed by two separate cross walls.
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10~~~~~~~1 FIG. 6. Longitudinal section of a sporulating hypha of S. erythraeus showing a complete sporulation septum (arrowhead). Note the two clearly separated cross walls and the typical structure of the cytoplasmic membrane. FIG. 7. Longitudinal section of a sporulating hypha of S. erythraeus showing several sporulation septa. Note the spiny appendages of the fibrous sheath. FIG. 8. Longitudinal section of a sporulating hypha of S. aureofaciens showing three completed sporulation septa (arrowheads). FIG. 9. Longitudinal section of a sporulating hypha of S. albus showing two completed sporulation septa. Note the two separate cross walls. FIG. 10. Longitudinal section of a chain of arthrospores of S. albus in an advanced stage of maturation. The thickness of the wall has increased to about 30 nm. Arthrospores remain attached by the fibrous sheath and the central zone of the sporulation septum, whose corners have rounded. The arthrospore cytoplasm contains vacuole-like spaces of low electron density that may be granules of poly-f3-hydroxybutyrate. FIG. 11. Section of a mature arthrospore of S. erythraeus. No structures may be seen within the cytoplasm. The wall has thickened to about 40 nm. Note the disintegrating fibrous sheath. 1445
1446
HARDISSON AND MANZANAL
J. BACTERIOL.
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VOL. 127, 1976
stage lasts from the initiation of sporogenesis to the formation of the sporulation septum. The second stage includes all the changes that take place from the delimitation of the future arthrospore to the formation of the mature arthrospore. Each stage has been subdivided by Hopwood et al. (4, 14). Sporulation-septum formation. Sporulation in Streptomyces starts with the simultaneous formation of specialized cross walls that Wildermuth and Hopwood (14) call sporulation septa, which divide the preexisting aerial hyphae. Results obtained show that according to the synthesis of the sporulation septum the Streptomyces genus can be divided into three groups. Type I (Fig. 1-11) is distinguished because the sporulation septum is originated by the deposit on the inner face of the hyphal wall of a material with similar electron density to that of the wall (Fig. 2-5). This deposit has the appearance of two separate annuli or cross walls, which grow centripetally until joining in the center of the hyphae, forming the sporulation septum (Fig. 6, 8, and 9). These cross walls remain as separate entities throughout the process. The cytoplasmic membrane invaginates as the double annulus grows (Fig. 1-5) and joins before the cross walls do. To this type belong S. erythraeus, S. albus, and S. aureofaciens. In type II (Fig. 12-33), previous to the synthe3is of the two annuli, material whose electron density is slightly less than that of the wall is deposited at regularly spaced intervals on the inside of the hyphal wall (Fig. 13, 14, 23, 24, 27,
SPORULATION IN STREPTOMYCES
1447
and 28). The cytoplasmic membrane invaginates as this deposit of material builds up, from which are subsequently formed two annuli or cross walls which by joining centrally constitute the sporulation septum. Between the hyphal wall and the deposited material the existence of a boundary or separation may be seen (Fig. 14, 24, and 28), as pointed out in a previous work on sporulation in Streptomyces coelicolor (14). In some instances, such as in S. flaveolus (Fig. 13-16) and S. ambofaciens (Fig. 23 and 24), the deposits have the form of a wedge and the cross walls are clearly visible, whereas in S. antibioticus (Fig. 28-31) the deposits are very large, constituting almost the whole sporulation septum (Fig. 29 and 30), and the cross walls that complete the septum (Fig. 31) are much smaller. The abundance of mesosomes in proximity to and in contact with the developing septum must be emphasized (Fig. 13-18, 23, and 24), as has been previously observed (1, 7, 8, 11, and 14). In type III, represented by S. cinnamonensis (Fig. 34-42), the sporulation septum is originated by a single deposit of electron-dense material between the hyphal wall and the plasma membrane which invaginates (Fig. 34 and 35). This material grows inwards until joining, as do the invaginated membranes (Fig. 36), to form the sporulation septum (Fig. 37). Lamellar and vesicular mesosomes are associated with the sporulation septum and the nuclear region (Fig. 36 and 37). The nuclear material undergoes remarkable changes during the process assuming a compact structure as septa-
