Microbiology (1999), 145, 1161-1 172
Printed in Great Britain
Possible involvement of cAMP in aerial
mycelium formation and secondary
metabolism in Streptomyces griseus
Dae-Kyung Kang,’ Xin-Ming Li,l Kozo Ochi* and Sueharu Horinouchi’
Author for correspondencc: Sueharu Horinouchi. Tel: + 8 l 3 3812 2111 ext. 5123. Fax: +81 3 5802 2931.
e-mail : asuhori@ hongo.ecc.u-tokyo.ac.jp
1
2
Department of
Biotechnology, Graduate
School of Agriculture and
Life Sciences, The
University of Tokyo,
Bunkyo-ku, Tokyo
1 13-8657, Japan
National Food Research
institute, Tsukuba, ibaraki
305-8642, Japan
In Strep tomyces griseus, A-f actor (2-isocapry1oyl-3R-hy droxymet hy I- ybutyrolactone) triggers secondary metabolism and morphogenesis b y binding
a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of
the A-factor-deficient mutant HH1 generated strain HO2, defective in the
synthesis of ArpA and therefore able to form aerial mycelium, spores and
streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus
in strain HO2 yielded a gene that suppressed aerial mycelium formation and
streptomycin production. Nucleotide sequencing and subcloning revealed that
the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2
production of cAMP was growth-dependent until the middle of the
exponential growth stage; the production profile was the same as in the wildtype strain. However, the amount of cAMP produced was five times larger
when mutant HO2 harboured cyaA on the high-copy-number plasmid plJ486.
Consistent with this, supplying cAMP exogenously a t a high concentration to
mutant H02 suppressed formation of both aerial mycelium and streptomycin.
On the other hand, some lower concentrations of cAMP stimulated or
accelerated aerial mycelium formation. No effects of exogenous cAMP on
morphogenesis and secondary metabolism were apparent in the wild-type
strain. In addition, disruption of the chromosomal cyaA gene in the wild-type
strain had almost no effect. Introducing cyaA cloned in either a low- or a highcopy-number plasmid suppressed morphogenesis and secondary metabolism
not only in mutant HO2 but also in other arpA mutants, implying that the
effects of cAMP became apparent in the arpA-defective background. When
mutant H02 carried cyaA on a plasmid, synthesis of the stringent response
factor ppGpp was greatly reduced; this may account for the observed
suppression by cAMP of morphogenesis and secondary metabolism. cAMP also
affected protein tyrosine phosphorylation, as determined with antiphosphotyrosine antibody.
Keywords : A-factor, A-factor receptor protein, protein tyrosine phosphorylation,
stringent response
INTRODUCTION
Members of the bacterial genus Streptomyces produce a
wide variety of secondary metabolites including antibiotics and other biologically active substances. Streptomyces spp. are also characterized by their complex
...........................,.,,,,.,......................,..,,.,...................................................,.....,...................,.,.
The GenBanWEMBUDDBJ accession number for the sequence reported in
this paper is AB018557.
0002-3095 0 1999 SGM
morphological differentiation culminating in sporulation (Chater, 1993). We have studied a diffusible
low-molecular-mass signalling molecule, A-factor
(2-isocapr y 1oy 1-3R-h y d r ox y me t h y 1- y- b u t y r o 1ac to ne) ,
which triggers formation of both streptomycin and
aerial mycelium in Streptomyces griseus (for reviews,
see Khokhlov, 1982, 1988; Horinouchi & Beppu, 1990,
1992,1994; Horinouchi, 1996). A-factor acts as a switch
by binding to DNA-bound A-factor receptor protein
(ArpA), dissociating it from the DNA, and releasing its
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D.-K. K A N G a n d O T H E R S
repressor function (Onaka & Horinouchi, 1997; Onaka
et al., 1997). The intracellular concentration of A-factor
thus determines the timing of secondary metabolism and
aerial mycelium formation. A series of S. griseus K M
mutants and S. griseus H01, which are defective in
ArpA, produce streptomycin and form aerial mycelium
and spores even in the absence of A-factor (Miyake et
al., 1990; Onaka et al., 1995). Introducing the intact
arpA gene on a plasmid into these strains represses both
streptomycin production and aerial mycelium
formation. However, mutant H02, which also has a
defect in the biosynthesis of ArpA, is not repressed by
introducing a r p A on a plasmid. These observations
prompted us to examine the characteristics of mutant
H02.
This paper describes properties of S. griseus mutant
strain H02, which has led us to notice the possible
involvement of cAMP in secondary metabolism and cell
differentiation in S. griseus. Ragan & Vining (1978)
observed a possible, but indirect, relationship between
cAMP and streptomycin biosyntheses by determining
the timing of cAMP production in relation to that of
streptomycin. We have confirmed their observations
and further demonstrated distinct phenotypic changes
dependent on cAMP by using mutant H 0 2 . Very
recently, Susstrunk et al. (1998) showed pleiotropic
effects of cAMP on germination, secondary metabolism
and cell differentiation in Streptomyces coelicolor A3(2).
