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Keywords:

  • Streptomyces;
  • rodlet;
  • aerial mycelium;
  • surface cultures

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Morphogenesis in streptomycetes is characterized by the formation of aerial hyphae that emerge from the substrate mycelium. Despite many years of study, a detailed picture of the events that occur during the transition from substrate to aerial mycelium has yet to be defined. In this paper, it was shown that a specific cell death event takes place during early growth of the substrate mycelium in Streptomyces coelicolor and Streptomyces lividans. Subsequently, a second mycelium starts to develop from the remaining viable segments of these substrate hyphae in the form of islands, which progressively cover the plate surface. Interestingly, the genes coding for the chaplin and rodlin proteins, which are involved in the formation of the hydrophobic layer characteristic of aerial structures, are specifically expressed in the second mycelium islands, strongly suggesting that this second mycelium should be considered the early precursor of the mature hydrophobic aerial mycelium.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Streptomycetes are filamentous bacteria that are characterized by their complex morphogenetic programme (Chater & Chandra, 2006). During growth on surfaces, a vegetative substrate mycelium is established, after which aerial hyphae emerge that develop into pigmented exospores. Morphogenesis involves a ‘multicellular’ behaviour pattern in which programmed cell death processes constitute developmental landmarks (Nicieza et al., 1999; Manteca et al., 2005a, b). As such, these bacteria have been proposed to be the source of some of the characteristic domains of proteins involved in apoptosis, such as eukaryote-type protein kinases of the PNK2 family, apoptotic Toll-interleukin-receptor domain (TIR) adaptors, caspase-like proteases, or apoptotic (AP-) ATPases or NTPases (Yarmolinsky, 1995; Hochman, 1997; Aravind et al., 1999; Koonin & Aravind, 2002).

The classical model for the developmental cycle of Streptomyces grown in the form of confluent agar cultures is based on the assumption that differentiation takes place only along the vertical axis (bottom-up): a vegetative (substrate) mycelium grows on top of and inside the agar until it undergoes a death process, after which hyphae begin to differentiate to form a reproductive (aerial) mycelium that grows into the air (Wildermuth, 1970; Mendez et al., 1985; Miguelez et al., 1999; Fernandez & Sanchez, 2002). This ‘vertical’ description therefore assumes that the onset of development is more or less homogeneous in all areas of the plate surface. Recently, this simplified developmental model has been extended (Manteca et al., 2005a, b, 2006) with a description of additional features. The first one refers to the existence of a young substrate mycelium, consisting of compartmentalized hyphae with alternating live and dead segments that die in an orderly pattern (Manteca et al., 2005a). The second one is the rapid development of the reproductive, noncompartmentalized mycelium (syncytial) in the form of ‘islands’ that progressively spread to cover the entire plate surface. Remarkably, this syncytial mycelium develops from the remaining live segments of the compartmentalized hyphae (Manteca et al., 2005a, b, 2006). In order to discriminate between both types of mycelia, the initial compartmentalized mycelium and the syncytial mycelium were named ‘first’ and ‘second’ mycelium, respectively (Manteca et al., 2005a). These phenomena demonstrate that differentiation in confluent agar cultures not only occurs along the vertical axis but also across the plane of the agar surface (‘horizontal heterogeneity’) (Manteca et al., 2005a). All the Streptomyces species/strains analysed to date exhibit the developmental features reported previously (Manteca et al., 2005a, b, 2006), the only differences between them lying in the time required to reach the different phases and the sizes of the second mycelium islands (Manteca et al., 2005a, b, 2006). However, it was not clear what the nature of the second mycelium was, or its developmental relationship with the aerial hyphae. It is demonstrated here that expression of the chaplin and rodlin genes (Claessen et al., 2002, 2003; Elliot et al., 2003), whose products are involved in the formation of the rodlet layer of aerial hyphae and spores, occurs early in the viable parts of the compartmentalized mycelium, demonstrating that these segments should be considered the early precursors of aerial hyphae.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Strains and media

Streptomyces lividans TK23 and Streptomyces coelicolor M145 were the species used in this study. To allow abundant sporulation to take place, cultures were grown on solid GYM medium (glucose, yeast, malt) (Novella et al., 1992) supplemented with Difco agar. Plates were inoculated with 100 μL of a spore suspension (1.5 × 107 viable spores mL−1) and incubated at 30°C.