FIG. 12-26. Sporulation of S. flaveolus and S. ambofaciens. Abbreviations: WS, Deposit of septum material; SW, spore wall; S, storage granules; B, boundary between the hyphal wall and the septum material. Other symbols as in previous figures. Bar equals 200 nm. FIG. 12. Longitudinal section of an aerial hypha of S. flaveolus, showing the fibrous sheath, the hyphal wall, the fibrillar nuclear region, and storage granules. FIG. 13. Longitudinal section of an aerial hypha of S. flaveolus showing the initial stage of sporulation septum formation. Note the deposit of septum material (arrowhead) between the hyphal wall and the invaginating membrane in association with tubular vesicular mesosomes. FIG. 14. Longitudinal section of a sporulating hypha of S. flaveolus showing the final stages.of sporulation septum completion. From the wedge-shaped deposited material (arrow head) two separate annuli which grow inward are formed (arrowhead). Note a large vesicular mesosome in association with the forming septum and a large lamellar mesosome in association with the nuclear region. FIG. 15. Longitudinal section of a sporulating hypha of S. flaveolus showing three complete sporulation septa (arrowheads). FIG. 16. Longitudinal section of a sporulating hypha of S. flaveolus showing a newly completed sporulation septum (arrowhead). Note the double cross wall between the wedge-shaped deposit material. FIG. 17. Longitudinal section of a sporulating hypha of S. flaveolus showing the initial stage of arthrospore maturation. Note three zones in the septum: a broad outer zone near the hyphal wall, and of similar electron-density, that starts to become rounded; a narrower and less dense intermediate zone; and a more electron-dense and thicker central zone. FIG. 18. Longitudinal section of a sporulating hypha of S. flaveolus showing a later stage of arthrospore maturation. The thickness of the cross walls increases, and the rounding of the corners becomes more evident. At the same time (arrowheads) lysis in the septum zones nearest the hyphal wall occurs.
1448
HARDISSON AND MANZANAL
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JOURNAL
OF BACTERIOLOGY, Sept. 1976, p. 1443-1454 Copyright C 1976 American Society for Microbiology
Ultrastructural Studies of Sporulation in Streptomyces CARLOS HARDISSON* ArD MANUEL B. MANZANAL
Departamento de Microbiologia, Universidad de Oviedo, Oviedo, Spain Received for publication 29 April 1976
This is the first study of sporogenesis in Streptomyces carried out on a relatively high number of species (seven) which allows us, using also previously published results, to establish a general picture of this process. In the sporogenesis of Streptomyces two basic stages can be considered: the sporulation septum synthesis and the arthrospore maturation. Our ultrastructural study of the sporulation septum formation suggests the existence within this genus of three basic types. Type I is distinguished because the septum is formed from the beginning by two separate cross walls. Within this type we include Streptomyces erythraeus, Streptomyces albus, and Streptomyces aureofaciens and also include Streptomyces venezuelae, Streptomyces griseus, and Streptomyces osteogriseus. Type II is distinguished because there is a deposit of material previous to the synthesis of the double annulus which completes the septum. This type can be divided into two subtypes. In the first the deposits are wedge-shaped and the double annulus is clearly visible, and to this group belong Streptomyces flaveolus, Streptomyces ambofaciens, and Streptomyces coelicolor. In the second the deposits, which have a different shape and are very well developed, constitute almost entirely the sporulation septum in which the double annulus is barely visible; Streptomyces antibioticus and also Streptomyces viridochromogenes belong to this group. Type III, represented by Streptomyces cinnamonensis, is distinguished because the septum is formed by a single cross wall.
The genus Streptomyces belonging to the order Actinomycetales shows a comparatively complex development cycle in which from a branched vegetative mycelium submerged in the substrate is formed an aerial mycelium with hyphae of greater diameter. Through the sporulation process these aerial hyphae give rise to chains of arthrospores, which placed under favorable conditions germinate and repeat the cycle. The term arthrospores has been proposed by Cross (5) to refer to the asexual spores produced by Streptomyces that are formed by the annular ingrowth of specialized, regularly spaced cross walls in existing hyphae enclosed in a fibrQus sheath. The arthrospores are differentiated from bacterial endospores both by the sporogenesis process and by their properties. In recent years great interest has been shown in the study of sporulation processes in bacteria, because they constitute a simple, and therefore an attractive, model for the study of a process of cellular differentiation. Although most of these studies are performed on species of two genera Bacillus and Clostridium, several works have been published on the different processes of sporulation within the Actinomycetes, particularly in the genera Streptomyces and Thermoactinomyces (6).
In this paper we present the results of the ultrastructural study of the sporulation in seven species of Streptomyces. We compare these results with those obtained previously by other authors and make a tentative classification of the genus Streptomyces according to the patterns of the sporulation process.
