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Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
Correspondence
Keith Chater
keith.chater{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Cybersense Biosystems Ltd, CEH Oxford, Mansfield Road, Oxford OX1 3SR, UK.
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INTRODUCTION |
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-1,4 linkages and 5 % -1,6 branching linkages. In bacteria, three enzymes typically catalyse glycogen biosynthesis from glucose 1-phosphate: ADP-glucose pyrophosphorylase (GlgC), glycogen synthase (GlgA) and glycogen branching enzyme (GlgB) (Preiss, 1984
).
Glycogen metabolism has a complex interplay with the colony development of streptomycetes. In these actinomycetes, spore germination is followed by the growth of a dense network of branched hyphae (the substrate mycelium), which after about 2 days gives rise to a fuzzy white growth of aerial hyphae (which are often coiled). The initially multigenomic apical compartments of aerial hyphae then undergo multiple septation, ultimately giving rise to chains of unigenomic spores (Chater & Losick, 1997).
Transmission electron microscopy (TEM) has revealed two localized phases of net glycogen deposition during colony development (Braña et al., 1986; Plaskitt & Chater, 1995; Bruton et al., 1995). Phase I deposits are found in a region of the substrate mycelium bordering the aerial mycelium, and phase II deposits are present in the apical compartments of the aerial mycelium as they are undergoing sporulation septation. Glycogen is virtually absent from three other cell-types: the young vegetative hyphae; the regions of aerial hyphae (‘stalks’) immediately below the sporulating apical compartment; and mature spores. It has been suggested that some of the carbohydrate that makes up glycogen is transferred from one phase to the next in the form of trehalose (-1,1-linked diglucose), which is diffusible, and metabolically inert compared with sugars that retain a reducing end (Schneider et al., 2000).
In Escherichia coli, distinct unbranched aggregations of polysaccharide have been observed by TEM of stained thin sections in glgB (branching enzyme) mutants (Lares et al., 1974). Bruton et al. (1995) observed similar structures when they disrupted either of two branching enzyme genes (glgBI and glgBII) in the model species Streptomyces coelicolor A3(2). Each S. coelicolor mutant was affected in just one phase of glycogen deposition. In the glgBI mutant, abnormal phase I deposits (small numbers of large irregular blobs instead of the large numbers of dispersed small granules typical of wild-type deposits) occupied much of the volume of the hyphae at the interface of the substrate and aerial mycelium, while phase II deposits in aerial hyphae appeared normal. On the other hand, the glgBII mutant displayed normal phase I deposition, but the phase II deposits were abnormal, occurring as small numbers of large blobs in the immature spore compartments of the aerial hyphae. The abnormal phase II deposits were less frequent, and much more regularly rounded in appearance, than the unbranched phase I deposits of the glgBI mutant (Bruton et al., 1995).
In this study, we have investigated further the interplay of glycogen and development in S. coelicolor, using a representative set of mutants in the ‘early’ whi developmental genes. These mutants develop abundant aerial mycelium, but lack the regular multiple sporulation septa typical of the wild-type, and produce no spore-like bodies (Hopwood et al., 1970; Chater, 1972; McVittie, 1974; Schwedock et al., 1997; Flärdh et al., 1999). The early whi genes are involved, directly or indirectly, in the regulation of later sporulation genes including those for the grey spore pigment (reviewed by Chater, 2001). The mutants therefore have white, instead of grey, aerial mycelium.
The most studied early whi gene, whiG, encodes a member of a specialized subgroup of sigma (
) factors, whose best-known members are involved in motility in various bacteria (Chater et al., 1989). The level of WhiG is critical in initiating sporulation (Chater et al., 1989), and whiG mutants have long, straight, undifferentiated aerial hyphae. Mutants in the remaining well-studied early whi genes display at least some coiling of aerial hyphal tips (Chater et al., 1989; Flärdh et al., 1999). Among theses genes, whiA encodes a protein of unknown function with orthologues in several other Gram-positive bacteria (Aínsa et al., 2000); whiB belongs to a gene group whose products code for actinomycete-specific small cysteine-rich regulatory proteins of unknown structure (Davis & Chater, 1992; Soliveri et al., 2000); whiH encodes a member of the GntR family of transcription factors (Ryding et al., 1998); and whiI encodes an atypical member of the kind of response regulator usually associated with histidine protein kinases in bacterial two-component systems (Aínsa et al., 1999). Both whiH and whiI are direct targets for RNA polymerase holoenzyme containing WhiG (Ryding et al., 1998; Aínsa et al., 1999).
