A putative regulatory element for carbon-source-dependent differentiation in Streptomyces griseusb

Kenji Ueda1, Kouichi Matsuda1, Hideaki Takano1 and Teruhiko Beppu1

Department of Applied Biological Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-8510, Japan1

Author for correspondence: Kenji Ueda. Tel: +81 466 84 3936. Fax: +81 466 84 3935. e-mail: ueda{at}brs.nihon-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To identify negative regulatory genes for cellular differentiation in Streptomyces griseus, DNA fragments repressing the normal developmental processes were cloned on a high-copy-number plasmid. One of these DNA fragments markedly repressed aerial mycelium and spore formation on solid media containing glucose or galactose, but not on media containing maltose or mannitol. The fragment contained three complete ORFs; precise subcloning revealed that a 249 bp fragment located in the promoter region between ORF1 and ORF3 was sufficient for repression. Quantification of the promoter activities by using a thermostable malate dehydrogenase gene as a reporter showed that the promoter for ORF3 (PORF3) maintained high activity in mycelia grown in the presence of glucose but lost activity rapidly in maltose medium. PORF3 activity increased markedly when the promoter sequence was introduced on a high-copy-number plasmid. The results suggested that carbon-source-dependent deactivation of PORF3 mediated by a transcriptional repressor may initiate differentiation in S. griseus.

Keywords: Streptomyces griseus, morphological differentiation, carbon source dependence, repressor, craA

Abbreviations: ARP, A-factor receptor protein; MDH, malate dehydrogenase

b The GenBank accession number for the sequence reported in this paper is AB023642.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptomycetes show remarkable morphological differentiation during growth. The developmental process is controlled by complex regulatory cascades involving multiple gene functions. Many mutant classes with defective morphological phenotypes, such as bld (defective in aerial mycelium formation) and whi (defective in spore formation), have been isolated from Streptomyces coelicolor A3(2). Investigation of the genes complementing these mutations has uncovered molecular aspects of the regulatory mechanisms of development (Chater, 1984 , 1989 , 1993 , 1998 ). We have analysed the regulation of differentiation in a streptomycin-producing species, Streptomyces griseus, in which A-factor (2-isocapryloyl-3R-hydroxymethyl-{gamma}-butyrolactone) plays a crucial role as a hormonal auto-regulator to activate signal transduction pathways that induce both morphological differentiation and streptomycin production (Hara & Beppu, 1982 ; Khokhlov et al., 1967 ). A-factor-deficient mutants of S. griseus lose both phenotypes simultaneously and recover them when A-factor is supplied at nM levels (Horinouchi et al., 1984 ). To identify positive regulators initiating differentiation of this organism, we cloned from the wild-type several suppressor genes that induced aerial mycelium formation in an A-factor-negative mutant. For example, a gene cluster consisting of amfA, amfB and amfR cloned on a high-copy-number plasmid restored aerial mycelium formation but not streptomycin production (Ueda et al., 1993 ). amfR, encoding a putative response regulator of two-component regulatory systems, is believed to act positively on the initiation of developmental processes; its disruption abolished aerial mycelium and spore formation completely (Ueda et al., 1998 ). amfC cloned separately on a high-copy-number plasmid had a similar suppressive effect, and null mutation also resulted in significant loss of sporulation efficiency (Kudo et al., 1995 ). These results indicate that introduction of a positive regulatory gene at a high copy number may compensate for the original defects in the signal transduction system and result in the wild phenotype.

