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Deprogrammed sporulation in Streptomyces
FEMS Microbiology Letters 216 (2002) 1^7
www.fems-microbiology.org
MiniReview
Deprogrammed sporulation in Streptomyces Yasuo Ohnishi, Jeong-Woo Seo, Sueharu Horinouchi
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Received 10 June 2002; received in revised form 4 September 2002; accepted 4 September 2002 First published online 2 October 2002
Abstract The bacterial genus Streptomyces forms chains of spores by septation at intervals in aerial hyphae and subsequent maturation on solid medium. Substrate hyphae undergo extensive lysis, liberating nutrients on which aerial hyphae develop. Some mutant strains, however, ectopically form spores by septation in substrate hyphae on solid medium or in vegetative hyphae in liquid medium, which suggests that all hyphae have the potential to differentiate into spores. A Streptomyces griseus mutant strain NP4, which has a mutation in the regulatory system for an ATP-binding cassette (ABC) transporter gene, forms ectopic spores in substrate hyphae only on glucosecontaining medium. In addition, overexpression of a substrate-binding protein of the ABC transporter in the wild-type strain causes ectopic septation in very young substrate hyphae and subsequent sporulation in response to glucose. These ectopic spores germinate normally. The ectopic sporulation is independent of A-factor, a microbial hormone that determines the timing of aerial mycelium formation during normal development. Thus, substrate hyphae of Streptomyces have a potential to develop into spores without formation of aerial hyphae. For programmed development, therefore, the strict repression of septum formation in substrate mycelium should be necessary, as well as the positive signal relay leading to aerial mycelium formation followed by septation and sporulation. 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Morphological development; Ectopic sporulation ; ATP-binding cassette transporter; A-Factor; Streptomyces
1. Introduction The Gram-positive, soil-inhabiting, ¢lamentous bacterial genus Streptomyces shows complex morphological development (Fig. 1), which makes this genus one of the model prokaryotes for studying multicellular di¡erentiation [1,2]. In an early stage of Streptomyces growth on solid medium, spores germinate to subsequently develop into branched, ¢lamentous, multinucleoidal hyphae (substrate hyphae) that contain relatively infrequent, thin, single-layer vegetative septa. Exponential growth is achieved by branching of the hyphae, resulting in the formation of an intricate mycelial network. The ¢rst step of morphological di¡erentiation is the emergence of aerial hyphae by reuse of material assimilated into substrate hyphae, such as proteins, DNA, and storage compounds. Subsequently synchronous septation occurs only in aerial hyphae to form chains of unigenomic compartments separated by
* Corresponding author. Tel. : +81 (3) 58 41 51 23; Fax : +81 (3) 58 41 80 21. E-mail address : [email protected] (S. Horinouchi).
double-layer, thick sporulation septa. At a later stage, chains of the unigenomic compartments are destined to become individual spores. Some Streptomyces strains produce spores in submerged culture when critical nutritional and environmental conditions are met. Streptomyces griseus B-2682, for example, produces abundant submerged spores in nutrient-depleted media [3]. The sporulation process in liquid culture includes the formation of specialized branches designated sporogenic hyphae, in which doublelayer sporulation septa are formed. The submerged spores of S. griseus are similar, but never identical, to aerial spores and are sensitive to lysozyme digestion, probably because of the thinness of the spore wall. Most studies of the Streptomyces development have been accomplished by characterizing developmental mutants of Streptomyces coelicolor A3(2). In principle, these mutants can be divided into two classes : bald (bld) mutants, which fail to produce fuzzy aerial mycelium, and white (whi) mutants, which produce aerial hyphae but cannot form gray-pigmented spores. By complementation experiments with these mutants, at least 15 regulatory genes for morphological di¡erentiation have been identi¢ed and studied at the molecular level, and some of their interac-
0378-1097 / 02 / $22.00 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 9 9 6 - 5
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tions have been characterized. These works were summarized in recent excellent reviews [4,5]. Some mutant strains of Streptomyces produce ectopic spores directly from substrate mycelium, because normal regulatory events blocking division are deprogrammed in the substrate hyphae. For example, S. griseus NP4 produces ectopic spores, which are indistinguishable from normally developed aerial spores, by septation in substrate hyphae. In this mini-review, we put an emphasis on the idea that, in addition to the positive signal relays leading to aerial mycelium formation, a mechanism to strictly inhibit septum formation in substrate hyphae is absolutely necessary for the programmed development of Streptomyces.