All of these observations suggest that cAMP exerts
general pleiotropic effects o n secondary metabolism and
morphological development in Streptomyces spp.
Bacterial strains, plasmids and culture media. S. griseus I F 0
13350 was obtained from the Institute of Fermentation, Osaka.
Streptomyces strains were grown in YMPD medium [yeast
extract (Difco), 0.2 '/o ; meat extract (Wako Pure Chemicals),
0.2 '/o ; Bacto-peptone (Difco), 0.4 '/o ; NaCl, 0.5 '/o ;
MgSO, .7H,O, 0.2 '/o ; glucose, 1'/o ; and glycine, 0.5 '/o ;
p H 7.21. YMPD agar medium buffered with 25 m M TES
( p H7 . 2; Sigma) was used for examining phenotypes of S.
griseus. Thiostrepton (15 pg ml-l) and neomycin (20 pg ml-l)
were added when necessary. R2YE medium (Hopwood et al.,
1985) containing 0.2 '/o asparagine instead of proline was used
to regenerate protoplasts of S. griseus. Plasmids pIJ486 and
pIJ487, both with a copy number of 40-100 (Ward et al.,
1986), and pKU209, with a copy number of 1-2 (Kakinuma et
al., 1991), were used as cloning vectors in S. griseus; they
conferred thiostrepton resistance. Plasmid pARPLl containing
arpA was previously described by Onaka et al. (1995).Plasmid
pUC19 was used for DNA manipulation in Escherichia coli
strains JM109 and JMl lO (Yanisch-Perron et al., 1985).E . coli
strains were routinely cultured in Luria broth (Sambrook et
al., 1989). When necessary, ampicillin (50 pg ml-') was added.
Recombinant DNA techniques. Restriction endonucleases, T 4
DNA ligase and DNA polymerase I (Klenow) were purchased
from Takara Shuzo. DNA fragments were purified from
agarose gels with a Gene Clean kit (Bio 101). Nucleotide
sequences were determined by the dideoxynucleotide chaintermination method (Sanger et al., 1977) combined with the
pUC19 cloning system (Yanisch-Perron et al., 1985) on an
1162
automated fluorescent DNA sequencer (Li-Cor model 4000L).
General techniques for recombinant DNA work were described by Sambrook et al. (1989), and those in Streptomyces
were described by Hopwood et al. (1985).
A-factor-binding assay. A-factor-binding activity in crude
lysates of S. griseus strains was determined by the method of
Miyake et al. (1989). Briefly, mycelium was harvested by
centrifugation from mutant H 0 2 and the wild-type strain I F 0
13350 grown in YMPD medium at 28 "C for 3 d, washed once,
and disrupted by sonication. The sonicate was centrifuged at
30000 g, and ammonium sulfate was added to the supernatant
to give a final concentration of 60% (w/v). The precipitate
was collected, dissolved in buffer A (50 m M triethanolamine
and 0.5 M KC1, p H 7.0) and dialysed against buffer A. The
assay mixture containing 200 p1 of the crude lysate (5 mg
protein) and 30 n M ["]A-factor [2-8 Ci mmol-' (103.6 GBq
mmol-l)] was incubated at 30 "C for 30 min in the presence or
absence of a 500-fold molar excess of nonlabelled A-factor and
immediately applied to a Sephadex G-25 column (1.2 by 6 cm;
Pharmacia PD10) for rapid separation of receptor-bound from
free [3H]A-factor. Fractions 3 and 4 always contain ArpAbound [3H]A-factor (Miyake et al., 1989; Onaka et al., 1995).
Shotguncloning of cyaA. Chromosomal DNA from S. griseus
I F 0 13350 was isolated and digested with BamHI; 2-20 kb
fragments were purified by agarose gel electrophoresis. The
fragments were ligated with BamHI-digested PI5486 and the
reaction mixture was used to transform protoplasts of S.
griseus H 0 2 . After protoplast regeneration on the modified
R2YE medium, transformants were selected by replica plating
on YMPD agar containing thiostrepton. From more than 6000
transformants, one colony showing a bald (Bld) phenotype
was picked for further study.
