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Journal of Bacteriology, March 2007, p. 2219-2225, Vol. 189, No. 6
0021-9193/07/$08.00+0     doi:10.1128/JB.01470-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Changes to the Cell Surface during the Life Cycle of Streptomyces coelicolor: Atomic Force Microscopy of Living Cells

Ricardo Del Sol,² Ian Armstrong,¹ Chris Wright,¹ and Paul Dyson^2*

Nanotechnology Centre, School of Engineering,¹ School of Medicine, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, United Kingdom²

Received 18 September 2006/ Accepted 18 December 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell surface changes that accompany the complex life cycle of Streptomyces coelicolor were monitored by atomic force microscopy (AFM) of living cells. Images were obtained using tapping mode to reveal that young, branching vegetative hyphae have a relatively smooth surface and are attached to an inert silica surface by means of a secreted extracellular matrix. Older hyphae, representing a transition between substrate and aerial growth, are sparsely decorated with fibers. Previously, a well-organized stable mosaic of fibers, called the rodlet layer, coating the surface of spores has been observed using electron microscopy. AFM revealed that aerial hyphae, prior to sporulation, possess a relatively unstable dense heterogeneous fibrous layer. Material from this layer is shed as the hyphae mature, revealing a more tightly organized fibrous mosaic layer typical of spores. The aerial hyphae are also characterized by the absence of the secreted extracellular matrix. The formation of sporulation septa is accompanied by modification to the surface layer, which undergoes localized temporary disruption at the sites of cell division. The characteristics of the hyphal surfaces of mutants show how various chaplin and rodlin proteins contribute to the formation of fibrous layers of differing stabilities. Finally, older spores with a compact rodlet layer develop surface concavities that are attributed to a reduction of intracellular turgor pressure as metabolic activity slows.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The model organism Streptomyces coelicolor represents a group of soil-dwelling filamentous bacteria responsible for synthesis of a wide range of bioactive secondary metabolites, in particular, antibiotics. A good understanding of streptomycete biology has been established, based on extensive studies of S. coelicolor over many years and, more recently, availability of this bacterium's complete genome sequence (1). Of particular interest is the complex streptomycete life cycle. After spore germination, vegetative growth leads to formation of a mycelium consisting of a ramifying network of syncytial hyphae that penetrate a moist substrate by extension of hyphal tips and subapical branching. Subsequent reproductive growth often coincides with the onset of antibiotic production and proceeds with the formation of filamentous aerial hyphae that eventually undergo differentiation into chains of unigenomic spores. Several genes that are critical to various stages of this morphological differentiation have been described in S. coelicolor (5, 15), including bld genes, required for the initial growth of aerial hyphae, and whi genes, needed for the subsequent development of spore chains. Growth into the air is accompanied by a change in cell surface properties: vegetative hyphae growing in moist substrates have hydrophilic cell surfaces, whereas the aerial hyphae and spores are hydrophobic. A developmental sigma factor, encoded by the bldN gene, influences expression of two types of hydrophobic surface-active proteins that decorate aerial hyphae (11). The chaplins consist of eight members, ChpA to ChpH, that self-assemble into amyloid fibrils (7, 11). In addition, the rodlin proteins, RdlA and RdlB, are believed to organize chaplin fibrils into "rodlets" (8). When grown on rich medium, S. coelicolor also secretes a lantibiotic-like peptide, SapB (17), which lowers the medium surface tension and is thought to enable hyphae to breach the medium-air interface to grow into the air (21). Whereas the bld regulatory cascade controls synthesis of SapB, the chaplin and rodlin genes are believed to belong to a "sky" regulon that operates between the bld and whi pathways (6).

For many years, electron microscopy has been the only technique available for examining microbial surfaces at high resolution (2, 3), and, combined with cryotechniques, this allows fine-structure imaging of vitrified cell structures in conditions close to the native hydrated state (18, 22). However, these procedures are technically demanding and do not offer the opportunity for direct observation of living cells. Atomic force microscopy (AFM) is an established imaging technique that is an important and flexible tool for characterizing biological materials. This technique has a number of advantages over other microscopies as biological surfaces can be studied in real time with high resolution in different environments, gaseous or aqueous, with very little surface preparation and no surface coating. Thus, AFM has been used to give some very detailed images of microorganisms, including bacterial cells (9, 10). Here, we exploit AFM to characterize the surfaces of growing hyphae and spores of S. coelicolor. This offers a unique insight into the changes to the cell surface that accompany growth and morphological differentiation of this organism.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria and growth conditions. The following strains were used in this study: S. coelicolor strain M145 (16), S. coelicolor rdlAB (8), S. coelicolor chpABCDH (7), S. coelicolor chpABCDEFGH (8), and Streptomyces avermitilis 12804 (NCIMB, United Kingdom). To visualize vegetative and aerial growth, the strains were grown in the acute angle of a coverslip inserted in a sporulation medium, mannitol soy agar, as previously described (4, 16). Inserted coverslips were removed at different times of growth to visualize different developmental stages.