2. A-Factor signaling pathway leading to morphological development in S. griseus 2.1. A-factor regulatory cascade that determines the timing of morphological di¡erentiation Many Streptomyces species produce Q-butyrolactones, most of which have been shown to act as chemical signaling molecules for the onset of antibiotic production. AFactor (2-isocapryloyl-3R-hydroxymethyl-Q-butyrolactone) is the most intensively and extensively studied Q-butyrolactone and is exceptionally required for not only antibiotic production but also morphological di¡erentiation in S. griseus [6,7] (Fig. 2). Possibly, during evolution Streptomyces species have developed Q-butyrolactones as ‘hormonal regulators’ to control di¡erent stages of physiological and/or morphological di¡erentiation in the regulatory hierarchy. A-Factor di¡uses freely in an individual hypha and into the neighboring hyphae across the cytoplasmic membrane and induces the transcription of adpA by binding to the A-factor receptor protein (ArpA), which has bound and repressed the adpA promoter, and dissociating it from the promoter [8]. Although AdpA was originally identi¢ed as an A-factor-dependent transcriptional activator for strR encoding the pathway-speci¢c transcriptional activator for streptomycin biosynthetic genes, AdpA has been shown to induce the transcription of a number of genes involved in secondary metabolism and morphogenesis, comprising an AdpA regulon. The intracellular concentration of A-factor thus determines the timing of adpA expression, which results in the switch-on of all the downstream genes in the regulatory cascade. In fact, an adpAdisruptant produces neither streptomycin nor a yellow pigment and shows a bald phenotype. Until now, we suppose that the A-factor signaling pathway is responsible for the onset of aerial mycelium formation and the e¡ects of A-factor on the later processes in the development would not be so signi¢cant (Fig. 2). The only exception we have observed is that AdpA appears to control the transcription of ssgA, which is involved in
spore septum formation in aerial hyphae (unpublished data). The following examples are the genes that are activated by AdpA at a speci¢c time and required for the onset of aerial mycelium formation. 2.2. Extracytoplasmic function (ECF) sigma factor adsA encoding an ECF sigma factor (cAdsA ) is one of the target genes of AdpA [9]. Transcription of adsA begins approximately at the time of aerial mycelium formation. Disruption of the chromosomal adsA gene results in loss of aerial hyphae formation but not streptomycin and yellow pigment production, indicating that cAdsA is involved only in morphological development and not in secondary metabolic function. S. coelicolor A3(2) bldN, the orthologue of adsA, plays the same role as adsA [10]. Interestingly, cBldN is responsible for the transcription from the p1 promoter of another bld gene, bldM, which encodes a response regulator-like protein. We assume that the e¡ect of A-factor on morphological di¡erentiation is mediated mainly by cAdsA , which would activate many genes required for aerial mycelium formation. 2.3. Metallo- and serine-proteases sgmA encoding an extracellular metalloendopeptidase (SGMP II) is another direct target of AdpA [11]. Disruption of the chromosomal sgmA gene results in a slight but distinct delay of aerial hyphae formation, suggesting that SGMP II is somehow involved in morphological development. Extracellular proteases, together with other hydrolytic enzymes, are involved in the cannibalistic reuse of material in substrate hyphae during aerial hyphae formation. It is noteworthy that several other extracellular serine-protease genes, such as sprA, sprB, sprD, and sprT, are also transcribed in an AdpA-dependent manner (our unpublished data). It is also possible that some of them work as processing enzymes for extracellular signaling molecules involved in morphological development. 2.4. AmfS, a peptidic morphogen A processed product of AmfS acts as an extracellular peptidic morphogen of S. griseus [12]. amfS encoding a 43amino acid peptide is a member of the amf gene cluster, which was previously identi¢ed as a regulator for the onset of aerial mycelium formation [13]. The amf genes contains ¢ve open reading frames: amfT encoding a transmembrane protein similar to protein Ser/Thr kinases; amfS; amfB and amfA both encoding an ATP-binding cassette (ABC) transporter; and amfR encoding a response regulator-like protein of two-component regulatory systems. The ram gene cluster, which has the same gene organization as amf, in S. coelicolor A3(2) and Streptomyces lividans appears to play the same role in aerial mycelium formation (see references in [4,5]). Mutational analyses
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and extracellular complementation tests demonstrated that AmfS is essential for aerial mycelium formation and secreted via the AmfAB transporter. A synthetic C-terminal octapeptide, but not the full-length peptide of AmfS, partially induced aerial mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen. AmfR is perhaps a transcriptional activator for the amf genes. An adpA-disruptant lacks extracellular AmfS activity, suggesting that the regulation by AmfS is under the control of A-factor and AdpA. We previously reported that amfR was co-transcribed with two upstream open reading frames (orf5 and orf4 in this order) and their transcription depended on A-factor [14]. However, our recent analysis has shown that the orf5 promoter is rather constant and the amfR promoter itself depends on A-factor (our unpublished results). Our preliminary experiments suggest that AdpA directly binds the upstream region of the amfR promoter, showing direct control of amfR expression by AdpA.
3. Ectopic, deprogrammed sporulation by Streptomyces mutants Spores of Streptomyces are usually formed only in aerial hyphae on solid medium and in sporogenic hyphae in liquid medium. However, some mutant strains sporulate ectopically in substrate hyphae on solid medium or in vegetative hyphae in liquid medium, suggesting that all hyphae, irrespective of substrate or aerial hyphae, have the potential to di¡erentiate into spores. A better understanding of these mutations will give us new insights into the morphological di¡erentiation of Streptomyces. 3.1. Overexpression of whiG whiG encodes a sigma factor of RNA polymerase, which speci¢cally initiates the development program that leads to spore formation from aerial hyphae [4]. In S. coelicolor A3(2), the RNA polymerase cWhiG holoenzyme transcribes regulatory genes, whiH and whiI, both of which consist of a DNA-binding domain and a domain usually involved in signal sensing. The copy number of whiG has a dramatic e¡ect on development of S. coelicolor A3(2); in the absence of cWhiG , the aerial hyphae develop no sporulation septa, whereas an excess amount of cWhiG causes hypersporulation in aerial hyphae on solid medium and occasional ectopic sporulation (ESP) in vegetative hyphae both on solid and in liquid medium [15]. Thus the activity of cWhiG determines the developmental fate of hyphae. 3.2. Deletion of DNA located close to the glkA locus A 7.4-kb deletion of DNA lying close to, but distinct from glkA, causes ectopic sporulation in substrate hyphae