Gene disruption. The chromosomal cyaA gene was disrupted
by the method of O h & Chater (1997).The BamHI ends of the
originally cloned 3 kb fragment were changed into EcoRI sites
by use of an 8-mer linker and cloned in the EcoRI site of
pUC19. The 654 bp Aor51HI fragment in the cyaA-coding
sequence was replaced with a 1.32 kb SmaI fragment containing a neomycin phosphotransferase (neo)gene from Tn5
(Beck et al., 1982) by standard DNA manipulation. The 3.6 kb
EcoRI fragment containing the neo-disrupted cyaA gene was
purified by agarose gel electrophoresis, alkali-denatured, and
introduced by transformation into S. griseus strains I F 0 13350
and H 0 2 . After neomycin (20 pg ml-')-resistant transformants
had been selected, true disruptants were chosen based on the
results from Southern hybridization of BamHI-digested
chromosomal DNA with the neo sequence and the 3 kb
fragment on pHCYA3 as probes. The probes were labelled
with horseradish peroxidase by using an ECL kit (code
RPN3000 ; Amersham).
Assay of streptomycin production in the presence of CAMP.
Freshly prepared spores of S. griseus strains were used to
inoculate 1 0 m l YMPD medium. cAMP at a final concentration of 0.25 or 2.5 m M was added at inoculation and at 9,
18, 23 and 34 h after inoculation. After 5 d cultivation, 200 pl
of the culture broth on a paper disc was placed on agar
medium seeded with Bacillus subtilis ATCC 6633. The yield of
streptomycin was estimated from the diameter of the inhibition zone (Horinouchi et al., 1984).
Quantification of extracellular CAMP. S. griseus H 0 2
harbouring pIJ486 or pHCYAl was grown at 28 "C for 3 d in
YMPD medium containing 15 pg thiostrepton ml-'. The
mycelium was disrupted with a glass homogenizer and 1 ml
was used to inoculate 100 ml of the same medium. Portions
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CAMP a n d physiology in S. griseus
20
15
10
5
n
"
x
m
z
2
4
6
8
1
0
1
2
20
15
10
5
n
-
2
4
6
8 1 0 1 2
Fraction number
Fig. 1. A-factor binding in 5. griseus mutant HO2. (a) A-factor-binding activities in lysates prepared from the wild-type S.
griseus I F 0 13350 (top) and mutant HO2 (bottom) are shown. Fractions 3 and 4 represent ArpA-bound [3H]A-factor,and
fractions 6-1 0 represent free [3H]A-factor. Specific binding is defined as the difference between binding of the
radioactive A-factor in the presence (0)
and absence ( 0 )of a 500-fold molar excess of nonlabelled A-factor. (b) Western
blot analysis of the cell lysate with the anti-ArpA antibody. ArpA in the mycelium of 5. griseus wild-type strain I F 0 13350
and mutant HO2 grown for 36 h (corresponding to the mid-exponential phase) was detected by Western blotting with
the anti-ArpA antibody. ArpA is detected in the mycelium prepared from wild-type strain IF0 13350 harbouring plJ486
( - ) or pHCYAl (+). Almost no ArpA is detected in the mycelium prepared from mutant HO2 harbouring either plJ486
( - ) or pHCYAl (+).
(5-10 ml) of the culture broth were taken out at intervals
during cultivation at 28 "C, and supernatants were obtained
by centrifugation at SO00 g for S min. The supernatants were
boiled for 3 min and stored at -20 "C before use. The
amounts of CAMPin the supernatants were measured with the
Amersham enzyme immunoassay kit (code RPN225).
fugation of the sonicates at 5000g for 10 min were used as
crude extracts. When proteins with phosphotyrosines were
detected, 100 pM Na,VO, and 20 m M NaF were added to the
sonication buffer to inhibit phosphatase activities. Protein
concentrations were determined by the method of Bradford
(1976) using BSA as a standard.
Quantification of intracellular ppGpp. Intracellular ppCpp
was determined using HPLC apparatus equipped with a 25 cm
Partisil PXS 10 SAX column (Whatman; void vol. 3.02 ml)
according to the method of Ochi (1986). Spores (approx.
2 x lo8) were spread on a UV-sterilized cellophane sheet
(diameter 8 cm) on the surface of YMPD agar medium. The
cellophane sheet with mycelium was removed at intervals and
quickly weighed. It was laid upside-down in 2 ml methanol in
a Petri dish and 10 m l 1 M formic acid was added immediately.
The entire procedure was completed within 10 s. After the
sample had been extracted at 4 "C for 1 h, a supernatant was
obtained by centrifugation and dried in vacuum; the amount
of ppGpp present was measured by HPLC.
Western blot assay for detection of ArpA. Polyclonal antibody for ArpA was prepared in rabbits as described previously
(Onaka & Horinouchi, 1997). For detection of ArpA in S.
griseus strains, the crude extracts described above were
electrophoresed in 0.1 '/o SDS-10 O/O polyacrylamide gels and
electroblotted to a PVDF membrane (Immobilon-P;
Millipore). The membrane was probed with the antibody and
then incubated with horseradish-peroxidase-conjugatedantirabbit IgG (Amersham). Signals were generated using the
enhanced chemiluminescence system (ECL ; Amersham).