Viability staining

Culture samples were obtained and processed for microscopy at different incubation times, as described previously (Fernandez & Sanchez, 2002; Manteca et al., 2005b). A viability assay, based on simultaneously staining cells in a population with propidium iodide and SYTO 9, was used to study cultures of various ages (Fernandez & Sanchez, 2001, 2002; Manteca et al., 2005a, b, 2006). Propidium iodide is specific for nucleic acids of damaged (leaky) cells, while SYTO 9 stains those of viable cells (Manteca et al., 2005a). To analyse the morphological differentiation along the vertical axis of the mycelium, solid blocks of agar cultures were cut manually with a microtome to obtain sections of c. 0.3 mm (cross section) (Fernandez & Sanchez, 2002). Differentiation along the horizontal axis (medium surface) was studied on plates overlaid with cellophane disks, which does not affect the differentiation processes (Fernandez & Sanchez, 2002; Manteca et al., 2006). The cellophane was removed from the culture, cut into 1.5 cm squares, and placed on slides before staining. Samples were observed under a Leica TCS-SP2-AOBS confocal laser-scanning microscope (CLSM) at wavelengths of 488 and 568 nm for excitation and 530 nm (green) or 630 nm (red) for emission. An overlay of the images was performed using leica confocal software.

eGFP visualization

To analyse the expression of the rodlin and chaplin genes, Streptomyces strains were transformed with the plasmids pIJ8630a or pIJ8630b containing the 262 bp S. coelicolor promoter region of rdlA or rdlB and the promoter region of chpG and chpH, respectively (Claessen et al., 2002, 2003), to guide eGFP gene expression. Recombinant strains were grown on GYM medium and processed using a CLSM as described above. In order to visualize eGFP, the mycelium was only stained with propidium iodide (red staining; Fig. 2), but not with SYTO 9. Samples were observed under a Leica TCS-SP2-AOBS CLSM and observed at wavelengths of 480 nm (absorption) and 560 nm (emission) for eGFP, and 568 nm (absorption) 630 nm (emission) for propidium iodide. An overlay of the images was carried out using the leica confocal Software.

image

Figure 2.  Confocal laser-scanning fluorescence microscopy analysis of rdlA expression in the surface cultures of Streptomyces lividans TK23. Samples were stained with propidium iodide. Numbers in brackets indicate growth times. Pictures correspond to horizontal sections, with the exception of (e) and (f), which show vertical sections. (a–c) Early culture times showing the beginning of the growth of small islands of aerial hyphae. (d) Islands of aerial hyphae spreading over the plate surface. (e) Vertical section displaying rodlet gene expression (green). (f) The same field observed under differential interference contrast mode shows the mycelium layer thickness. (g) A detail of the plate surface covered with aerial mycelium. (h) Sporulated hyphae. (i) The same field observed under differential interference contrast mode.