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of S. coelicolor A3(2) on solid medium [16]. The ESP phenotype associated with the 7.4-kb deletion depends on the whiG function, since introduction of a whiG mutation into the deletion mutant blocks sporulation in both aerial and substrate hyphae. Because whiG is activated posttranscriptionally, a simple explanation for this is that some protein that represses the cWhiG activation in substrate hyphae is encoded in the 7.4-kb region. The genome sequencing denies the possibility that an anti-sigma factor for cWhiG is encoded in this region. The open reading frame(s) responsible for the ESP phenotype remains to be identi¢ed. 3.3. Overexpression of ssgA ssgA encoding a 15-kDa acidic protein is involved in spore septum formation in both S. griseus [17] and S. coelicolor A3(2) [18]. In both strains, an ssgA-disruptant shows a white phenotype. In S. coelicolor A3(2), overexpression of ssgA results in formation of occasional septa in substrate hyphae and subsequent spore-like compartments in liquid culture. In S. griseus, a point mutation in the regulatory region for ssgA results in fragmentation of mycelium and formation of spore-like compartments, although the e¡ect of the mutation on ssgA transcription remains to be elucidated. The function of SsgA in septum formation is unknown. 3.4. Overexpression of a substrate-binding protein of an ABC transporter We recently isolated and analyzed a bald mutant, NP4, derived by UV mutagenesis from the wild-type S. griseus IFO13350 [19]. Mutant NP4 forms ectopic septa at regular intervals along substrate hyphae in response to glucose, and each of the compartments develops into a spore (Fig. 1). The ectopic septa are formed only on medium containing glucose, but not on medium containing other carbon sources. This is why NP4 shows a bald and wrinkled colony morphology. The ectopic spores are indistinguishable in size and shape, and thickness of spore cortex from the aerial spore of the parental S. griseus, as determined by transmission electron microscopy. The ESP occurs also in liquid medium, and we say that a mutation(s) in NP4 can change Streptomyces into a ‘nocardioform’ bacterium. The septum in substrate hyphae in NP4 is formed at a similar timing to that when the aerial septum is formed by the wild-type strain (Fig. 3). This is a vivid contrast to the septation in substrate hyphae by the wild-type strain carrying multiple copies of dasA (see below). Shotgun cloning experiments with a library of chromosomal DNA of the parental strain and mutant NP4 as the host yielded DNA fragments giving two di¡erent phenotypes ; one (dasR) complementing the bald phenotype of the host, and the other (dasA) causing a much more severe wrinkled morphology in the host. dasR encodes a tran-
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Fig. 2. A-Factor regulatory cascade leading to aerial mycelium formation in S. griseus. A-Factor is gradually accumulated in a growth-dependent manner. AfsA, a probable A-factor synthesis enzyme, is involved in A-factor production. When the concentration of A-factor reaches a certain critical level, it binds the A-factor receptor protein ArpA, which has bound the promoter region of adpA, and dissociates ArpA from the promoter, thus leading to transcription and translation of adpA. AdpA is a pleiotropic activator essential for both morphological development and secondary metabolism. AdpA activates adsA encoding an ECF sigma factor (cAdsA ) and sgmA encoding a metalloendopeptidase (SGMP II). cAdsA is essential for the onset of aerial mycelium formation, probably activating multiple genes involved in aerial mycelium formation. SGMP II is also suggested to be involved in morphological development. AdpA also activates transcription of other protease genes. Transcription of amfR, which encodes a response regulator-like protein and is essential for AmfS production, is directly controlled by AdpA. AmfS is an extracellular peptidic morphogen that is essential for aerial mycelium formation.
scriptional repressor belonging to the GntR family and dasA encodes a lipoprotein probably serving as a substrate-binding protein in an ABC transport system. These genes, together with dasB and dasC, both encoding a membrane-spanning protein, are members in the gene cluster for an ABC transporter. DasR represses the transcription of dasA by directly binding to its promoter (our
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unpublished data). Nucleotide sequencing and transcriptional analysis of the dasR^dasA region in NP4 suggested that NP4 has a defect in the regulatory system to control the expression of these genes, but not in the dasR or dasA. The transcription of both dasA and dasR is greatly enhanced in mutant NP4. The presence of multiple copies of dasA in the wild-type strain causes the host to form ectopic septa in very young substrate hyphae after only 1 day of growth and subsequent spores in response to glucose (Fig. 3). The timing is a vivid contrast with that of ectopic sporulation by mutant NP4 that forms ectopic septa in substrate hyphae at days 3^4 under the same conditions. The ectopic spores formed by the wild-type containing multiple copies of dasA have a thinner wall than those of mutant NP4 (Fig. 1), in agreement with the observation that the former is sensitive to lysozyme and heat. This implies that some of the gene products necessary for the architecture of aerial spores are absent during the maturation of the ectopic spores. These ectopic spores, however, germinate at more than 90% frequency. The ectopic septation and sporulation in the wild-type strain carrying multicopies of dasA is independent of A-factor, since an A-factor-de¢cient mutant HH1 carrying multicopies of dasA also shows the ESP phenotype. This is also supported by the observation that the wild-type strain carrying multicopies of dasA forms ectopic septa after only 1 day of growth, when the concentration of A-factor is still low. As described above, A-factor is essential for the onset of aerial mycelium formation. No dependence of ectopic sporulation in substrate hyphae of the dasA-overexpressing strains on A-factor clearly indicates no obligatory link between aerial mycelium formation and sporulation. How is the ABC transporter system involved in the ESP phenotype? A simple explanation is that some signal molecule for morphological development is imported via the ABC transporter. The e¡ect of glucose on the ectopic sporulation of mutant NP4 and on the wild-type strain containing multicopies of dasA tempts us to speculate that DasA recognizes and binds glucose or a glucose derivative and imports it via the Das system, thus inducing the ESP phenotype. However, it is likely to us that DasA interacts with some protein in the membrane, such as a sensor kinase of a two-component regulatory system and a receptor of certain signal molecules, because overexpression of only the substrate-binding protein (DasA) of the
6 Fig. 1. Life cycle of S. griseus and ectopic spores in mutant NP4 and the wild-type strain carrying multiple copies of dasA. Spores germinate to form substrate (vegetative) mycelium, which grows by cell wall extension at hyphal tips and branches, forming a coherent mat. A-Factor produced in a growth-dependent manner reaches a certain critical concentration and triggers aerial mycelium formation and secondary metabolite production. Aerial hyphae are eventually septated, which results in the generation of many unigenomic compartments, each destined to become a spore. S. griseus mutant NP4 forms ectopic septa at intervals along substrate hyphae, and each of the compartments develops into a spore. The spores were indistinguishable in size and shape, and thickness of spore coat from the aerial spore of the parental S. griseus strain. The timing of spore septum formation is almost similar to that of aerial spore formation in the wild-type strain. The wild-type strain carrying multicopies of dasA forms ectopic septa in very young substrate hyphae after only 1 day of growth. The main wall of the ectopic spore of the dasA-overexpressing strain is thinner than that of the aerial spore of the wild-type strain.