Preparation of cell extracts for Western blotting. S. griseus
strains grown under similar conditions at 28 "C in YMPD
medium were sampled (5-10 ml) at intervals and the mycelium
was collected by centrifugation at 15000 g for 20 min. T h e
mycelium was suspended in 2 ml 20 m M Tris/HCl ( p H 7-0),
1 m M EDTA, 1 m M D T T and 10% (v/v) glycerol, and
disrupted by sonication. The supernatants obtained by centri-
Western blotting with anti-phosphotyrosine antibody. For
immunological detection of phosphotyrosine-containing
proteins, the crude extracts (20 pg protein) were electrophoresed in 0.1 '/o SDS-10% polyacrylamide gels (7 x 9 cm).
Western blotting was carried out as described by Matsudaira
(1987) with the following modifications. The proteins were
electro-transferred from the gel to a PVDF membrane at
200 mA for 2 h in CAPS buffer ( p H 11.0). The blot was
blocked overnight at 4 "C with 10% blocking reagent
(Boehringer Mannheim) in TBS containing 10 m M Tris/HCI
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1163
(pH 7.2) and 140 mM NaCl. T h e blot was then incubated with
the p e rox i d a se-coi i j ~ i agted a tit i-ph osp h ot y ro sine anti body
(clone 3-36.5-10; Boehringer Mannheini), and diluted 1: 1000
in TBS containing 5 % blocking reagent for 2 h at room
temperature. T h e blot was thoroughly washed with five
changes of TBS containing 1% blocking reagent and developed with the BM chemiluminescent system (Boehringer
Mannheini) according t o the protocol of the manufacturer.
RESULTS
Properties of 5. griseus mutant H 0 2
S. griseus mutant H 0 2 was derived as a sporulationp osit i v e a n d st r ept om y cin - p rod u c i ng mutant by UV -
mutagenesis from the A-factor-deficient mutant strain
HH1. Strain HH1 neither produces streptomycin nor
forms aerial mycelium because of a deletion in afsA,
which encodes a key enzyme for A-factor biosynthesis
(Hara et al., 1983; Horinouchi et al., 1984; Ando et al.,
1997a). The same strategy was previously used to isolate
mutants defective in ArpA, such as a series of K M
mutants (Miyake et al., 1990) and mutant H 0 1 (Onaka
et a[.,1997). Examination of an extract of mutant H 0 2
showed very low, but detectable, A-factor-binding
activity (Fig. l a ) , suggesting that it contained a mutation
that almost completely abolished either the A-factorbinding activity of ArpA or ArpA biosynthesis. Immunoblotting with anti-ArpA antibody showed that mutant
H 0 2 produced a very small amount of ArpA (Fig. Ib).
Although ArpA in mutant H 0 2 is slightly larger than in
the wild-type strain, this tendency has been observed for
all arpA-defective mutants so far examined, probably
due to lack of processing at the carboxy-terminus. In
wild-type S. griseus, ArpA seems to be processed at its
carboxy-terminus (Onaka et al., 1995). A faint ArpA
signal in mutant H 0 2 was assumed to reflect the very
low A-factor-binding activity. Mutant H 0 1 , in which a
mutated a r p A encodes a protein with serine replacing
proline-115 (Onaka et al., 1997), produced the altered
ArpA protein from the early stage of growth. Mutants
KM5, KM7 and KM12 also produced proteins reactive
with the anti-ArpA antibody (data not shown), suggestive of a mutation in the arpA-coding sequence. It was
therefore apparent that, in contrast to mutant H 0 1 and
the KM mutants, mutant H 0 2 had a defect in the
biosynthesis of ArpA. Aerial mycelium formation and
streptomycin production by these a r p A mutants in the
absence of A-factor was explained in terms of the
repressor-type behaviour of ArpA (Miyake et al., 1990;
Onaka et al., 1995, 1997). However, introduction of
plasmid pARPHl containing arpA on the high-copynumber vector pIJ487, or of pARPLl containing arpA
on the low-copy-number vector pKU209, into mutant
H 0 2 did not repress aerial mycelium formation or
streptomycin production (data not shown). This result
contrasts with that for other arpA mutants such as H 0 1
and KM7, where a r p A on the plasmids almost completely represses both phenotypes (Onaka et al., 1995,
1997). Mutant H 0 2 was thus assumed to contain a
defect(s) that abolished both ArpA biosynthesis and the
signal relay from ArpA t o the downstream genes.