We set out to investigate the dependence of the two glgB genes on whiA, B, G, H and I, using three isogenic sets of whi mutants. These were wild-type for the glgB genes, mutant in glgBI, or mutant in glgBII. Their patterns of glycogen deposition were analysed by TEM. The mutant glycogen branching phenotypes presented cytological markers that could be used to ascertain whether and where the phase I and/or phase II glycogen branching enzymes were active in a particular whi mutant. The results showed that phase II deposition is closely dependent on whiG, and that glycogen present throughout the aerial hyphae of a whiG mutant is branched by the action of GlgBI. This observation has suggested that there may be an additional, sporulation-independent, and ecologically meaningful role for aerial growth.
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METHODS |
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) was the origin of all strains utilized in this study. Media and conditions for Streptomyces culture, mating and protoplast transformation were as described in Kieser et al. (2000).
Construction of whi mutants.
To introduce hyg-disrupted versions of whi genes into J1508, DNA from previously constructed disruptants of S. coelicolor M145 (Ryding et al., 1998; Flärdh et al., 1999; Aínsa et al., 2000) was denatured and used to transform J1508 protoplasts as described by Oh & Chater (1997). Details of the constructed strains are given in Table 1.
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). Recombinants resistant to both hygromycin and thiostrepton were readily obtained, and Southern blotting was used to confirm the disruption of the relevant whi and glgB genes.
Electron microscopy.
Preparation of samples and transmission electron microscopy of thin sections were as previously described by Thiéry (1967) and Plaskitt & Chater (1995). Colonies were grown for 4 days on minimal medium (MM) containing mannitol (0·5 %) as carbon source. In order to recognize aerial hyphae of whi mutants in thin sections (in the absence of stages of sporulation) we used three kinds of information (as in Plaskitt & Chater, 1995): position in the colony; the absence of a surrounding matrix of agar, which showed as a weakly stained and somewhat granular background; and the occurrence, where appropriate and available, of curved hyphal compartments. The examples shown in the figures are chosen to illustrate the morphology of the deposits, and are not necessarily representative of the full range of glycogen abundance levels.
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RESULTS |
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found that glycogen was present in the aerial hyphae of a series of whi gene point mutants obtained by mutagenesis of the wild-type S. coelicolor A3(2). In the whiA, whiB, whiH and whiI mutants these deposits were spatially separated from phase I (substrate mycelium associated) glycogen, suggesting that the developmentally distinct phases were undisturbed in these mutants. In contrast, in the whiG mutant glycogen was present in nearly all cell compartments of the colony, other than young vegetative mycelium, making it impossible to recognize the two discrete phases of glycogen deposition. Here we first set out to verify these observations using a series of constructed null whi mutants, all containing the hygromycin-resistance cassette (hyg), in an isogenic genetic background (J1508 was chosen because it accumulated comparatively large amounts of glycogen: Schneider et al., 2000). Thin sections of colonies that had been grown on solid medium were stained with silver proteinate to reveal polysaccharide deposits, and examined by TEM. After this treatment, glycogen granules are seen as many small dark spots dispersed in the cytoplasm (sometimes densely packed), and unbranched polyglucose (in branching enzyme mutants) is seen as large blobs, typically one per hyphal compartment.
The previously observed glycogen deposition phenotypes of the parental strain J1508 and its glgBI- and glgBII-disrupted strains (J1861 and J1859 respectively) (Bruton et al., 1995; Plaskitt & Chater, 1995) were confirmed. J1508 (Fig. 1) and its whiA, B, H and I derivatives (Fig. 2) all showed phase I glycogen granules in many hyphal compartments of the substrate mycelium just below the air–agar interface, with a glycogen-free zone in the aerial mycelium above it. The upper parts of the aerial mycelium of J1508 contained phase II glycogen granules associated particularly with the initiation of spore compartments. Thick-walled mature spores contained little or no glycogen. Likewise, the whiA, B, H and I mutants contained some glycogen in the upper parts of the aerial hyphae. Compared with the parental strain J1508 and with each other, the amounts were low in the whiA and whiB mutants, fairly low in the whiI mutant, and quite abundant in the aerial mycelium of whiH. In contrast to all the other strains, the whiG mutant displayed large amounts of glycogen dispersed throughout the undifferentiated aerial mycelium (Fig. 2). All these observations were similar to those of Plaskitt & Chater (1995), though in general we found relatively few curled aerial hyphae in the J1508-derived whiA, B, H and I mutants compared with previous observations on equivalent A3(2)- and M145-derived mutants. This indicated that neither the newly constructed mutants nor the earlier mutants had unsuspected secondary mutations influencing glycogen deposition.