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Electron microscopy

Mycelia isolated at different time points were sandwiched between two copper platelets using a 400-mesh gold grid as a spacer. The samples were then frozen by liquid propane immersion at −189°C and fractured at −150°C and 10−8 mbar in a Bal-Tec BAF 060 freeze-etching system (BAL-TEC, Liechtenstein). Replicas were obtained by unidirectional shadowing with 2 nm of Pt/C at 45° and 20 nm of C at 90°, floated on household bleach for 5 h, and observed using a Jeol 1010 electron microscope at 80 kV.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Streptomyces lividans TK23 and S. coelicolor M145 were used for the purposes of this study. Cultures were grown in a medium that allowed abundant sporulation to take place (see ‘Materials and methods’). After staining with propidium iodide and SYTO 9 as described in ‘Materials and methods’, the developmental cycle was followed under a CLSM. As reported previously for Streptomyces antibioticus ATCC11891, Streptomyces cinereoruber DSM 40012 (formerly S. antibioticus ETH7451) (Fernandez & Sanchez, 2001, 2002), and Streptomyces glaucescens ETH22794 (Manteca et al., 2005a, b, 2006), a death process was observed in S. lividans and S. coelicolor after 9 h that affected most of the substrate mycelium (Manteca et al., 2005b). At this point, when the mycelium had not yet covered the entire plate surface, single hyphae undergoing the death process exhibited characteristic alternating live (green) and dead (red) segments (Fig. 1a). The death event gradually spread and eventually involved the entire substrate mycelium, which covered the whole plate surface after 10–11 h (Manteca et al., 2005a, b, 2006). Consistent with other Streptomyces species (Manteca et al., 2005a, b, 2006), some of the live segments began to enlarge asynchronously (after 14–17 h) in certain areas of the plate (forming what are called ‘islands’; Fig. 1b–d), while red segments progressively disintegrated until they disappeared (not shown) (Manteca et al., 2005b). This disintegration was accompanied by the appearance of cellular debris in between the second mycelium hyphae (not shown) (Manteca et al., 2005a, b, 2006). Further growth of the live segments within the areas located in between the original islands gave the plate surface a more or less homogeneous appearance at later time points (30–48 h, Fig. 1e). As already pointed out, these phenomena are identical to the developmental cycle described previously in other Streptomyces species (Manteca et al., 2005a, b, 2006), albeit with slight, temporal differences. The developmental time points and the differentiation cycle are identical in S. lividans and S. coelicolor (not shown).

image

Figure 1.  Confocal laser-scanning fluorescence microscopy analysis of Streptomyces lividans TK23 surface cultures at different times and developmental stages. Samples were stained with SYTO 9 and propidium iodide (see text). The numbers in brackets indicate growth times. Pictures (a) and (b) show cross sections; pictures (c), (d), (e), and (f) show longitudinal sections. (a) Substrate mycelium live–dead hyphae, with live and dead segments alternating regularly. (b) Detail of the early ‘aerial hyphae’ (second mycelium); red segments of dead substrate mycelium are visible in the emerging mycelium. (c, d) Early development of ‘aerial hyphae’ from the substrate mycelium in the form of small islands. (e) Plate surface covered with aerial hyphae; the pattern of islands originating at early times remains visible. (f) Sporulation phase.

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Aerial hyphae are characterized by a hydrophobic surface layer, called the rodlet layer, which has the typical ultrastructure of a mosaic of 8–10-nm-wide parallel rods (Wildermuth et al., 1971; Smucker & Pfister, 1978; Claessen et al., 2002, 2003, 2004). It has been proposed that these rods are composed of chaplin fibrils (Claessen et al., 2003), which are organized into rodlets by the homologous rodlin proteins RdlA and RdlB (Claessen et al., 2004). The rdl genes are expressed in growing aerial hyphae, but not in spores (Claessen et al., 2002). To analyse the expression of the rodlin genes, S. lividans TK23 was transformed with the plasmids pIJ8630a or pIJ8630b containing the 262 bp S. coelicolor promoter region of rdlA or rdlB, respectively, guiding the expression of the eGFP gene (Claessen et al., 2002). Recombinant strains were grown on GYM medium and processed using a CLSM as described above. In order to visualize eGFP, the mycelium was stained only with propidium iodide (in red; Fig. 2), but not with SYTO 9. The eGFP expression pattern coincided with the enlargement/spreading of the viable segments of the first mycelium in the wild-type strain at 14 h (Fig. 1). In fact, hyphae expressing the rodlin genes developed specifically from the viable segments of the compartmentalized mycelium, as they did not stain with propidium iodide (Fig. 2b and c). Vertical sections revealed that rdlA expression was located and limited to the hyphae in the upper areas of the mycelium layer; i.e., those that were in direct contact with the air (Fig. 2e). Identical results were obtained with the rdlB promoter (not shown). This confirmed that rdl expression in differentiating cultures is spatially confined to those regions that are in direct contact with the air (Claessen et al., 2006).