--
1164
______
Fig. 2. Repression by cyaA of morphological differentiation and
streptomycin production in 5. griseus mutant HO2. (a)
Formation of aerial mycelium and spores. Mutant HO2
harbouring cyaA on plJ486 (plasmid pHCYAl), grown on YMPD
medium a t 28°C for 7 d, does not form aerial mycelium,
whereas HO2 harbouring plJ486 alone forms spores. (b)
Streptomycin production after 3 d growth was bioassayed with
B. subtilis as an indicator. Mutant HO2 harbouring plJ486 gives
an inhibitory zone comparable t o that given by the wild-type
strain IF0 13350, whereas mutant HO2 harbouring pHCYAl
gives a much smaller zone.
Cloning a gene that repressed sporulation of S.
griseus H 0 2
To reveal the defect in mutant H 0 2 we tried to clone a
gene that repressed aerial mycelium formation and
streptomycin production. By shotgun-cloning BamHIdigested chromosomal DNA from the wild-type S.
griseus I F 0 13350 into mutant H 0 2 with a high-copynumber vector, pI 5486, we isolated a tramformant that
showed a Bld phenotype. Fig. 2(a) shows the Bld
phenotype of mutant H 0 2 harbouring pHCYAl (see
below). This transformant produced a much smaller
amount of streptomycin than did mutant H 0 2
harbouring the vector pIJ486 (Fig. 2b). Production of a
diffusible yellow pigment, one of the secondary
metabolites produced by S. griseus, was also repressed
(data not shown). The cloned gene thus appeared to
repress or inhibit both cellular differentiation and
secondary metabolism in mutant H 0 2 . However, as
described below, introduction of pHCYAl into the
wild-type strain I F 0 13350 did not cause any detectable
phenotypic changes.
The recombinant plasmid in the Bld colony contained a
3 k b BamHI fragment in the BamHI site of pIJ486
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CAMP and physiology in S. griseus
-
~ 1 1 1 1 1 1
Orfl (CyaA)
Orf3
Not1
Nru I
BamHI
I
0
NCOI
NCOI
Aor51HI
NruI AorSlHI
I
I
1
I
2
I
pHCYAl
pHCYA2
pHCYA3
pLCYAl
Barn
m
3 (kb)
Fig. 3. Restriction map, sequence analyses
and subcloning of the 3.0 kb BamHl
fragment cloned on plJ486. The location and
direction of ORFs were determined by
analysis of the nucleotide sequence using
the FRAME program (Bibb et a/., 1984) with a
Repression sliding window of 100 codons. The
subcloned fragments are on a high-copynumber plasmid, plJ486 (pHCYA series), and
a
low-copy-number plasmid,
pKU209
(pLCYA1). The results of subcloning of the
fragments are assessed from their ability to
+
repress aerial mycelium formation by 5.
+
griseus mutant H02.
Subcloning of orfl
200
c
(b)
pHCYA1
n
Cultivation time (h)
Fig. 4. cAMP production by S. griseus mutant HO2 harbouring
pHCYAl as a function of cultivation time. (a) Growth measured
as wet cell weight. Mutant H 0 2 harbouring plJ486 (a),as a
control, or pHCYAl (0)
was grown in YMPD liquid medium. (b)
Amounts of cAMP in the culture broth determined by the
immunological method. The peaks of cAMP biosynthesis a t 40
and 48 h appear t o coincide with the decision points of mutant
HO2 harbouring plJ486 and pHCYA1, respectively.
(plasmid pHCYA3; Fig. 3).We purified the recombinant
plasmid DNA from the transformant and constructed a
restriction map of the insert (Fig. 3). The nucleotide
sequence of the cloned fragment revealed the presence of
two complete ORFs (Orfl with 399 amino acids and
Orf2 with 249 amino acids) and one truncated ORF
(Orf3).
A 1-6kb NruI-BamHI fragment was excised from
pHCYA3 and subcloned between the HincII and BamHI
sites of pUC19. The fragment was then excised as an
EcoRI-Hind111 fragment and inserted between the
EcoRI and HindIII sites of pIJ486 to give pHCYA1.
For construction of pLCYA1, the same NruI-BamHI
1-6kb fragment was blunt-ended with Klenow fragment
and 8-mer EcoRI linkers were attached to the ends.
The EcoRI fragment thus generated was inserted in the
EcoRI site of pKU209. For construction of pHCYA2, the
1.9 kb SacI fragment was first cloned into the SacI site of
pUC19. The fragment was then excised as an EcoRIHindIII fragment and inserted between the EcoRI and
HindIII sites of pIJ486. O n the basis of these subcloning
experiments (Fig. 3), we identified orfl in either the
high-copy-number plasmid PI5486 (plasmid pHCYA1)
or the low-copy-number plasmid pKU209 (plasmid
pLCYA1) to be responsible for repression of aerial
mycelium formation and streptomycin production in
mutant H 0 2 .