In this paper, we identify a putative negative regulatory gene for cellular differentiation in S. griseus by an approach opposite to the one that led to the isolation of amfR. We used wild-type S. griseus as a host and cloned genes that, on a high-copy-number plasmid, repressed normal cellular differentiation. This allowed us to clone a DNA fragment that significantly repressed aerial mycelium and spore formation in the presence of glucose and galactose but not in the presence of maltose. Sequence responsible for the repression was identified in the promoter region of a putative negative regulatory gene possibly involved in carbon-source-dependent regulation of cellular differentiation in S. griseus.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
S. griseus IFO 13350 (wild-type) was obtained from the Institute of Fermentation, Osaka, Japan. An A-factor-deficient mutant, S. griseus HH1, defective in aerial mycelium formation and streptomycin production, was derived from strain IFO13350 by incubation at 37 °C (Horinouchi et al., 1984 ). A-factor-receptor-deficient strains S. griseus KM7 (Miyake et al., 1990 ) and HO1 (Onaka et al., 1997 ) were derived from strain HH1 by UV mutagenesis. Streptomyces lividans TK21 was obtained from D. A. Hopwood, John Innes Institute, Norwich, UK. Plasmid pIJ702 (carrying thiostrepton resistance and melanin biosynthesis) (Katz et al., 1983 ) and pIJ486 (carrying thiostrepton resistance and promoter-less neomycin resistance) (Ward et al., 1986 ) have a copy number of 40–100 per genome. Each of these plasmids was presumed to have the same copy number in S. griseus as in S. lividans. Plasmid pIJ922 (carrying thiostrepton resistance) and pTMA1 [carrying thiostrepton resistance and a promoter-less thermostable malate dehydrogenase (MDH) gene] (Vujaklija et al., 1991 ) have a copy number of one per genome (Hopwood et al., 1985 ). DNA was cloned in pUC18 and pUC19 (Yanisch-Perron et al., 1985 ) and manipulated in Escherichia coli JM109 [(lac–pro) thi-1 endA1 gyrA96 hsdR17 relA1 recA1/F' traD36 proAB lacIq lacZ M15]. S. griseus strains were grown in Bennett’s glucose medium, containing (g l-1): yeast extract (Difco), 1; meat extract (Kyokuto), 1; NZ amine type A (Wako Pure Chemical), 2; and glucose, 10 (pH 7·2); in YMP/sugar medium, containing (g l-1): yeast extract (Difco), 2; meat extract (Kyokuto), 2; Bacto peptone (Difco), 4; NaCl (Kokusan), 5; MgSO4 (Kokusan), 2; and an appropriate sugar, 10 (pH 7·2); and in nutrient agar (Difco). Solid media were prepared by addition of 1·5% agar (Kokusan). Growth conditions for E. coli strains were as described by Maniatis et al. (1982 ).

General recombinant DNA techniques.
Restriction endonucleases and other modifing enzymes were purchased from Takara Shuzo. Thiostrepton was a gift from Asahi Chemical Industry. DNA was manipulated in E. coli as described by Maniatis et al. (1982 ), and in Streptomyces as described by Hopwood et al. (1985 ). Nucleotide sequence was determined with an automated DNA sequencer (Licor, model L4000) and a Thermo Sequenase cycle sequencing kit (Amersham).

Shotgun cloning.
Chromosomal DNA isolated from the mycelium of wild-type S. griseus was partially digested with BamHI and ligated to pIJ702 at its BglII site. The ligation mixture was used to transform the wild-type strain, and transformants showing thiostrepton resistance were screened for differentiation on YMP/glucose agar. Colonies showing sporulation-negative phenotypes were cultured in 100 ml YMP/glucose liquid medium. Plasmids were extracted and used to retransform the wild-type to confirm that the phenotype was plasmid-linked.