To analyse the expression of the chaplin genes, S. lividans TK23 and S. coelicolor M145 were transformed with the plasmids pIJ8630a or pIJ8630b containing the S. coelicolor promoter region of chpG and chpH, respectively, guiding the expression of the eGFP gene (Claessen et al., 2003). Recombinant strains were grown and processed as described above for the rodlin genes. As shown for the rodlins, the GFP expression pattern coincided with the development of viable segments developing from the second mycelium in the wild-type strain at early time points (Fig. 3b and c). Furthermore, the initial growth also occurs in the form of small islands on the mycelium surface (Fig. 3a and d). These mycelium islands expressing the chpH promoter gradually extend until they completely cover the mycelium layer surface (Fig. 3e and f). Analogous to the rodlins, the vertical sections illustrated that chpH expression (an early time expression chaplin gene; see below) was limited to the hyphae located in the upper areas of the mycelium layer (Fig. 3g). At later time points (48 h), the appearance of the hyphae is lost in some areas of the colonies and the green fluorescence appears as a mass lacking a definite shape (Fig. 3i) as a consequence of the lytic processes described previously during Streptomyces differentiation (Manteca et al., 2005a, b, 2006). This phenomenon is particularly apparent at later time points (72 h; Fig. 3j). Similar results were obtained with the chpG promoter (a late time expression chaplin gene, data not shown).

image

Figure 3.  Confocal laser-scanning fluorescence microscopy analysis of chpH expression in surface cultures of Streptomyces coelicolor M145. Samples were stained with propidium iodide. The numbers in the brackets indicate growth times. Pictures correspond to horizontal sections with the exception of (g) and (h), which show vertical sections. (a–c) Early culture times showing the beginning of the growth of small islands of aerial hyphae. (d–e) Islands of aerial hyphae spreading over the plate surface. (f) Detail of aerial hyphae expressing chpH. (g) Vertical section showing chaplin gene expression in the upper areas of the mycelium layer. (h) The same field observed under differential interference contrast mode. (i, j) Detail of the plate surface covered with aerial mycelium; lytic processes affecting the eGFP-expressing hyphae cause these structures to lose their shape.

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A significant difference between chpH and chpG kinetics concerns the time at which expression extends until it covers the entire culture surface (24 and 48 h, respectively). Also, the loss of the hyphal morphology that accompanies the lytic processes takes place later during the course of development for the eGFP-expressing chpG (72 h as opposed to 24 h for the chpH, see above). These data corroborate former Northern blot expression experiments that otherwise allowed two types of chaplins to be defined: chpE and chpH, both of which express early in the developmental processes (24 h), as well as a group of six late expressed chaplins (36 h), namely, chpA, chpB, chpC, chpD, chpF, and chpG (Claessen et al., 2003).

The developmental cycle was also analysed in three S. coelicolor chaplin mutants with the vital stains propidium iodide and SYTO 9: the fivefold (ΔchpABCDH), sixfold (ΔchpABCDEH), and eightfold (chp null) mutant (ΔchpABCDEFGH). When observed under the confocal scanning microscope, development is normal with respect to the existence of the early compartmentalized mycelium and the first and second programmed cell death rounds (not shown). Phenotypic differences among these mutants concern mainly the extension of development of the white appearance on the plate surface and sporulation (Claessen et al., 2004, 2006, data not shown).