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4. Repression of septum formation in substrate hyphae is important for the normal development pathway
Fig. 3. Time-course of DasA-mediated ESP. The wild-type S. griseus IFO13350 forms septa in aerial hyphae and matures each of the compartments. Mutant NP4 forms spores directly from substrate hyphae, but with almost the same time-course as the wild-type. The wild-type strain carrying multiple copies of dasA forms septa in very young substrate hyphae and matures each of the compartments in only a day.
ABC transporter causes the phenotype and because DasA is expressed at the timing of aerial mycelium formation when the glucose in medium is supposedly consumed. Some substrate-binding proteins of ABC transporters have multiple functions. For example, ChvE in Agrobacterium is a multifunctional glucose/galactose-binding protein that participates in the uptake of speci¢c monosaccharides, chemotaxis to these sugars, and virulence gene induction. For induction of virulence genes to form crown gall tumors, monosaccharide-bound ChvE interacts with the periplasmic region of VirA, a sensor kinase in the VirA^VirG two-component signal transduction system [20,21]. For chemotaxis, ChvE is supposed to interact with chemotaxis receptors such as Tar and Trg [20]. Therefore, it is possible that substrate-bound or free DasA commences a regulatory pathway for morphological development by interacting with other regulatory proteins in the membrane, although how glucose is involved in this hypothesis is not explained. Investigation of the das operon in S. coelicolor A3(2), which contains a very similar gene cluster (open reading frames CAB94616 to 394619; www.sanger.ac.uk/Projects/S_coelicolor/), may yield useful information, since the genome sequence has been determined and DNA microarray studies are possible.
The deprogrammed sporulation of mutant NP4 and the wild-type strain containing multicopies of dasA implies that once septa are formed, even in substrate hyphae, in response to some signal, each compartment is inevitably destined to develop into a spore. This is also suggested by the above-described mutants showing ESP. In substrate hyphae of the wild-type S. griseus, some signals must block the commitment to septation and subsequent sporulation. What does repress the septation in substrate hyphae in the normal development pathway? A-Factor does not release the block, because exogenous addition of an appropriate amount of A-factor to substrate hyphae of the A-factor-de¢cient mutant HH1 causes no septum formation but normal formation of aerial spores. A-Factor, as described above, is concerned mainly with the onset of aerial mycelium formation and not with later steps of the development. An excess amount of DasA appears to release the block, since introduction of multicopies of dasA into mutant HH1 results in ESP. It is very surprising that overexpression of only one gene (dasA) can easily release the block and cause ESP. The transcription of dasA in the wild-type strain begins just after commitment of aerial mycelium formation and increases during sporulation. Therefore the repression of dasA transcription is absolutely required for the inhibition of septation in substrate hyphae. Considering the mutation of NP4 that has a defect in the regulation of dasA^dasR, we assume that the amount of DasA that is controlled by dasR and the regulatory system controlling the amount of DasR are important. For normal development, an ordered expression of bld and whi genes in the regulatory hierarchy is necessary [4,5]. Many bld mutants of S. coelicolor A3(2) show their phenotypes only on glucose-containing medium and, without glucose, can develop normally [22]. On the other hand, the DasA-mediated ectopic development occurs only in the presence of glucose. The study of the das genes, as well as the bld genes, will give information on the glucose repression in morphological development. The glucose repression in Streptomyces is not adequately understood, although glucose kinase itself, but not its product, glucose 6-phosphate, is known to play crucial roles, probably by interacting with multiple regulatory proteins [23].
5. Concluding remarks Substrate hyphae of Streptomyces have a potential to develop into spores without formation of aerial hyphae. However, sporulation in substrate hyphae is strictly repressed in the normal developmental process. Overproduction of DasA, a substrate-binding protein of the ABC transporter, in the wild-type S. griseus causes a very strong
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ESP phenotype in response to glucose. The ESP is independent of A-factor, implying that there is no obligatory link between aerial mycelium formation and sporulation. Analysis on the regulatory mechanism of dasR transcription would provide some clues to the signals that repress the septation in substrate hyphae, in relation to the availability of glucose. Identi¢cation of an open reading frame(s) that is responsible for the ESP phenotype in S. coelicolor A3(2) carrying the 7.4-kb deletion near the glkA locus would also provide important information for the signals that repress the septation in substrate hyphae.
[10]
[11]
[12]
[13]
Acknowledgements [14]
The work from this laboratory was supported by the Nissan Science Foundation, by the Asahi Glass Foundation, and by the Bio Design Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan (BDP-02-VI2-6).