AFM. The AFM instrument used was a Dimension 3100 (Nanoscope IV controller; Thermomicroscopes). S. coelicolor cells grown attached to glass coverslips were imaged in air (at 20°C and 40% rH). Hyphae at specific stages of development were first identified using the optical microscope attached to the AFM. AFM images were obtained using the intermittent contact mode of the instrument. Standard tapping mode tips (Olympus) were used (cantilever nominal spring constant, k = 0.064 N m^–1). A scan rate of 1 Hz was employed with resolution of 512 by 512 or 1,024 by 1,024 lines. Height and phase images were simultaneously recorded. Height images provide quantitative information on sample surface topography whereas phase images, although they do not represent the true topography of the sample, reveal a higher sensitivity to small surface features, resulting in images with greater detail, as previously observed (19). Phase images are derived from the phase angle between the actual vibration and the applied signal used to oscillate the AFM cantilever so that the tip intermittently taps the surface and reduces the lateral force that is applied during imaging. In this study we present largely phase images, except where stated. To quantify dimensions, multiple measurements were made on at least six independently grown biological samples. Unless otherwise stated, a mean value was derived; standard deviations were 17% of these mean values.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vegetative growth. Figure 1 shows representative AFM images of vegetative hyphae recorded in tapping mode. As seen in Fig. 1A, a "germ tube" is 0.62 µm wide along its length but slightly wider (0.91 µm) where it emerges from the spore, with a rather smooth cell surface. A 1.2-µm-wide extracellular film deposited on the glass surface apparently forms an adhesive substrate for the growing germ tube (see below). A higher-resolution image (Fig. 1B) reveals a uniform, slightly roughened surface to this young hypha. A similar surface is evident in older (12 h postgermination), branching hyphae, as represented in Fig. 1C. In this image, a branch has newly emerged from the main hypha, some 1.85 µm distal from the position of an indentation (shown in higher resolution in Fig. 1D) that we interpret as the position of a cross-wall. The branch has emerged from the center of the reflex angle of an approximate 70° bend in the main hypha. At the point of emergence from the main hypha, the branch is only 0.42 µm wide but attains a normal width of 0.62 µm some 2 µm distal from the branch site. A remarkable feature revealed by AFM is an extracellular matrix up to 1.5 µm wide that is deposited on the glass as the hyphae grow. Two such layers are evident in Fig. 1C: a relatively thick (15 to 20 nm), wide matrix deposited by the main hypha and a thinner (3 to 5 nm) and narrower layer formed around the younger, emerging branch. A higher-resolution image of the thicker matrix (Fig. 1D) reveals a granulated surface, with individual granules measuring 20 to 40 nm in diameter. Hyphae grown in submerged culture and transferred to glass immediately prior to imaging lack such an extracellular matrix (results not shown).

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FIG. 1. Progression of surface detail changes during vegetative growth of S. coelicolor. All images are phase images. (A) Germinating spore and germ tube. (B) Surface detail from panel A, displaying some roughness of the germ tube surface. (C) Vegetative hypha and growing branch, surrounded by extracellular matrix. (D) Higher magnification of the vegetative septum and extracellular matrix from panel C. (E and F) Initial stages of assembly of a fibrous layer, prior to aerial growth, with evident differences in the degree of fiber density. (G) High magnification of fibers of panel F, showing single and presumptive end-to-end, longer fibers. Scale bars are indicated in each panel.

Imaging older hyphae (20 h or more postgermination but before macroscopic evidence of aerial development) reveals that the cell surface away from the growing tip is decorated with irregularly spaced fibers of differing dimensions (Fig. 1E, F, and G). We observed that the abundance of these rods increased progressively (compare Fig. 1E, 20 h postgermination, and F, 22 h postgermination). The maximum length of the fibers measured was 140 nm, with the majority being 60 to 68 nm long (Fig. 1G). The longer fibers are likely to consist of two subunits arranged end to end. The width of the fibers ranged from 17 nm to 22 nm. Some are evidently shed during growth and contribute to the complexity of the extracellular matrix deposited on the glass surrounding the hyphae.