Subcloning experiments.
The 3·7 kb BamHI fragment originally cloned in pKM284 was at the BglII site of pIJ702. Low-copy-plasmid pKM284L was constructed as follows: the 3·7 kb BamHI region was recovered together with partial sequences from pIJ702 that included the C-terminal half of the thiostrepton resistance gene (tsr) as a 5·9 kb fragment by digesting pKM284 with PstI and EcoRV. This fragment was then inserted between the PstI and EcoRV sites of pIJ922 with the correct junction of the tsr gene. pKM284-1 was constructed by inserting the BamHI–SphI region between the BglII and SphI sites of pIJ702. pKM284-2, pKM284-2H (see also the next section for promoter assay) and pKM284-2L were constructed by inserting the BamHI–FbaI fragment into the BglII site of pIJ702, the BamHI site of pIJ486 and the BamHI site of pIJ922, respectively. pKM284M1 and pKM284M3 were constructed by inserting the BamHI–FbaI fragment into the BamHI site of pTMA1 followed by confirmation of their correct orientation by digestion with a combination of BamHI, EcoRI and HindIII. pKM284-3 was constructed by inserting the FbaI fragment containing ORF3 with a partial sequence from pIJ702 into the BglII site of pIJ702. Similarly, the ORF2-containing FbaI fragment was cloned at the BglII site of pIJ702 to generate pKM284-4. To construct pKM284-5, the BalI–EcoRI fragment was blunt-ended by treatment with the Klenow fragment, attached to an 8-mer BglII linker at both ends so that it could be recovered as a BglII fragment and cloned at the BglII site of pIJ702. To construct pKM284-6, pKM284 was digested with PmaCI and ligated to an 8-mer PstI linker. The PmaCI–KpnI region was recovered as a PstI–KpnI fragment and inserted between the PstI and KpnI sites of pIJ702. pKM284-7 was constructed by inserting the PstI–KpnI fragment from pKM284-6 between the PstI and KpnI sites of pKM284-4. To construct pKM284-8, the BalI–FbaI region was excised as a BglII–FbaI fragment from pKM284-5 and cloned at the BglII site of pIJ702. To construct pKM284-9, the BamHI–PmaCI region was excised as a BamHI–PstI fragment from pKM284-6 and inserted between the BglII and PstI sites of pIJ702. To construct pKM284-10, the BalI–MluI region was excised as a BglII–MluI fragment from pKM284-8 and inserted between the BglII and MluI sites of pIJ702. To construct pKM284-11, the BalI–PmaCI fragment was attached to an 8-mer BglII linker, recovered as a BglII fragment and ligated to the BglII site of pIJ702. pKM284-12 was constructed by cloning a trimmed BglII fragment from pKM284-11 at the BglII site of pIJ702. The trimming was done as follows: pKM284-11 was cleaved with SphI and digested with a combination of exonuclease III and mungbean nuclease. After blunt-end formation with the Klenow fragment, an 8-mer BglII linker was attached; the product was digested with BglII and inserted at the BglII site of pIJ702. The trimmed BglII fragment was also cloned at the BamHI site in pUC19 and its nucleotide sequence was determined. To construct pKM284{Delta}1 and pKM284{Delta}3, pKM284 was partially digested with MluI and self-ligated after treatment with the Klenow fragment. The ligation mixture was used to transform the wild-type strain and transformants were screened for colonies harbouring plasmids with the correct frameshifts at the MluI sites.

Promoter assay.
Promoter activities were measured by using a thermostable MDH gene as a reporter, following the method described previously (Vujaklija et al., 1991 ). Plasmids pKM284M1 and pKM284M2, carrying the promoter-less MDH gene preceded by promoters in the direction of ORF1 and ORF3, respectively, were used to transform the S. griseus wild-type strain; MDH activities expressed by those promoters during growth on several media were measured. The strain for assaying the titration effect was constructed by introducing pKM284-2H into a thiostrepton-resistant transformant harbouring pKM284M3, taking advantage of their compatibility. Because of the aphII gene downstream from the inserted promoter, pKM284-2H conferred kanamycin resistance. Transformants showing thiostrepton and kanamycin resistance were screened for their retention of the two kinds of plasmid. Wet weight of mycelium in each culture was measured to assess growth.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning DNA fragments that repress differentiation in S. griseus
To isolate negative regulators of differentiation, we used shotgun cloning and targeted sporulation-negative transformants as described in Methods. Among approximately 6 x 103 transformants, we obtained 11 sporulation-negative colonies carrying DNA fragments with different plasmids (indicated by restriction patterns) inserted at the BglII site of pIJ702. Phenotypes conferred by these plasmids were checked for reproducibility by reintroduction into the wild-type.

One of these plasmids, pKM284, caused significant repression of aerial mycelium and spore formation in wild-type S. griseus grown on YMP/glucose or YMP/galactose agar (Fig. 1a, left). In contrast, it resulted in normal differentiation on YMP/maltose agar, YMP agar without carbon sources (Fig. 1a) or YMP/mannitol agar (not shown). Neither streptomycin production nor A-factor production was affected on these media. On YMP/glucose agar (Fig. 1b) pKM284 also repressed aerial mycelium and spore formation by A-factor receptor protein (ARP)-negative mutants of S. griseus HO1 (Onaka et al., 1997 ) and KM7 (Miyake et al., 1990 ). ARP is a receptor that negatively regulates both cellular differentiation and streptomycin production in S. griseus. Binding of A-factor to ARP results in derepression (Miyake et al., 1990 ; Onaka et al., 1997 ). The bald phenotype of an A-factor-deficient mutant of S. griseus strain HH1 was not affected by introducing pKM284 (Fig. 1b, left end). This plasmid also significantly reduced spore formation in S. lividans TK21 on glucose medium (Fig. 1b, right end).