A computer-aided search with the CLUSTAL w program
showed that orfl encoded a protein with end-to-end
similarity in amino acid sequence to the adenylate
cyclase from S. coelicolor A3(2) (Danchin et al., 1993).
The carboxy-terminal portion of Orfl also showed
great similarity to the catalytic domains of adenylate
cyclases from a prokaryote, Brevibacterium liquefaciens
(Peters et al., 1991),and two eukaryotes, Saccharomyces
cerevisiae (Kataoka et al., 1985) and Schixosaccharomyces pombe (Yamawaki-Kataoka et al., 1989). CyaAs
of S. griseus and S. coelicolor A3(2) showed no sequence
similarity to those from E. coli (Aiba et al., 1984),
Bacillus anthracis (Escuyer et al., 1988) or Erwinia
chrysanthemi (Danchin & Lenzen, 1988). Supporting
the identification of orfl as an adenylate cyclase (cyaA)
gene, the transformants harbouring orfi on a plasmid
produced large amounts of CAMP, as described below.
Thus, the S. griseus CyaA showing end-to-end similarity
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D.-K. K A N G a n d O T H E R S
growth hesitation at mid-exponential phase (Neumann
et af., 1996; Holt et af., 1992). Mutant H 0 2 harbouring
pLCYAl produced 82-98 pmol cAMP ml-l with its
peak at the decision point. The wild-type strain I F 0
13350 produced cAMP in the same pattern and in almost
the same amount as mutant H 0 2 harbouring pIJ486.
O u r attempts to measure cAMP accumulated within the
mycelium failed to show a detectable amount.
Effects of exogenously added cAMP on
morphogenesis and secondary metabolism
Fig. 5. Acceleration (bottom) and inhibition (top) of aerial
mycelium and spore formation by exogenously supplemented
cAMP in 5. griseus mutant HO2. Spore suspensions of mutant
H 0 2 were spread on YMPD agar and paper discs containing
15 pmol or 5 pmol cAMP were placed on the agar surface a t the
indicated times after inoculation. The photographs were taken
after 2 d. At 7 d after inoculation, sporulation occurred over all
of the bottom Petri dish, and in the area outside the
accelerated zone in the upper Petri dish. In the area close t o
the paper discs containing 15 pmol CAMP, where aerial
mycelium formation was repressed, only substrate mycelium
was observed, even a t 10 d after inoculation.
to those of S. coelicofor A3(2) and B. liquefaciens
belongs to a family of eukaryotic adenylate cyclases (see
also discussion by Danchin et af., 1993 and Peters et al.,
1991), rather than to the two types of prokaryotic
enzymes.
Orf2 exhibits 77 ‘!o identity in amino acid sequence to B.
subtilis BirA, which regulates biotin synthesis (Bower et
af., 1995). The truncated Orf3 has strong sequence
similarity to the B chain of the propionyl-CoA carboxylase (Donadio et al., 1996), which participates in
the catabolic pathway of odd-chain fatty acids, isoleucine, threonine, methionine and valine. These two
ORFs seem, therefore, to be functionally unrelated to
CyaA.
Production of cAMP by mutant H 0 2 carrying cyaA on
a plasmid
We measured by enzyme-immunoassay the amount of
cAMP produced by mutant H 0 2 harbouring pHCYAl
(Fig. 4). Culture broths accumulated up to 192 pmol
ml-l in a growth-dependent manner until midexponential phase ; the cAMP then rapidly disappeared,
presumably due t o cyclic phosphodiesterase activity
(Terry & Springham, 1981). Mutant H 0 2 harbouring
pIJ486 produced only 36 pmol cAMP ml-l in the same
time frame. T h e timing of the cAMP peak in both strains
coincided with the ‘decision point’ associated with the
1166
Contrary to our expectations, adding 5 pmol cAMP
exogenously on a paper disc to S. griseus mutant H 0 2 at
inoculation resulted in rapid aerial mycelium formation
around the disc in 2 d as observed by microscopy (Fig.
5). cAMP presumably diffused rapidly into the agar, and
by 4 d, aerial mycelium was present throughout the Petri
dish. Accelerated aerial mycelium formation, and accordingly sporulation, was seen with cAMP in the range
of 0-5-5 pmol per paper disc, and was more evident
around the discs applied in the early stages of growth;
when the discs were applied a long time after inoculation, the areas where aerial mycelium formed were
smaller. These observations suggested that cAMP at a
certain concentration caused precocious aerial mycelium
formation. Because a decrease in p H due to acid
accumulation in a cyaA-disruptant of S. coeficofor A3(2)
disturbed antibiotic biosynthesis and morphological
development (Susstrunk et al., 1998), we examined the
effect of buffering the medium. However, the same
phenotypic changes caused by exogenous cAMP were
observed on medium supplemented with 25 m M TES.