MATERIALS AND METHODS Microorganisms. The following strains of the Streptomyces genus were used: Streptomyces albus, CMI 52766; Streptomyces ambofaciens, ATCC 23877; Streptomyces antibioticus ATCC 11891; Streptomyces aureofaciens, ATCC 13304; Streptomyces cinnamonensis, ATCC 12308; Streptomyces erythraeus, ATCC 11635; Streptomyces flaveolus, ATCC 3319. Media and culture conditions. The microorganisms were inoculated by streaking on GAE medium (glucose, 1%; asparagine, 0.1%; yeast extract, 0.05%; K2HPO4, 0.05%; MgSO4 - 7H20, 0.05%; FeSO4, 0.001%; and agar, 2%) contained in petri plates. After incubation at 28°C well-isolated colonies whose stage of development was checked by direct observation in the electron microscope were selected for the ultrastructural study of the sporulation. Electron microscopy. Selected whole colonies at different incubation times were cut out of the medium on small blocks of agar and fixed by the RyterKellemberger method (13). The dehydration was carried out according to Burdett and Rogers (2). Samples were embedded in Araldite. The sectioning 1443
1444
J. BACTERIOL.
HARDISSON AND MANZANAL
was carried out in a LKB III ultramicrotome and stained with 2% uranyl acetate. The Philips EM300 was operated at 40 and 80 kV with instrumental magnifications from 3,000 to 30,000. For direct observation, the surface of the colonies was touched gently with Formvar-coated copper grids. Aerial mycelium and arthrospores adhere to the surface of the grids and can be observed directly in the electron
microscope. Usually colonies with abundant aerial mycelium and only a few arthrospores give good samples for the study of sporulation.
RESULTS In the sporulation process of Streptomyces two basic stages may be considered. The first
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FIG. 1-11. Sporulation ofS. erythraeus, S. aureofaciens, and S. albus. Abbreviations: AA, Double edge of ingrowing annulus; FS, fibrous sheath; SS, sporulation septum. Bar equals 200 nm. FIG. 1. Longitudinal section of a sporulating hypha ofS. erythraeus showing a partially complete sporulation septum. Note the constriction of the dividing nuclear material and the invagination of the plasma membrane. FIG. 2. Longitudinal section of a sporulating hypha of S. erythraeus showing two complete sporulation septa and, at the center, another in the process of being formed (arrowhead). FIG. 3. Longitudinal section of a sporulating hypha of S. aureofaciens showing a partially complete sporulation septum (arrowhead). Note the two separate cross walls and the plasma membrane invagination. FIG. 4. Longitudinal section of an aerial hypha of S. albus at the onset of sporulation showing three forming sporulation septa (arrows). FIG. 5. Enlarged detail of the above micrograph. Note the invagination of the plasma membrane and that the sporulation septum is formed by two separate cross walls.
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10~~~~~~~1 FIG. 6. Longitudinal section of a sporulating hypha of S. erythraeus showing a complete sporulation septum (arrowhead). Note the two clearly separated cross walls and the typical structure of the cytoplasmic membrane. FIG. 7. Longitudinal section of a sporulating hypha of S. erythraeus showing several sporulation septa. Note the spiny appendages of the fibrous sheath. FIG. 8. Longitudinal section of a sporulating hypha of S. aureofaciens showing three completed sporulation septa (arrowheads). FIG. 9. Longitudinal section of a sporulating hypha of S. albus showing two completed sporulation septa. Note the two separate cross walls. FIG. 10. Longitudinal section of a chain of arthrospores of S. albus in an advanced stage of maturation. The thickness of the wall has increased to about 30 nm. Arthrospores remain attached by the fibrous sheath and the central zone of the sporulation septum, whose corners have rounded. The arthrospore cytoplasm contains vacuole-like spaces of low electron density that may be granules of poly-f3-hydroxybutyrate. FIG. 11. Section of a mature arthrospore of S. erythraeus. No structures may be seen within the cytoplasm. The wall has thickened to about 40 nm. Note the disintegrating fibrous sheath. 1445