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Fig. 1. Repression of cellular differentiation by pKM284. (a) Colony surfaces of S. griseus wild-type harbouring pIJ702 (control) or pKM284 on YMP solid media with or without various carbon sources. Colonies of the wild-type harbouring low-copy-number plasmids pIJ922 (control) or pKM284L on YMP/glucose are also shown (right end). (b) Colony surfaces of S. griseus HH1, HO1, KM7 and S. lividans TK21 harbouring plasmids pIJ702 or pKM284 on YMP/glucose media. All patches were photographed after 4 d growth.

 
Nucleotide sequence of the cloned fragment
The above observations suggested that the DNA fragment cloned on pKM284 plays a regulatory role in carbon-source-dependent control of cellular differentiation in S. griseus. The size of the cloned BamHI fragment was approximately 3·7 kb, and subsequent nucleotide sequencing followed by frame analysis (Bibb et al., 1984 ) detected three complete ORFs (ORF1–3) in the internal 3613 bp KpnI–SphI region (Fig. 2). ORF1 and ORF3 were preceded by potential ribosome-binding sites, GGAAAGG and GAGAA (Gold et al., 1981 ) (Fig. 3c). ORF1 encoded a polypeptide of 259 amino acids with a molecular mass of 29·0 kDa. A database search revealed that this protein is a sigma factor belonging to the {sigma}B family (Hecker & Volker, 1998 ) (Fig. 3a), and showing 94% identity to CrtS, which was previously identified in Streptomyces setonii by its activity in restoring carotenoid synthesis (Kato et al., 1995 ). ORF2 encoded a protein with 289 amino acids (31·4 kDa) containing a putative helix–turn–helix motif in its N-terminal half (Fig. 3b), implicating a possible function as a DNA-binding protein. ORF3 potentially encoded a protein of 246 amino acids (26·6 kDa) without significant homology to other proteins.



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Fig. 2. Restriction map and subcloning of the cloned DNA fragment with a GC-plot of the nucleotide sequence. Plasmid pKM284 contains the originally cloned 3·7 kb BamHI fragment at the BglII site of pIJ702. The other plasmids were constructed as described in Methods. The estimated repression of aerial mycelium (AM) formation conferred by each subclone is summarized on the right. The nucleotide sequence of the 3613 bp KpnI–SphI fragment was analysed by the FRAME program (Bibb et al., 1984 ) with a moving window of 100.

 


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Fig. 3. Multiple alignments of ORF1 and ORF2 products and nucleotide sequence of the promoter region between ORF1 and ORF3. (a) Multiple alignment of the ORF1 product with various sigma factors: CrtS from S. setonii (GenBank accession no. D17466); RpoF from S. coelicolor A3(2) (P37971); and {sigma}B (SigB, P06574), {sigma}F (SigF, P07860) and {sigma}G (SigG, P19940) from Bacillus subtilis. A polymerase-binding domain and helix–turn–helix motif are underlined. (b) Multiple alignment of the putative helix–turn–helix motif of the ORF2 product with several transcriptional regulators: BetI (P17446) and AcrR (P34000) from E. coli; and LuxR (P21308) from Vibrio harveyi. In (a) and (b), identical and semi-conserved amino acids are indicated by asterisks and dots, respectively. (c) Nucleotide sequence of the promoter region between ORF1 and ORF3 with the deduced N-terminal amino acid sequences below. The 249 bp region conferring repression of differentiation is boxed with a solid line. Potential ribosome-binding sites are underlined, and an inverted repeat sequence is shown by converging arrows.