Aerial development. In comparison to the vegetative hyphae, there are two striking differences in all images of aerial hyphae and spores obtained by tapping mode AFM; Fig. 2 shows representative images. First, for young aerial hyphae imaged after 30 h growth, there is an absence of the granulated extracellular matrix secreted around the vegetative hyphae, even though the aerial hyphae attach to the glass coverslip as they grow (they did not require attachment with poly-L-lysine prior to imaging, although, compared to the vegetative hyphae, aerial hyphae were reproducibly easier to dislodge by movement of the AFM tip). Second, the cell surfaces are composed entirely of a dense mosaic of short fibers. Assuming that the fibers are comprised of hydrophobic chaplin and/or rodlin proteins (see below), the layer surrounding the tip (Fig. 2B) can presumably help a young growing aerial hypha break the surface tension at the interface between a moist substrate and the air. Initially, the fibrous layer appears to be relatively disorganized (Fig. 2A and B). The disorganized fibrous material is evidently sloughed off during growth (Fig. 2D), implying that it is only loosely attached to the hyphal surface, leaving a more organized mosaic layer on the surface of older hyphae. A fibrous layer is maintained during subsequent differentiation of an aerial hypha as a spore chain develops (Fig. 2C to H) but appears much more compact and organized on spores (compare Fig. 2A and H). Development of spore compartments occurs after growth arrest of an aerial hypha. Staining hyphae with probes to detect either chromosomal partitioning or cell wall synthesis, coupled with fluorescence microscopy, has revealed synchronous multiple septum formation along aerial hyphae as uninucleoid spore compartments are formed (20). The first indications of cell wall changes accompanying spore formation revealed by AFM are regularly spaced surface indentations in apical regions of hyphae (Fig. 2D); the spacing between indentations is on average 1.2 µm, consistent with the spacing between sporulation septa detected by fluorescence microscopy (20). Subsequent modification of the fibrous layer occurs during cell division, presumably as a consequence of changes in the underlying cell wall (Fig. 2E and F). At this stage the mosaic of fibers is temporarily disrupted in a ring above the point of septation, revealing a smoother surface that we assume is the cell wall. Some of the disassembled fibrous material is deposited adjacent to the hyphae (Fig. 2F). However, as the spores subsequently round up, the mosaic layer is restored to completely cover the surface (Fig. 2H).

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FIG. 2. Heterogeneity of the fibrous layers during aerial development of S. coelicolor. All images are phase images unless otherwise stated. (A) Disorganized fibrous layer of presporogenic aerial hypha. (B) An aerial hypha prior to sporulation septation, showing complete coverage of the tip by the fibrous layer. (C) Presporogenic hypha shedding fibrous material. (D) An aerial hypha shedding fibrous material and undergoing sporulation septation revealed by regular indentations on the surface. (E) A later stage of sporulation septation with localized disassembly of the fibrous layer above the position of cell division. (F) Lower magnification of localized disassembly of the fibrous layer at positions of cell division. (G) Three-dimensional image of a mature spore chain, displaying concavities and differences in rotational planes between spores. (H) Phase image of a spore chain showing a more compact rodlet layer (compared to panels A to C), concavities, and reassembly of the rodlet layer at the junctions between spores. Scale bars are indicated in each panel.

A final change evident in mature spore chains is the appearance of concavities in the surface of individual spores (Fig. 2G and H). This is entirely reproducible and is observed in spore chains of living cultures grown for 96 h or more. We believe the spore indentations arise due to a slowing in metabolic activity and a resultant loss of internal turgor due to partial dehydration. Although the plane of the surfaces that undergo indentation changes in individual spores along the length of a spore chain (Fig. 2G), we have not observed any regularity to suggest rotation of spores along a chain.

Characteristics of the fibrous layers are determined by rodlins and chaplins. The rodlet layer observed on the surface of spores and aerial hyphae using electron microscopy is believed to consist of fibrils of chaplin proteins organized by the two rodlin proteins, RdlA and RdlB, into individual rodlets (8). Using AFM, we examined the surface of 5-day-old hyphae of both a mutant lacking all eight chaplin genes and a mutant expressing only three chaplins, ChpEFG. The former resembles the immature substrate hyphae of the wild type in that no fibrous surface decoration is apparent (data not shown). The mutant expressing ChpEFG is covered in long fibrils 120 to 250 nm in length and 15 to 20 nm wide (Fig. 3A). However, a very large amount of this material is deposited on the glass surrounding the hyphae, suggesting that the missing chaplin proteins promote adhesion of the fibrous layer to the cell wall. A mutant expressing only ChpF and ChpG has similar characteristics (data not shown).