[15]
[16]
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sential for morphological development in Streptomyces griseus. J. Bacteriol. 182, 4596^4605. Bibb, M.J., Molle, V. and Buttner, M.J. (2000) cBldN , an extracytoplasmic function RNA polymerase sigma factor required for aerial mycelium formation in Streptomyces coelicolor A3(2). J. Bacteriol. 182, 4606^4616. Kato, J., Suzuki, A., Yamazaki, H., Ohnishi, Y., Horinouchi, S. (2002) Control by A-factor of a metalloendopeptidase gene involved in aerial mycelium formation in Streptomyces griseus. J. Bacteriol. 184, in press. Ueda, K., Oinuma, K., Ikeda, G., Hosono, K., Ohnishi, Y., Horinouchi, S. and Beppu, T. (2002) AmfS, an extracellular peptidic morphogen in Streptomyces griseus. J. Bacteriol. 184, 1488^1492. Ueda, K., Miyake, K., Horinouchi, S. and Beppu, T. (1993) A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators. J. Bacteriol. 175, 2006^2016. Ueda, K., Hsheh, C.-W., Tosaki, T., Shinkawa, H., Beppu, T. and Horinouchi, S. (1998) Characterization of an A-factor-responsive repressor for amfR essential for onset of aerial mycelium formation in Streptomyces griseus. J. Bacteriol. 180, 5085^5093. Chater, K.F., Bruton, C.J., Plaskitt, K.A., Buttner, M.J., Mendez, C. and Helmann, J.D. (1989) The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B. subtilis. Cell 59, 133^143. Kelemen, G.H., Plaskitt, K.A., Lewis, C.G., Findlay, K.C. and Buttner, M.J. (1995) Deletion of DNA lying close to the glkA locus induces ectopic sporulation in Streptomyces coelicolor A3(2). Mol. Microbiol. 17, 221^230. Jiang, H. and Kendrick, K.E. (2000) Characterization of ssfR and ssgA, two genes involved in sporulation of Streptomyces griseus. J. Bacteriol. 182, 5521^5529. van Wezel, G.P., van der Meulen, J., Kawamoto, S., Luiten, R.G., Koerten, H.K. and Kraal, B. (2000) ssgA is essential for sporulation of Streptomyces coelicolor A3(2) and a¡ects hyphal development by stimulating septum formation. J. Bacteriol. 182, 5653^5662. Seo, J.-W., Ohnishi, Y., Hirata, A. and Horinouchi, S. (2002) An ATP-binding cassette transport system involved in the regulation of morphological di¡erentiation in response to glucose in Streptomyces griseus. J. Bacteriol. 184, 91^103. Peng, W.-T., Lee, Y.-W. and Nester, E.W. (1998) The phenolic recognition pro¢les of the Agrobacterium tumefaciens VirA protein are broadened by a high level of the sugar binding protein ChvE. J. Bacteriol. 180, 5632^5638. Winans, S.C. (1992) Two-way chemical signaling in Agrobacteriumplant interactions. Microbiol. Rev. 56, 12^31. Pope, M.K., Green, B.D. and Westpheling, J. (1996) The bld mutants of Streptomyces coelicolor are defective in the regulation of carbon utilization, morphogenesis and cell-cell signalling. Mol. Microbiol. 19, 747^756. Stu«lke, J. and Hillen, W. (1999) Carbon catabolite repression in bacteria. Curr. Opin. Microbiol. 2, 195^201.
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MiniReview
Deprogrammed sporulation in Streptomyces Yasuo Ohnishi, Jeong-Woo Seo, Sueharu Horinouchi
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Received 10 June 2002; received in revised form 4 September 2002; accepted 4 September 2002 First published online 2 October 2002
Abstract The bacterial genus Streptomyces forms chains of spores by septation at intervals in aerial hyphae and subsequent maturation on solid medium. Substrate hyphae undergo extensive lysis, liberating nutrients on which aerial hyphae develop. Some mutant strains, however, ectopically form spores by septation in substrate hyphae on solid medium or in vegetative hyphae in liquid medium, which suggests that all hyphae have the potential to differentiate into spores. A Streptomyces griseus mutant strain NP4, which has a mutation in the regulatory system for an ATP-binding cassette (ABC) transporter gene, forms ectopic spores in substrate hyphae only on glucosecontaining medium. In addition, overexpression of a substrate-binding protein of the ABC transporter in the wild-type strain causes ectopic septation in very young substrate hyphae and subsequent sporulation in response to glucose. These ectopic spores germinate normally. The ectopic sporulation is independent of A-factor, a microbial hormone that determines the timing of aerial mycelium formation during normal development. Thus, substrate hyphae of Streptomyces have a potential to develop into spores without formation of aerial hyphae. For programmed development, therefore, the strict repression of septum formation in substrate mycelium should be necessary, as well as the positive signal relay leading to aerial mycelium formation followed by septation and sporulation. 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Morphological development; Ectopic sporulation ; ATP-binding cassette transporter; A-Factor; Streptomyces
1. Introduction The Gram-positive, soil-inhabiting, ¢lamentous bacterial genus Streptomyces shows complex morphological development (Fig. 1), which makes this genus one of the model prokaryotes for studying multicellular di¡erentiation [1,2]. In an early stage of Streptomyces growth on solid medium, spores germinate to subsequently develop into branched, ¢lamentous, multinucleoidal hyphae (substrate hyphae) that contain relatively infrequent, thin, single-layer vegetative septa. Exponential growth is achieved by branching of the hyphae, resulting in the formation of an intricate mycelial network. The ¢rst step of morphological di¡erentiation is the emergence of aerial hyphae by reuse of material assimilated into substrate hyphae, such as proteins, DNA, and storage compounds. Subsequently synchronous septation occurs only in aerial hyphae to form chains of unigenomic compartments separated by
* Corresponding author. Tel. : +81 (3) 58 41 51 23; Fax : +81 (3) 58 41 80 21. E-mail address : [email protected] (S. Horinouchi).
double-layer, thick sporulation septa. At a later stage, chains of the unigenomic compartments are destined to become individual spores. Some Streptomyces strains produce spores in submerged culture when critical nutritional and environmental conditions are met. Streptomyces griseus B-2682, for example, produces abundant submerged spores in nutrient-depleted media [3]. The sporulation process in liquid culture includes the formation of specialized branches designated sporogenic hyphae, in which doublelayer sporulation septa are formed. The submerged spores of S. griseus are similar, but never identical, to aerial spores and are sensitive to lysozyme digestion, probably because of the thinness of the spore wall. Most studies of the Streptomyces development have been accomplished by characterizing developmental mutants of Streptomyces coelicolor A3(2). In principle, these mutants can be divided into two classes : bald (bld) mutants, which fail to produce fuzzy aerial mycelium, and white (whi) mutants, which produce aerial hyphae but cannot form gray-pigmented spores. By complementation experiments with these mutants, at least 15 regulatory genes for morphological di¡erentiation have been identi¢ed and studied at the molecular level, and some of their interac-
0378-1097 / 02 / $22.00 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 9 9 6 - 5
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tions have been characterized. These works were summarized in recent excellent reviews [4,5]. Some mutant strains of Streptomyces produce ectopic spores directly from substrate mycelium, because normal regulatory events blocking division are deprogrammed in the substrate hyphae. For example, S. griseus NP4 produces ectopic spores, which are indistinguishable from normally developed aerial spores, by septation in substrate hyphae. In this mini-review, we put an emphasis on the idea that, in addition to the positive signal relays leading to aerial mycelium formation, a mechanism to strictly inhibit septum formation in substrate hyphae is absolutely necessary for the programmed development of Streptomyces.