 
Subcloning of the cloned fragment
To identify more precisely the region responsible for repressing aerial mycelium and spore formation, subcloning experiments were performed (summarized in Fig. 2). We had noticed that the pKM284-2 carrying the BamHI–FbaI fragment containing N-terminal partial sequences of ORF1 and ORF3 together with their promoter region repressed aerial mycelium and spore formation to the same level as the original fragment. This implied that the promoter region might be responsible for the activity. Other results with pKM284-3 to pKM284-10 showed that the promoter region for ORF1 and ORF3 commonly caused slightly leaky but still significant repression of aerial mycelium formation. Moreover pKM284-11, carrying the BalI–PmaCI fragment in which no coding sequences were present (Fig. 3c) also showed significant repression. We suspected that introducing the promoter region on a high-copy-number plasmid might cause repression by titrating a regulatory protein(s) that binds to an operator sequence in the promoter region. Further trimming experiments revealed that the internal 249 bp region (Fig. 3c, boxed) was sufficient to cause the repression (pKM284-12).

Neither the low-copy-number plasmid carrying the original fragment (pKM284L; Fig. 1a, right end) nor other plasmids containing the promoter region on low-copy-number vectors (pKM284-2L, pKM284M1 and pKM284M2; data not shown) caused repression, supporting the idea that the loss of aerial mycelium and spore formation was a multi-copy effect conferred by the high copy number of pIJ702 (40–100 copies per genome). Using pIJ486 (40–100 copies per genome) to clone the BamHI–FbaI region (pKM284-2H) gave the same phenotype as pKM284, indicating that the repressive effect is not specific to pIJ702. Frameshift mutations in ORF1 or ORF3 on pKM284 (pKM284{Delta}1 and pKMK284{Delta}3) did not abolish the repressive effect of the original plasmid, indicating that the intact forms of neither ORF1 nor ORF3 are essential for repression. We do not have a clear explanation for the slightly leaky repression exhibited by several plasmids, including pKM284-12.

Quantitative analysis of promoter activity and its dependence on carbon source
The above results implying a titration effect of the promoter fragment, along with its dependence on carbon source, prompted us to quantify the promoter activity in vivo. The BamHI–FbaI fragment containing the promoter region was cloned in pTMA1, a promoter-probe vector carrying a thermostable MDH gene as a reporter (Vujaklija et al., 1991 ), and the transcriptional activities in the directions of both ORF1 (PORF1) and ORF3 (PORF3) were measured as MDH activities.

As shown in Fig. 4, PORF1 showed relatively low activity throughout the 7 d of growth in YMP liquid media, irrespective of the carbon source. On the other hand, the activity of PORF3 depended markedly on the carbon source: in YMP/glucose, it increased to its highest level after 4 d cultivation and was maintained for a further 3 d. In contrast, in YMP/maltose, it increased during the first 3 d and then dropped sharply. The level during the later cultivation was far lower than that in YMP/glucose. Growth in these cultures was almost identical, with maximum mycelium wet weight at day 5 (Fig. 4a). With galactose as a sole carbon source the profile was similar to that with glucose, while YMP/mannitol and YMP without additional carbon sources gave similar patterns to that obtained with maltose (not shown). MDH was confirmed to stably reflect promoter activity irrespective of the carbon source in the medium (our unpublished control experiment). Therefore these results suggest that promoter activity in the direction of ORF3 is regulated in a carbon-source-dependent manner.



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Fig. 4. Time course of MDH production directed by the promoters subcloned onto pTMA1 in the wild-type strain. Strains harbouring pKM284M1 (PORF1) (squares) or pKM284M3 (PORF3) (circles) were cultured in YMP/glucose (Glc, solid lines and filled symbols) or YMP/maltose (Mal, dashed lines and open symbols); wet weight of mycelium (a) and MDH activities (b) are plotted as a function of cultivation time. Enzyme activities of the co-transformed strain harbouring pKM284M3 and pKM284-2H cultured in YMP/glucose were similarly calculated and are plotted as filled triangles. Experiments were repeated three times and representative results are shown.