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FIG. 3. Comparison of the fibrous layers of S. coelicolor chaplin and rodlin mutants and of S. avermitilis. All images are phase images unless otherwise stated. (A) Unstable fibrous layer of an aerial hypha of the chpABCDH mutant. (B) Scaly fibrous layer covering an aerial hypha of an rdlAB mutant. (C) Three-dimensional image of nondecorated spores of the rdlAB mutant. (D and E) Fibrous filaments coating presporogenic aerial hyphae of S. avermitilis. (F) Higher-magnification height image of extruded filaments from panel D, showing subunit detail. (G) Detail of the fibrous layer coating a spore chain of S. avermitilis. Scale bars are indicated in each panel.

To assess the role of the rodlin proteins, we examined a mutant in which both rdlA and rdlB were deleted. In this mutant, presporogenic aerial hyphae are coated in semispherical scales of various dimensions rather than fibrils or rodlets (Fig. 3B). The scales are evidently shed onto the surrounding glass but not to the same extent as the extensive fibrous deposits of the mutant expressing only ChpEFG. However, instability of this scaly fibrous layer is suggested by the observation that the surfaces of spore chains lack substantial decoration (Fig. 3C).

Finally, again to assess the role of rodlin proteins, we turned our attention to another streptomycete, S. avermitilis. The genome of this organism has also been fully sequenced, and subsequent annotation revealed the presence of four chaplin genes but no orthologs of the S. coelicolor rodlin genes. The fibrous surface layer of this organism is quite distinct: developing hyphae extrude very long (up to 3 µm in length) filaments, 25 nm wide, that coat the hyphae and form networks on the surrounding substrate (Fig. 3D and E). These filaments appear to consist of shorter fibrous subunits, approximately 70 nm in length, arranged end to end (Fig. 3F). Obtaining high-resolution images of older sporulating hyphae proved problematic, possibly due to a mucous layer overlying the fibrous layer. However, the images obtained of spore chains revealed a surface consisting of a dense network of filaments (Fig. 3G).

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have illustrated how AFM can be used to characterize changes to the cell surface accompanying development of S. coelicolor. The ability to obtain high-resolution images of living cells with minimal sample preparation has revealed novel aspects of growth of this organism, including the attachment of vegetative hyphae to an inert substrate and the progressive development of the hydrophobic fibrous layer and its subsequent modification during growth and cell division. We are currently attempting to define the nature of the extracellular matrix secreted by growing vegetative hyphae. The appearance of the matrix suggests that it is secreted as a fluid and then dehydrates to form a granulated film. We believe that the matrix allows the vegetative hyphae to adhere to glass and other inert silica-based substrates, for example, clay particles in soil. Consistent with this idea is that vegetative hyphae grown in submerged culture and transferred to a glass slide have a far greater tendency to move when subjected to tapping by the AFM probe and must be attached to the glass with poly-L-lysine for detailed analysis. An interesting observation is that the width of the matrix surrounding a hypha is constant; indeed, this is clearly evident in Fig. 1C, where the matrix secreted by a newly emerging branch partly overlaps the preexisting matrix of the older main hypha. An implication of this is that the matrix material is secreted only from the advancing tip; this itself implies a new functional aspect of the growing tip that differentiates it from the rest of the apical compartment. In contrast, the assembly of fibers that decorate the older vegetative hyphae progresses a little distal from the tip (Fig. 1E and F). The hyphae in these images represent a transition stage in the life cycle, when chaplin and rodlin proteins are expressed in insufficient amounts to form the dense layers present on aerial hyphae and when fibers are largely absent from the growing tip itself. Indeed, expression of only the chpE and chpH genes has been detected during vegetative growth (7). Our data indicate that these chaplin proteins do assemble into decorative fibers rather than simply being secreted into the surrounding medium as monomers, as previously suggested (7). However, as both of these chaplins lack "sorting signals" to permit covalent attachment to the cell wall, their association with the hyphal wall is likely to be weak; indeed, we observed many fibers shed onto the surrounding glass. As a consequence of their sparse decoration, at this stage the hyphae and, in particular, the tips may be insufficiently hydrophobic in nature to transit the medium-air interface.

