Microbiology (1999), 145, 2265–2271
Printed in Great Britain
A putative regulatory element for
carbon-source-dependent differentiation
in Streptomyces griseus
Kenji Ueda, Kouichi Matsuda, Hideaki Takano and Teruhiko Beppu
Author for correspondence : Kenji Ueda. Tel : j81 466 84 3936. Fax : j81 466 84 3935.
e-mail : ueda!brs.nihon-u.ac.jp
Department of Applied
Biological Sciences, Nihon
University, 1866 Kameino,
Fujisawa-shi, Kanagawa
252-8510, Japan
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-sourcedependent deactivation of PORF3 mediated by a transcriptional repressor may
initiate differentiation in S. griseus.
Keywords : Streptomyces griseus, morphological differentiation, carbon source
dependence, repressor, craA
INTRODUCTION
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-γbutyrolactone) plays a crucial role as a hormonal autoregulator to activate signal transduction pathways that
induce both morphological differentiation and streptoAbbreviations : ARP, A-factor receptor protein ; MDH, malate dehydrogenase.
The GenBank accession number for the sequence reported in this paper is
AB023642.
mycin 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.
0002-3342 # 1999 SGM
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K. U E D A a n d O T H E R S
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
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 mC (Horinouchi et al., 1984). A-factorreceptor-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\Fh traD36 proAB lacIq lacZ
M15]. S. griseus strains were grown in Bennett’s glucose
medium, containing (g l−") : yeast extract (Difco), 1 ; meat
extract (Kyokuto), 1 ; NZ amine type A (Wako Pure Chemical), 2 ; and glucose, 10 (pH 7n2) ; in YMP\sugar medium,
containing (g l−") : yeast extract (Difco), 2 ; meat extract
(Kyokuto), 2 ; Bacto peptone (Difco), 4 ; NaCl (Kokusan), 5 ;
MgSO (Kokusan), 2 ; and an appropriate sugar, 10 (pH 7n2) ;
and in%nutrient agar (Difco). Solid media were prepared by
addition of 1n5 % 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 3n7 kb BamHI fragment orig-
inally cloned in pKM284 was at the BglII site of pIJ702. Lowcopy-plasmid pKM284L was constructed as follows : the
3n7 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 5n9 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 ORF2containing 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 8mer 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∆1
and pKM284∆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
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Regulation of differentiation in S. griseus
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.
pIJ922
(a)
pIJ702
pKM284
Glucose
RESULTS
Galactose Maltose
None
Glucose
pKM284L
(b)
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 i 10$ 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).
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 3n7 kb, and subsequent
nucleotide sequencing followed by  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
pIJ702
pKM284
HH1
HO1
KM7
S. lividans
TK21
.................................................................................................................................................
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.
with a molecular mass of 29n0 kDa. A database search
revealed that this protein is a sigma factor belonging to
the σB 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 (31n4 kDa)
containing a putative helix–turn–helix motif in its Nterminal half (Fig. 3b), implicating a possible function as
a DNA-binding protein. ORF3 potentially encoded a
protein of 246 amino acids (26n6 kDa) without significant homology to other proteins.
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 re2267
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GC content (mol%)
K. U E D A a n d O T H E R S
ORF3
ORF1
ORF2
Plasmid
Vector
Repression
of AM
.................................................................................................................................................................................................................................................................................................................
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 3n7 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.
sponsible 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-copynumber 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 lowcopy-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∆1 and
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Regulation of differentiation in S. griseus
(a)
ORF1
CrtS
RpoF
SigF
SigG
SigB
ORF1
CrtS
RpoF
SigF
SigG
SigB
(b)
ORF2
BetI
LuxR
AcrR
(c)
.................................................................................................................................................................................................................................................................................................................
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 σB (SigB, P06574), σF (SigF, P07860) and σG (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 semiconserved 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.
pKMK284∆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 promoterprobe vector carrying a thermostable MDH gene as a
reporter (Vujaklija et al., 1991), and the transcriptional
activities in the directions of both ORF1 (PORF ) and
ORF3 (PORF ) were measured as MDH activities."
$
As shown in Fig. 4, PORF 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 PORF 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.
To examine the PORF activity in the presence of the
promoter sequence at$ a high-copy-number, we con2269
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Mycelium wet wt
(g ml–1)
K. U E D A a n d O T H E R S
DISCUSSION
(a)
0·1
PORF3 (Mal)
PORF3 (Glc)
2
10
8
6
4
4
6
(b)
PORF3 (high copy, Glc)
MDH activity [U (mg protein)–1]
1·2
PORF3 (Glc)
1·0
0·8
0·6
PORF3 (Mal)
0·4
PORF1 (Mal)
0·2
PORF1 (Glc)
2
4
6
Cultivation time (d)
.................................................................................................................................................
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.
structed a strain harbouring two compatible plasmids,
pKM284M3 (PORF -MDH in low copy) and pKM2842H (PORF 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 PORF activity throughout
$ strongly suggested
the culture period (Fig. 4). The result
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.
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 (PORF )
$
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
PORF was enhanced in a carbon-source-dependent
$ exactly in parallel with repression of cellular
manner
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 PORF 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 carbonsource-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 PORF 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-sourcedependent 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 PORF
activity between an A-factor-deficient mutant and a$
wild-type strain of S. griseus (data not shown). This
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Regulation of differentiation in S. griseus
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 (Su$ sstrunk et al., 1998), are known to show
restored aerial mycelium on mannitol medium. In wildtype 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.
Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors
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Received 3 March 1999 ; revised 26 May 1999 ; accepted 15 June 1999.
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