No significant change of p H of the medium could be
detected with a p H indicator strip. Dibutyryl CAMP,
which is known to penetrate membranes readily
(Posternak et al., 1962),gave the same effects at the same
concentrations as CAMP, suggesting that dibutyryl
cAMP and cAMP are incorporated at similar rates in
Streptomyces.
In amounts larger than 5 pmol per disc, cAMP had an
inhibitory effect on aerial mycelium formation by
mutant H 0 2 without affecting cell growth per se. Aerial
mycelium formed in a doughnut-like area around paper
discs containing 15 pmol cAMP applied at inoculation,
indicating that stimulation of aerial mycelium formation
was concentration-dependent. In 5 d, aerial mycelium
and spore formation were observed in the area outside
the doughnut zone, but even after 7 d little or no aerial
mycelium formed inside the doughnut zone. This implies
that cAMP at higher concentrations inhibits aerial
mycelium formation. T h e inhibitory effect is more
pronounced when the cAMP is supplied earlier, as
judged by the larger diameters of inhibition zones. The
same effects of cAMP were seen on the medium buffered
with 25 m M TES.
When cAMP at a final concentration of 0.25 m M was
added at inoculation and at 9, 18, 23 and 34 h after
inoculation to cultures of mutant H 0 2 grown in YMPD
medium, streptomycin titres were similar to those of
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cAMP and physiology in S. griseus
controls cultured in the absence of cAMP (data not
shown). When 2.5 m M cAMP was added, however,
almost no streptomycin was produced, irrespective of
the timing of cAMP addition (data not shown). Thus
although a low concentration of cAMP may not have
stimulated streptomycin production to a detectable
level, a high concentration clearly inhibited production.
microscope also showed no measurable difference ; both
germinated with almost 100% frequency. Thus, disruption of cyaA did not appreciably affect germination
of the S. griseus spores.
Negligible effect of cAMP or overexpression of cyaA
on the wild-type strain
Morphological and physiological differentiation is often
a response to nutrient limitation. One of the bacterial
regulatory systems coupled to nutrient limitation is the
stringent response, which amongst other cellular
reactions causes cessation of RNA synthesis (Cashel,
1975; Gallant, 1979). ppGpp is one of the compounds
responsible for the stringent response. Its occurrence in
S. griseus and possible relationship to streptomycin
biosynthesis were studied by An & Vining (1978). Ochi
(1987a) proposed that its intracellular accumulation
triggers streptomycin biosynthesis and that a decrease in
the G T P pool leads to aerial mycelium formation. T o
explore these possibilities we measured changes in the
amount of ppGpp in mutant H 0 2 containing cyaA on a
plasmid.
When pHCYA1 was introduced by transformation into
S. griseus strain I F 0 13350, little change was detected in
the timing and abundance of aerial mycelium formation,
or in the timing and yield of streptomycin production
(data not shown). Furthermore, exogenous supplementation from paper discs containing 5 and 15 pmol cAMP
caused only minor apparent phenotypic consequences.
By very careful comparison of aerial hyphae around
paper discs containing 5 pmol and 15 pmol cAMP with
hyphae from areas far from the discs, stimulatory and
inhibitory effects, respectively, could be detected. This
implies that cAMP does cause subtle effects on morphogenesis and secondary metabolism in the wild-type
strain.
No detectable alteration of phenotypes in cyaAdisrupted strains
We constructed a cyaA null mutant from strain I F 0
13350 by replacing the wild-type gene with a cyaA
sequence disrupted by the neo gene sequence. We
obtained three disruptants that gave the expected
Southern hybridization pattern with two different
probes (data not shown). These cyaA mutants produced
no detectable amount of cAMP when assayed by the
enzyme-immunological method (data not shown), indicating that strain I F 0 13350 contains a single cyaA gene.
However, the growth profile showing a decision point,
the timing and abundance of aerial mycelium formation,
and the timing and yield of streptomycin production by
the disruptants were the same as those of the wild-type
strain.
We also derived cyaA-disruptants from mutant H 0 2 .
Morphological development and secondary metabolism
in these disruptants were not detectably different from
those of mutant H 0 2 . Exogenously supplied cAMP
exerted the same effects on these disruptants as on
mutant H 0 2 (data not shown). We also constructed
cyaA-disruptants from the A-factor-deficient mutant
strain HH1, but these mutants showed only a Bld and
streptomycin-negative phenotype, just like strain HH1.
Because Susstrunk et al. (1998) observed a much lower
frequency of germination in cyaA-disrupted S . coelicolor
A3(2) spores, we examined the germination of spores
from cyaA-disruptants of S . griseus I F 0 13350. When
freshly prepared spore suspensions from the wild-type
strain and one of the cyaA-disruptants were spread on
YMPD agar and the numbers of colonies appearing at
intervals were compared, no difference was observed.