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If, as Plaskitt & Chater (1995) suggested, phase II glycogen deposition is positively regulated, directly or indirectly, by the whiG gene product (WhiG), all glycogen present in a whiG mutant, even that in the uppermost parts of the developmentally defective aerial hyphae, would have been processed by the phase I branching enzyme GlgBI. In a whiG glgBI colony all granules of glycogen should therefore be replaced by large blobs of unbranched polysaccharide throughout the colony. This was indeed observed (Fig. 3), and no ‘normal’ glycogen was observed anywhere in the whiG glgBI colony. Thus, in the whiG mutant, all glycogen branching, even in the aerial mycelium, depends on the activity of GlgBI, showing not only that GlgBII is inactive in the whiG mutant, but also that the usual shut-off of net glycogen deposition in the growing aerial hyphae does not take place. On the other hand, since two spatially separate phases of glycogen deposition had been found in the other whi mutants examined, it seemed likely that both branching enzymes were active in a spatially appropriate manner in those mutants. Thus, we predicted that the substrate mycelium of double mutants of glgBI with whiA, whiB, whiH and whiI should contain large irregular aggregations of polysaccharide, while normal dispersed granules of glycogen, reflecting activity of GlgBII, were expected in the aerial hyphae. This prediction was borne out (Fig. 3).
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). On the other hand, a whiG glgBII mutant was expected to be indistinguishable from a whiG glgB+ mutant, since glgBI, which had been implicated in branching of all the glycogen throughout the colony of a whiG mutant, was still present. Data such as those in Fig. 4 confirmed this prediction.
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DISCUSSION |
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Different results were obtained with whiG mutants, in which we found no evidence of any GlgBII activity; instead the aerial hyphae contained abundant glycogen whose branching depended specifically on GlgBI. The expression of glgBII therefore depends on whiG, but not on whiH or I (both of which are themselves directly and completely whiG-dependent; Ryding et al., 1998; Aínsa et al., 2000) or whiA or B (which are whiG-independent; Aínsa et al., 1999). The abundance of aerial-mycelium-associated glycogen in whiG mutants may reflect the absence of WhiH from such mutants, if WhiH does indeed influence the quantity of glycogen in aerial hyphae (see above).
A search of the DNA sequence upstream of the glgBII operon (as far as the diverging coding sequence SCO7337, and including SCO7336, an uncharacterized small gene that may also be part of the operon: Schneider et al., 2000) revealed some regions similar to the consensus ‘–10’ sequence for WhiG-dependent promoters, but we were not successful in attempts to identify the transcript of the glgBII operon, so we have yet to establish a role for these sequences. None of the potential promoter sequences was conserved in the genome of Streptomyces avermitilis (Ikeda et al., 2003). This indicates that the WhiG-dependence of glgBII is likely to be indirect, perhaps via a whiG-dependent regulator that is not essential for sporulation, and which has therefore not been revealed by isolating sporulation-defective mutants.
It seems that in a whiG mutant, glycogen is deposited in the aerial hyphae as if they were physiologically equivalent to the part of the substrate mycelium from which aerial hyphae emerge. A plausible interpretation of this observation is that, in the wild-type, emerging aerial hyphae are initially like those of the whiG mutant but, as they extend, the WhiG form of RNA polymerase may become active and then cause commitment to a sporulation-specific cell fate. In the absence of WhiG, this developmental decision cannot be made, and an alternative non-reproductive kind of aerial growth continues.
Interestingly, scanning electron micrographs of soil-growing streptomycetes (Wellington et al., 1990) clearly indicate that hyphae can extend across air spaces to form bridges between soil surfaces. This observation makes us wonder if the extended non-reproductive aerial growth seen in whiG mutants might ever occur and have a role in the life of the wild-type S. coelicolor. We suggest that, in some soil microenvironments, some aerial hyphae may fail to activate WhiG, giving a whiG mutant phenocopy, and take on an alternative exploratory role, which could lead to the colonization of nearby soil surfaces (Fig. 5). The repeated deposition of glycogen in newly formed compartments during the extension of such hyphae may help to fuel further growth into the air. The occurrence of these deposits implies some form of transport of glycogen precursors from the substrate mycelium. Because such exploratory growth would not be useful unless other colonizable surfaces were nearby, a sensing mechanism based on the increased accumulation of volatile signals in partially enclosed small spaces could be imagined, which might account for the tufts of long white hyphae that are often seen to emerge from sporulating agar surface cultures of streptomycetes on prolonged incubation on Petri dishes or slants.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 June 2004; revised 3 November 2004; accepted 11 November 2004.
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