As shown in this work, the onset of chaplin and rodlin gene expression (14–17 h) takes place at early time points that coincide with the development of the second syncytial mycelium from the viable segments of the first compartmentalized mycelium. However, not all hyphae of the early second mycelium express the rodlin and chaplin genes, although their number increased progressively until the aerial mycelium became macroscopically visible (compare Fig. 1e with Fig. 2d or Fig. 1d with Fig. 3d). Expression of the S. lividans rodlin genes in surface cultures, measured by specific mRNA production, was previously detected on the second day of cultivation coinciding with the full development of aerial hyphae (Claessen et al., 2002). The apparent lack of expression of chaplin and rodlin genes at earlier time points can be explained by the existence of a horizontal variability as discussed above (Manteca et al., 2005b). In fact, when a sample of mycelium is processed at these early times, the small islands of second mycelium contribute only minimally to the more predominant substrate mycelium; hence, they go unnoticed. By contrast, at later times, when the aerial mycelium covers the entire plate (Claessen et al., 2002), the expression of chaplin and rodlin genes was readily detected. Thus, it can be concluded that the expression of these genes begins well before aerial hyphae become macroscopically visible.

As the chaplin and rodlin genes are expressed after 14–17 h, an interesting question was to analyse whether the surfaces of these hyphae were dotted with rodlets at these times (Claessen et al., 2002). As shown in Fig. 4a, some of the hyphae (those growing in small islands) began to display well-defined structures on their surface after 17 h, which could be early rodlet precursors. When the islands grew in size and became more numerous (Figs. 1e, 2d, 3e), these structures became more conspicuous (Fig. 4b). At the time when the plate was covered with an abundant layer of aerial hyphae (46 h; Fig. 3c), the characteristic fibrous rodlet layer was clearly observed. The formation and structuring of this hydrophobic layer has been proven to occur on the hyphal surface (Claessen et al., 2002, 2003, 2004). Accordingly, a delay between protein expression and the full organization of the rodlet sheet could conceivably take place, as in fact, we have observed in our work.

image

Figure 4.  Freeze-fracturing electron microscopy analysis of the Streptomyces lividans TK23 hyphae. Hydrophobic rodlet-like structures present a less organized appearance at earlier developmental times (a, b; arrows) than at later time points (c). Numbers in brackets indicate growth times.

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Figures 5 and 6 outline the main steps that take place during Streptomyces growth in surface cultures (Manteca et al., 2005b), including the rodlet-related features described in this work. The viable segments of the young compartmentalized mycelium, which grow near the medium/air interface, should be considered the early precursor of the mature hydrophobic aerial mycelium. When these segments begin to enlarge in the form of small islands and express chaplin and rodlin genes, they gradually spread until they cover the entire plate surface. Consequently, the formation of rodlets, which is one of the most characteristic structures of aerial mycelium, begins at time points before those considered previously and comprises most of the developmental cycle. Further molecular and functional studies will clarify additional differences, if any, between both types of second mycelia, i.e., those expressing the hydrophobic structures and the adjacent syncytial hyphae that do not form these proteins.

image

Figure 5.  Schematic representation of the Streptomyces developmental model (horizontal sections). Culture times are indicated. The most significant events are highlighted. Red represents dead cells; green, live cells; and grey lines represent rodlet proteins. Round shapes in phase 6 correspond to mature, differentiated spores.

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image

Figure 6.  Schematic representation of the Streptomyces developmental model (vertical sections). As in Fig. 5, the most significant events are highlighted. Time points are not indicated, as they vary over the horizontal surface culture. Red represents dead cells; green, live cells; and grey lines represent rodlet proteins. The circular shapes along the hyphae in phase (g) correspond to the second syncytial mycelium in which the nucleoids begin to divide. Dotted yellow lines indicate the agar border. Black areas in (g) and (h) indicate a total absence of fluorescence below the presporulated and sporulated mycelium layer, due to the disintegration of the dead hyphae and the degradation of nucleic acids (Manteca et al., 2005b). See text for details.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

The authors wish to thank Priscilla Aio Chase for revising the text and Sonia Ruiz from the Services Cientifico-Tecnicos, Universidad de Barcelona, for the help in electron microscopy sample preparation. This research was funded by grant BIO2004-06089 from the MEC, DGI, Programa Nacional de Biotecnologia, Spain.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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