2. A-Factor signaling pathway leading to morphological development in S. griseus 2.1. A-factor regulatory cascade that determines the timing of morphological di¡erentiation Many Streptomyces species produce Q-butyrolactones, most of which have been shown to act as chemical signaling molecules for the onset of antibiotic production. AFactor (2-isocapryloyl-3R-hydroxymethyl-Q-butyrolactone) is the most intensively and extensively studied Q-butyrolactone and is exceptionally required for not only antibiotic production but also morphological di¡erentiation in S. griseus [6,7] (Fig. 2). Possibly, during evolution Streptomyces species have developed Q-butyrolactones as ‘hormonal regulators’ to control di¡erent stages of physiological and/or morphological di¡erentiation in the regulatory hierarchy. A-Factor di¡uses freely in an individual hypha and into the neighboring hyphae across the cytoplasmic membrane and induces the transcription of adpA by binding to the A-factor receptor protein (ArpA), which has bound and repressed the adpA promoter, and dissociating it from the promoter [8]. Although AdpA was originally identi¢ed as an A-factor-dependent transcriptional activator for strR encoding the pathway-speci¢c transcriptional activator for streptomycin biosynthetic genes, AdpA has been shown to induce the transcription of a number of genes involved in secondary metabolism and morphogenesis, comprising an AdpA regulon. The intracellular concentration of A-factor thus determines the timing of adpA expression, which results in the switch-on of all the downstream genes in the regulatory cascade. In fact, an adpAdisruptant produces neither streptomycin nor a yellow pigment and shows a bald phenotype. Until now, we suppose that the A-factor signaling pathway is responsible for the onset of aerial mycelium formation and the e¡ects of A-factor on the later processes in the development would not be so signi¢cant (Fig. 2). The only exception we have observed is that AdpA appears to control the transcription of ssgA, which is involved in
spore septum formation in aerial hyphae (unpublished data). The following examples are the genes that are activated by AdpA at a speci¢c time and required for the onset of aerial mycelium formation. 2.2. Extracytoplasmic function (ECF) sigma factor adsA encoding an ECF sigma factor (cAdsA ) is one of the target genes of AdpA [9]. Transcription of adsA begins approximately at the time of aerial mycelium formation. Disruption of the chromosomal adsA gene results in loss of aerial hyphae formation but not streptomycin and yellow pigment production, indicating that cAdsA is involved only in morphological development and not in secondary metabolic function. S. coelicolor A3(2) bldN, the orthologue of adsA, plays the same role as adsA [10]. Interestingly, cBldN is responsible for the transcription from the p1 promoter of another bld gene, bldM, which encodes a response regulator-like protein. We assume that the e¡ect of A-factor on morphological di¡erentiation is mediated mainly by cAdsA , which would activate many genes required for aerial mycelium formation. 2.3. Metallo- and serine-proteases sgmA encoding an extracellular metalloendopeptidase (SGMP II) is another direct target of AdpA [11]. Disruption of the chromosomal sgmA gene results in a slight but distinct delay of aerial hyphae formation, suggesting that SGMP II is somehow involved in morphological development. Extracellular proteases, together with other hydrolytic enzymes, are involved in the cannibalistic reuse of material in substrate hyphae during aerial hyphae formation. It is noteworthy that several other extracellular serine-protease genes, such as sprA, sprB, sprD, and sprT, are also transcribed in an AdpA-dependent manner (our unpublished data). It is also possible that some of them work as processing enzymes for extracellular signaling molecules involved in morphological development. 2.4. AmfS, a peptidic morphogen A processed product of AmfS acts as an extracellular peptidic morphogen of S. griseus [12]. amfS encoding a 43amino acid peptide is a member of the amf gene cluster, which was previously identi¢ed as a regulator for the onset of aerial mycelium formation [13]. The amf genes contains ¢ve open reading frames: amfT encoding a transmembrane protein similar to protein Ser/Thr kinases; amfS; amfB and amfA both encoding an ATP-binding cassette (ABC) transporter; and amfR encoding a response regulator-like protein of two-component regulatory systems. The ram gene cluster, which has the same gene organization as amf, in S. coelicolor A3(2) and Streptomyces lividans appears to play the same role in aerial mycelium formation (see references in [4,5]). Mutational analyses
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and extracellular complementation tests demonstrated that AmfS is essential for aerial mycelium formation and secreted via the AmfAB transporter. A synthetic C-terminal octapeptide, but not the full-length peptide of AmfS, partially induced aerial mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen. AmfR is perhaps a transcriptional activator for the amf genes. An adpA-disruptant lacks extracellular AmfS activity, suggesting that the regulation by AmfS is under the control of A-factor and AdpA. We previously reported that amfR was co-transcribed with two upstream open reading frames (orf5 and orf4 in this order) and their transcription depended on A-factor [14]. However, our recent analysis has shown that the orf5 promoter is rather constant and the amfR promoter itself depends on A-factor (our unpublished results). Our preliminary experiments suggest that AdpA directly binds the upstream region of the amfR promoter, showing direct control of amfR expression by AdpA.