A dense fibrous layer completely coats aerial hyphae. Previous analysis has indicated that all eight chaplin genes and the two rodlin genes are expressed specifically during aerial development (7, 8, 11). Our AFM analysis indicates that the nature of the outermost fibrous layer changes during aerial development. Initially, the layer is relatively disorganized (Fig. 2A and B) and comprises a heterogeneous assembly of rod-like fibers and more spherical scales. The latter, in particular, resemble the scales coating the aerial hyphae of an rdlAB mutant (Fig. 3B). We assume that this heterogeneous layer consists of assemblies of the different chaplin proteins, some of which may be associated with the rodlin proteins, as the hyphae of a mutant lacking all eight chaplin genes are devoid of any decorative fibers, as are hyphae of several bld mutants (results not shown). Consistent with a loose assembly of fibers on the surface of wild-type hyphae, as aerial development proceeds fibrous material is shed onto the surrounding glass during growth. This sloughing off is most extreme in the mutant expressing only ChpEFG proteins (Fig. 3A). This is not unexpected due to the absence in these fibrils of "long" chaplins (ChpABC) that contain sorting signals that are targets for sortases (7, 11). Sortases cleave between the threonine and glycine residues in a conserved motif and mediate covalent attachment of the C-terminal end of the target protein to the peptidoglycan cross-bridge. The instability of the fibrous layer may help to explain the delay in aerial development that is characteristic of the quintuple chaplin mutant (7, 11).

The most tightly organized fibrous surface layer is evident on the surface of mature spores. This is likely to comprise the mosaic rodlet layer described in the early years of ultrastructure studies of S. coelicolor spores (13, 23) and subsequently investigated more recently at the molecular level (8). In both cases, electron microscopy of vitrified samples has revealed rodlets that measure 60 to 150 nm in length, in good agreement with the lengths of fibers we have observed. Individual rodlets were estimated to have a width of 12 nm, but they are often described as being organized in pairs with members of the pairs separated by a groove 20 nm wide (23). The hydrated fibers coating spores that we observed have a width of between 17 to 22 nm. Pairs of parallel tightly joined fibers are also apparent in the AFM images. We believe that the differences in the width and appearance of the rodlets imaged using the two different techniques are a consequence of the vitrification techniques used for electron microscopy. A further indication that the fibrous mosaic coating wild-type spores we observed is, indeed, the rodlet layer is its absence on the surface of spores of the rdlAB mutant. The rodlin proteins, together with chaplin proteins, are essential for correct formation of this layer (8), and the smooth spore surface of the mutant spores indicates that the fibrous scales of younger aerial hyphae have been shed during maturation.

Extensive disruption of the rodlet layer at the site of spore separation has been observed in electron micrographs (13). AFM reveals much more localized modification of the layer during this stage of development (Fig. 2E and F). The difference in the observed extent of disruption could be a consequence of relatively aggressive sample preparation techniques prior to electron microscopy that remove elements of the layer rendered unstable at the site of cell division due to remodeling of the underlying peptidoglycan. Moreover, whereas the rodlet layer is described as a highly stable insoluble surface layer (7), surrounding layers of nonanchored chaplin fibers are also likely to be removed by the sample preparation techniques preceding electron microscopy of cell surfaces. This may explain why electron microscopy of freeze-etched samples of nonseptated aerial hyphae has previously revealed only the rodlet layer (13, 23). The AFM images suggest that as an aerial hypha grows, less stable surface fibrils are continuously shed, and the rodlet layer is only then revealed as a spore chain matures. Indeed, transmission electron microscopy of thin sections of aerial hyphae suggests a relatively thick fibrous layer (12), likely to be composed of more than a single rodlet layer. We propose that the stable rodlet layer is composed of the rodlin proteins together with the long chaplins A to C that are anchored to the cell wall. The short chaplins, encoded by genes that are expressed in 10- to 25-fold greater amounts than the long chaplins (7), are likely to contribute to the less stable fibrous layer on the surface of growing aerial hyphae. The surface layer of aerial hyphae of S. avermitilis is also relatively unstable, with a lot of very long fibrous material being shed during growth. This species has orthologues encoding the two long chaplins, ChpB and ChpC, and two short chaplins, ChpE and ChpH, but no rodlin genes (14). Electron micrographs of S. avermitilis spores have previously indicated a relatively smooth surface with no evidence of a rodlet layer (8). This contrasts with the network of filaments we observed coating spore chains, again suggesting that this layer is unstable and is lost during sample preparation preceding electron microscopy.

 
R.D.S. was funded by grants from the BBSRC (58/EGI18395) and European Commission (LSHM-CT-2004-005224).

We are grateful to Dennis Claessen, Wouter de Jong, and Lubbert Dijkhuizen for providing the chaplin and rodlin mutants.

 
* Corresponding author. Mailing address: School of Medicine, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, United Kingdom. Phone: 44 1792 295667. Fax: 44 1792 295447. E-mail: p.j.dyson{at}swansea.ac.uk.

Published ahead of print on 28 December 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, March 2007, p. 2219-2225, Vol. 189, No. 6
0021-9193/07/$08.00+0     doi:10.1128/JB.01470-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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