1446
HARDISSON AND MANZANAL
J. BACTERIOL.
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VOL. 127, 1976
stage lasts from the initiation of sporogenesis to the formation of the sporulation septum. The second stage includes all the changes that take place from the delimitation of the future arthrospore to the formation of the mature arthrospore. Each stage has been subdivided by Hopwood et al. (4, 14). Sporulation-septum formation. Sporulation in Streptomyces starts with the simultaneous formation of specialized cross walls that Wildermuth and Hopwood (14) call sporulation septa, which divide the preexisting aerial hyphae. Results obtained show that according to the synthesis of the sporulation septum the Streptomyces genus can be divided into three groups. Type I (Fig. 1-11) is distinguished because the sporulation septum is originated by the deposit on the inner face of the hyphal wall of a material with similar electron density to that of the wall (Fig. 2-5). This deposit has the appearance of two separate annuli or cross walls, which grow centripetally until joining in the center of the hyphae, forming the sporulation septum (Fig. 6, 8, and 9). These cross walls remain as separate entities throughout the process. The cytoplasmic membrane invaginates as the double annulus grows (Fig. 1-5) and joins before the cross walls do. To this type belong S. erythraeus, S. albus, and S. aureofaciens. In type II (Fig. 12-33), previous to the synthe3is of the two annuli, material whose electron density is slightly less than that of the wall is deposited at regularly spaced intervals on the inside of the hyphal wall (Fig. 13, 14, 23, 24, 27,
SPORULATION IN STREPTOMYCES
1447
and 28). The cytoplasmic membrane invaginates as this deposit of material builds up, from which are subsequently formed two annuli or cross walls which by joining centrally constitute the sporulation septum. Between the hyphal wall and the deposited material the existence of a boundary or separation may be seen (Fig. 14, 24, and 28), as pointed out in a previous work on sporulation in Streptomyces coelicolor (14). In some instances, such as in S. flaveolus (Fig. 13-16) and S. ambofaciens (Fig. 23 and 24), the deposits have the form of a wedge and the cross walls are clearly visible, whereas in S. antibioticus (Fig. 28-31) the deposits are very large, constituting almost the whole sporulation septum (Fig. 29 and 30), and the cross walls that complete the septum (Fig. 31) are much smaller. The abundance of mesosomes in proximity to and in contact with the developing septum must be emphasized (Fig. 13-18, 23, and 24), as has been previously observed (1, 7, 8, 11, and 14). In type III, represented by S. cinnamonensis (Fig. 34-42), the sporulation septum is originated by a single deposit of electron-dense material between the hyphal wall and the plasma membrane which invaginates (Fig. 34 and 35). This material grows inwards until joining, as do the invaginated membranes (Fig. 36), to form the sporulation septum (Fig. 37). Lamellar and vesicular mesosomes are associated with the sporulation septum and the nuclear region (Fig. 36 and 37). The nuclear material undergoes remarkable changes during the process assuming a compact structure as septa-
FIG. 12-26. Sporulation of S. flaveolus and S. ambofaciens. Abbreviations: WS, Deposit of septum material; SW, spore wall; S, storage granules; B, boundary between the hyphal wall and the septum material. Other symbols as in previous figures. Bar equals 200 nm. FIG. 12. Longitudinal section of an aerial hypha of S. flaveolus, showing the fibrous sheath, the hyphal wall, the fibrillar nuclear region, and storage granules. FIG. 13. Longitudinal section of an aerial hypha of S. flaveolus showing the initial stage of sporulation septum formation. Note the deposit of septum material (arrowhead) between the hyphal wall and the invaginating membrane in association with tubular vesicular mesosomes. FIG. 14. Longitudinal section of a sporulating hypha of S. flaveolus showing the final stages.of sporulation septum completion. From the wedge-shaped deposited material (arrow head) two separate annuli which grow inward are formed (arrowhead). Note a large vesicular mesosome in association with the forming septum and a large lamellar mesosome in association with the nuclear region. FIG. 15. Longitudinal section of a sporulating hypha of S. flaveolus showing three complete sporulation septa (arrowheads). FIG. 16. Longitudinal section of a sporulating hypha of S. flaveolus showing a newly completed sporulation septum (arrowhead). Note the double cross wall between the wedge-shaped deposit material. FIG. 17. Longitudinal section of a sporulating hypha of S. flaveolus showing the initial stage of arthrospore maturation. Note three zones in the septum: a broad outer zone near the hyphal wall, and of similar electron-density, that starts to become rounded; a narrower and less dense intermediate zone; and a more electron-dense and thicker central zone. FIG. 18. Longitudinal section of a sporulating hypha of S. flaveolus showing a later stage of arthrospore maturation. The thickness of the cross walls increases, and the rounding of the corners becomes more evident. At the same time (arrowheads) lysis in the septum zones nearest the hyphal wall occurs.
1448
HARDISSON AND MANZANAL
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J. BACTERIOL.
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