3. Ectopic, deprogrammed sporulation by Streptomyces mutants Spores of Streptomyces are usually formed only in aerial hyphae on solid medium and in sporogenic hyphae in liquid medium. However, some mutant strains sporulate ectopically in substrate hyphae on solid medium or in vegetative hyphae in liquid medium, suggesting that all hyphae, irrespective of substrate or aerial hyphae, have the potential to di¡erentiate into spores. A better understanding of these mutations will give us new insights into the morphological di¡erentiation of Streptomyces. 3.1. Overexpression of whiG whiG encodes a sigma factor of RNA polymerase, which speci¢cally initiates the development program that leads to spore formation from aerial hyphae [4]. In S. coelicolor A3(2), the RNA polymerase cWhiG holoenzyme transcribes regulatory genes, whiH and whiI, both of which consist of a DNA-binding domain and a domain usually involved in signal sensing. The copy number of whiG has a dramatic e¡ect on development of S. coelicolor A3(2); in the absence of cWhiG , the aerial hyphae develop no sporulation septa, whereas an excess amount of cWhiG causes hypersporulation in aerial hyphae on solid medium and occasional ectopic sporulation (ESP) in vegetative hyphae both on solid and in liquid medium [15]. Thus the activity of cWhiG determines the developmental fate of hyphae. 3.2. Deletion of DNA located close to the glkA locus A 7.4-kb deletion of DNA lying close to, but distinct from glkA, causes ectopic sporulation in substrate hyphae
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of S. coelicolor A3(2) on solid medium [16]. The ESP phenotype associated with the 7.4-kb deletion depends on the whiG function, since introduction of a whiG mutation into the deletion mutant blocks sporulation in both aerial and substrate hyphae. Because whiG is activated posttranscriptionally, a simple explanation for this is that some protein that represses the cWhiG activation in substrate hyphae is encoded in the 7.4-kb region. The genome sequencing denies the possibility that an anti-sigma factor for cWhiG is encoded in this region. The open reading frame(s) responsible for the ESP phenotype remains to be identi¢ed. 3.3. Overexpression of ssgA ssgA encoding a 15-kDa acidic protein is involved in spore septum formation in both S. griseus [17] and S. coelicolor A3(2) [18]. In both strains, an ssgA-disruptant shows a white phenotype. In S. coelicolor A3(2), overexpression of ssgA results in formation of occasional septa in substrate hyphae and subsequent spore-like compartments in liquid culture. In S. griseus, a point mutation in the regulatory region for ssgA results in fragmentation of mycelium and formation of spore-like compartments, although the e¡ect of the mutation on ssgA transcription remains to be elucidated. The function of SsgA in septum formation is unknown. 3.4. Overexpression of a substrate-binding protein of an ABC transporter We recently isolated and analyzed a bald mutant, NP4, derived by UV mutagenesis from the wild-type S. griseus IFO13350 [19]. Mutant NP4 forms ectopic septa at regular intervals along substrate hyphae in response to glucose, and each of the compartments develops into a spore (Fig. 1). The ectopic septa are formed only on medium containing glucose, but not on medium containing other carbon sources. This is why NP4 shows a bald and wrinkled colony morphology. The ectopic spores are indistinguishable in size and shape, and thickness of spore cortex from the aerial spore of the parental S. griseus, as determined by transmission electron microscopy. The ESP occurs also in liquid medium, and we say that a mutation(s) in NP4 can change Streptomyces into a ‘nocardioform’ bacterium. The septum in substrate hyphae in NP4 is formed at a similar timing to that when the aerial septum is formed by the wild-type strain (Fig. 3). This is a vivid contrast to the septation in substrate hyphae by the wild-type strain carrying multiple copies of dasA (see below). Shotgun cloning experiments with a library of chromosomal DNA of the parental strain and mutant NP4 as the host yielded DNA fragments giving two di¡erent phenotypes ; one (dasR) complementing the bald phenotype of the host, and the other (dasA) causing a much more severe wrinkled morphology in the host. dasR encodes a tran-
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Fig. 2. A-Factor regulatory cascade leading to aerial mycelium formation in S. griseus. A-Factor is gradually accumulated in a growth-dependent manner. AfsA, a probable A-factor synthesis enzyme, is involved in A-factor production. When the concentration of A-factor reaches a certain critical level, it binds the A-factor receptor protein ArpA, which has bound the promoter region of adpA, and dissociates ArpA from the promoter, thus leading to transcription and translation of adpA. AdpA is a pleiotropic activator essential for both morphological development and secondary metabolism. AdpA activates adsA encoding an ECF sigma factor (cAdsA ) and sgmA encoding a metalloendopeptidase (SGMP II). cAdsA is essential for the onset of aerial mycelium formation, probably activating multiple genes involved in aerial mycelium formation. SGMP II is also suggested to be involved in morphological development. AdpA also activates transcription of other protease genes. Transcription of amfR, which encodes a response regulator-like protein and is essential for AmfS production, is directly controlled by AdpA. AmfS is an extracellular peptidic morphogen that is essential for aerial mycelium formation.
scriptional repressor belonging to the GntR family and dasA encodes a lipoprotein probably serving as a substrate-binding protein in an ABC transport system. These genes, together with dasB and dasC, both encoding a membrane-spanning protein, are members in the gene cluster for an ABC transporter. DasR represses the transcription of dasA by directly binding to its promoter (our
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unpublished data). Nucleotide sequencing and transcriptional analysis of the dasR^dasA region in NP4 suggested that NP4 has a defect in the regulatory system to control the expression of these genes, but not in the dasR or dasA. The transcription of both dasA and dasR is greatly enhanced in mutant NP4. The presence of multiple copies of dasA in the wild-type strain causes the host to form ectopic septa in very young substrate hyphae after only 1 day of growth and subsequent spores in response to glucose (Fig. 3). The timing is a vivid contrast with that of ectopic sporulation by mutant NP4 that forms ectopic septa in substrate hyphae at days 3^4 under the same conditions. The ectopic spores formed by the wild-type containing multiple copies of dasA have a thinner wall than those of mutant NP4 (Fig. 1), in agreement with the observation that the former is sensitive to lysozyme and heat. This implies that some of the gene products necessary for the architecture of aerial spores are absent during the maturation of the ectopic spores. These ectopic spores, however, germinate at more than 90% frequency. The ectopic septation and sporulation in the wild-type strain carrying multicopies of dasA is independent of A-factor, since an A-factor-de¢cient mutant HH1 carrying multicopies of dasA also shows the ESP phenotype. This is also supported by the observation that the wild-type strain carrying multicopies of dasA forms ectopic septa after only 1 day of growth, when the concentration of A-factor is still low. As described above, A-factor is essential for the onset of aerial mycelium formation. No dependence of ectopic sporulation in substrate hyphae of the dasA-overexpressing strains on A-factor clearly indicates no obligatory link between aerial mycelium formation and sporulation. How is the ABC transporter system involved in the ESP phenotype? A simple explanation is that some signal molecule for morphological development is imported via the ABC transporter. The e¡ect of glucose on the ectopic sporulation of mutant NP4 and on the wild-type strain containing multicopies of dasA tempts us to speculate that DasA recognizes and binds glucose or a glucose derivative and imports it via the Das system, thus inducing the ESP phenotype. However, it is likely to us that DasA interacts with some protein in the membrane, such as a sensor kinase of a two-component regulatory system and a receptor of certain signal molecules, because overexpression of only the substrate-binding protein (DasA) of the
6 Fig. 1. Life cycle of S. griseus and ectopic spores in mutant NP4 and the wild-type strain carrying multiple copies of dasA. Spores germinate to form substrate (vegetative) mycelium, which grows by cell wall extension at hyphal tips and branches, forming a coherent mat. A-Factor produced in a growth-dependent manner reaches a certain critical concentration and triggers aerial mycelium formation and secondary metabolite production. Aerial hyphae are eventually septated, which results in the generation of many unigenomic compartments, each destined to become a spore. S. griseus mutant NP4 forms ectopic septa at intervals along substrate hyphae, and each of the compartments develops into a spore. The spores were indistinguishable in size and shape, and thickness of spore coat from the aerial spore of the parental S. griseus strain. The timing of spore septum formation is almost similar to that of aerial spore formation in the wild-type strain. The wild-type strain carrying multicopies of dasA forms ectopic septa in very young substrate hyphae after only 1 day of growth. The main wall of the ectopic spore of the dasA-overexpressing strain is thinner than that of the aerial spore of the wild-type strain.