 
To examine the PORF3 activity in the presence of the promoter sequence at a high-copy-number, we constructed a strain harbouring two compatible plasmids, pKM284M3 (PORF3-MDH in low copy) and pKM284-2H (PORF3 in high copy) (see Methods section and Fig. 2). The MDH activity of this strain showed that placing the promoter fragment on a high-copy-number plasmid caused marked elevation of PORF3 activity throughout the culture period (Fig. 4). The result strongly suggested that the high-copy-number of the promoter fragment titrates out a negative regulatory element, probably a transcriptional repressor protein; this results in strong transcriptional activity and thus overexpression of ORF3.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Most genes involved in regulating cellular differentiation in Streptomyces have been identified and characterized through complementation of mutants with morphological defects such as bld or whi (Chater, 1998 ). However, regulators with negative functions are apparently difficult to obtain, because mutants showing ‘enhanced’ differentiation due to deficiencies in negative regulators must be recognized by their rapid or abundant formation of aerial mycelium or spores. The strategy adopted in this study, cloning regulatory genes on a multi-copy plasmid, should allow such genes or sequences to be recognized as transformants showing bald or bald-like phenotypes. Similar methods will be useful to screen a variety of regulatory genes for differentiation.

Introducing the promoter region of ORF3 (PORF3) on high-copy-number plasmids caused significant repression of aerial mycelium and spore formation in the presence of glucose or galactose, but not in the presence of maltose or mannitol. Transcriptional activity of PORF3 was enhanced in a carbon-source-dependent manner exactly in parallel with repression of cellular differentiation by pKM284. Elevated transcription from the promoter was maintained until the death phase in the glucose medium, while it dropped sharply during exponential growth in the maltose medium. We assume that this is caused by carbon-source-dependent regulation of PORF3 activity, and that the ORF3 product plays a negative role during initiation of differentiation. ORF3 protein may be produced during vegetative growth to block onset of differentiation, and its carbon-source-dependent repression may initiate differentiation. We propose the name craA to designate the ORF3 gene possibly involved in carbon-source-dependent regulation of aerial mycelium formation. For further characterization of craA as a negative regulator we need to overexpress this gene and disrupt it.

The elevated activities of PORF3 in the presence of pKM284M3 co-existing with pKM284-2H strongly suggested that a transcriptional repressor protein directly binding to the promoter sequence is involved. We detected a DNA-binding protein by gel retardation (data not shown). Binding of this protein to the 249 bp fragment was postulated to block the onset of differentiation during vegetative growth; its carbon-source-dependent transcriptional repression caused by the DNA-binding protein initiates aerial mycelium formation. The concentration of glucose in the medium might also be a key factor affecting the onset of differentiation through repression of craA transcription.

pKM284 repressed aerial mycelium and spore formation of ARP-negative mutants. This indicates that the regulatory point of craA is not directly related to ARP function, but is probably located in a downstream regulatory pathway specific to morphological differentiation. We could not detect a difference in PORF3 activity between an A-factor-deficient mutant and a wild-type strain of S. griseus (data not shown). This suggests that craA-mediated regulation acts independently of the A-factor cascade and integration of the signals from both systems may initiate differentiation. Evidence of significant repression by pKM284 in S. lividans (Fig. 1b) as well as in S. griseus suggests that craA-mediated regulation may be generally distributed among streptomycetes.

Carbon-source-dependent regulation of the initiation of differentiation has been suggested through the phenotypic features of bld mutants of S. coelicolor A3(2). Several classes of bld mutants, including bldA and bldD (Chater, 1989 ), as well as cya, an adenylate cyclase mutant (Süsstrunk et al., 1998 ), are known to show restored aerial mycelium on mannitol medium. In wild-type S. griseus, we observed that the efficiency of aerial mycelium and spore formation depends on carbon sources in the media. S. griseus shows abundant sporulation on maltose or mannitol media, and relatively poor and delayed sporulation on glucose media. Availability of carbon sources may be one of the factors that determine the timing of cellular differentiation in streptomycetes, and the putative regulatory system revealed in this study could be directly involved in one such carbon-source-dependent control mechanism.


   ACKNOWLEDGEMENTS
 
We thank Sir D. A. Hopwood and Professor S. Horinouchi for their kind permission to use various strains and plasmids. This study was supported by the Research for the Future Program of the Japan Society for the Promotion of Science, and the High-tech Research Center Project of The Ministry of Education, Science, Sports and Culture, Japan.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 3 March 1999; revised 26 May 1999; accepted 15 June 1999.



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