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4. Repression of septum formation in substrate hyphae is important for the normal development pathway
Fig. 3. Time-course of DasA-mediated ESP. The wild-type S. griseus IFO13350 forms septa in aerial hyphae and matures each of the compartments. Mutant NP4 forms spores directly from substrate hyphae, but with almost the same time-course as the wild-type. The wild-type strain carrying multiple copies of dasA forms septa in very young substrate hyphae and matures each of the compartments in only a day.
ABC transporter causes the phenotype and because DasA is expressed at the timing of aerial mycelium formation when the glucose in medium is supposedly consumed. Some substrate-binding proteins of ABC transporters have multiple functions. For example, ChvE in Agrobacterium is a multifunctional glucose/galactose-binding protein that participates in the uptake of speci¢c monosaccharides, chemotaxis to these sugars, and virulence gene induction. For induction of virulence genes to form crown gall tumors, monosaccharide-bound ChvE interacts with the periplasmic region of VirA, a sensor kinase in the VirA^VirG two-component signal transduction system [20,21]. For chemotaxis, ChvE is supposed to interact with chemotaxis receptors such as Tar and Trg [20]. Therefore, it is possible that substrate-bound or free DasA commences a regulatory pathway for morphological development by interacting with other regulatory proteins in the membrane, although how glucose is involved in this hypothesis is not explained. Investigation of the das operon in S. coelicolor A3(2), which contains a very similar gene cluster (open reading frames CAB94616 to 394619; www.sanger.ac.uk/Projects/S_coelicolor/), may yield useful information, since the genome sequence has been determined and DNA microarray studies are possible.
The deprogrammed sporulation of mutant NP4 and the wild-type strain containing multicopies of dasA implies that once septa are formed, even in substrate hyphae, in response to some signal, each compartment is inevitably destined to develop into a spore. This is also suggested by the above-described mutants showing ESP. In substrate hyphae of the wild-type S. griseus, some signals must block the commitment to septation and subsequent sporulation. What does repress the septation in substrate hyphae in the normal development pathway? A-Factor does not release the block, because exogenous addition of an appropriate amount of A-factor to substrate hyphae of the A-factor-de¢cient mutant HH1 causes no septum formation but normal formation of aerial spores. A-Factor, as described above, is concerned mainly with the onset of aerial mycelium formation and not with later steps of the development. An excess amount of DasA appears to release the block, since introduction of multicopies of dasA into mutant HH1 results in ESP. It is very surprising that overexpression of only one gene (dasA) can easily release the block and cause ESP. The transcription of dasA in the wild-type strain begins just after commitment of aerial mycelium formation and increases during sporulation. Therefore the repression of dasA transcription is absolutely required for the inhibition of septation in substrate hyphae. Considering the mutation of NP4 that has a defect in the regulation of dasA^dasR, we assume that the amount of DasA that is controlled by dasR and the regulatory system controlling the amount of DasR are important. For normal development, an ordered expression of bld and whi genes in the regulatory hierarchy is necessary [4,5]. Many bld mutants of S. coelicolor A3(2) show their phenotypes only on glucose-containing medium and, without glucose, can develop normally [22]. On the other hand, the DasA-mediated ectopic development occurs only in the presence of glucose. The study of the das genes, as well as the bld genes, will give information on the glucose repression in morphological development. The glucose repression in Streptomyces is not adequately understood, although glucose kinase itself, but not its product, glucose 6-phosphate, is known to play crucial roles, probably by interacting with multiple regulatory proteins [23].
5. Concluding remarks Substrate hyphae of Streptomyces have a potential to develop into spores without formation of aerial hyphae. However, sporulation in substrate hyphae is strictly repressed in the normal developmental process. Overproduction of DasA, a substrate-binding protein of the ABC transporter, in the wild-type S. griseus causes a very strong
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ESP phenotype in response to glucose. The ESP is independent of A-factor, implying that there is no obligatory link between aerial mycelium formation and sporulation. Analysis on the regulatory mechanism of dasR transcription would provide some clues to the signals that repress the septation in substrate hyphae, in relation to the availability of glucose. Identi¢cation of an open reading frame(s) that is responsible for the ESP phenotype in S. coelicolor A3(2) carrying the 7.4-kb deletion near the glkA locus would also provide important information for the signals that repress the septation in substrate hyphae.
[10]
[11]
[12]
[13]
Acknowledgements [14]
The work from this laboratory was supported by the Nissan Science Foundation, by the Asahi Glass Foundation, and by the Bio Design Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan (BDP-02-VI2-6).
[15]
[16]
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