Counting the number of the germinated spores under a
Change of the intracellular ppGpp pool in mutant
H02
In mutant H 0 2 containing the vector pIJ486, ppGpp
synthesis on YMPD medium began with the onset of
aerial hyphae formation and continued until the onset of
sporulation (Fig. 6). The time course of ppGpp production relative to the developmental stages matched
that in wild-type strain I F 0 13350 (data not shown) and
in S. griseus I F 0 13189 (Ochi, 1987a), although morphological development in mutant H 0 2 started slightly
later than in the wild-type strain. Mutant H 0 2
harbouring pHCYA1 accumulated a severely reduced
amount of ppGpp. This could mean that the failure of
mutant H 0 2 to produce streptomycin in the presence of
pHCYAl and probably in the presence of a large amount
of exogenously added cAMP is caused by insufficient
accumulation of ppGpp.
Phosphotyrosine proteins in mutant H 0 2
Waters et al. (1994) detected protein tyrosine kinases in
various Streptomyces species and observed changes in
the phosphorylation pattern depending on the growth
stage and culture conditions. We also observed that
protein serine/threonine and tyrosine kinase inhibitors
such as staurosporine and K-252a inhibit aerial mycelium formation and antibiotic production in S.
coeficolor A3(2) (Hong & Horinouchi, 1998) and in S.
griseus (Hong et al., 1993). Attempts to examine protein
phosphorylation patterns in mutant H 0 2 with and
without pHCYAl by in vitro phosphorylation with [ y "PIATP and cell lysates (Hong et al., 1993) failed
because the phosphorylation patterns were not reproducible. Although phosphorylation of several proteins
did appear to be affected by the presence of pHCYA1,
we abandoned this method in favour of using antiphosphotyrosine antibody to detect differences in protein phosphorylation on tyrosine residues. This procedure directly detects proteins with phosphotyrosine
residues in the cell lysate. In S. griseus, changes in the
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1167
AM
40 I
S
t
t
1
1000
500
12
I
1
I
I
I
I
2
3
4
5
Time of cultivation (d)
24
36
48
60
72
84
6
Fig. 6. Changes in the intracellular pool of ppGpp in S. griseus
H02. Mycelium of mutant HO2 harbouring plJ486 or pHCYAl
was spread on YMPD agar medium and incubated a t 28.5 "C. (a)
Growth observed visually. (b) ppGpp analysis by the
intracellular ppGpp assay described in Methods of whole cells
from one Petri dish. ppGpp accumulation begins concomitantly
with aerial hyphae formation in mutant HO2 harbouring
plJ486. Aerial mycelium (AM) and spores (S) were formed by
mutant HO2 harbouring plJ486 a t the points indicated. The
amount of ppGpp accumulated in mutant HO2 harbouring
pHCYA1 is much smaller than in mutant HO2 harbouring
plJ486.
.~
tyrosine phosphorylation pattern depended on the
growth phase (Fig. 7 ) , as has been reported in other
Streptomyces species (Waters et al., 1994). In addition,
as described below, phosphorylation patterns of the
wild-type strain and mutant H 0 2 were totally different.
This prompted us to examine protein phosphorylation
in detail with the anti-phosphotyrosine antibody. In the
wild-type S. griseus strain, Western blotting detected
more than nine proteins (bands 1-9) in the wild-type S.
gyiseus strain, all of which were clearly visible before the
decision point. However, some of them (bands 4 , 5 , 6 , 7
and 9) rapidly disappeared after that point, indicating
that phosphorylation of these proteins was strictly under
decision-point control.
In mutant H02, on the other hand, phosphorylation of
tyrosine residues in most of these proteins was severely
repressed before the decision point (Fig. 8). Only
proteins 1 and 3 were visible. At the decision point and
thereafter, the phosphorylation of most of the proteins
was apparent. I n addition, two new phosphorylated
proteins, x and y, appeared. Since the same
phosphorylation patterns were observed in other arpA
mutants (e.g. a series of KM mutants; data not shown),
the absence of tyrosine phosphorylation on these
Fig. 7. Changes in the profile of proteins with phosphotyrosine
residues in S. griseus IF0 13350. The wild-type strain IF0 13350
was grown in YMPD medium and mycelium was taken a t the
indicated times (points A-F) to detect proteins with
phosphotyrosine residues by Western blotting with antiphosphotyrosine antibody. Growth was measured as wet cell
weight. The decision point apparently corresponds to point C.
Nine reactive phosphorylated proteins (1-9) are clearly visible
before the decision point.
proteins appeared to be due to the mutation in arpA.
This contrasts sharply with phosphorylation of these
proteins before the decision point and their subsequent
dephosphorylation in the wild-type strain (Fig. 7). In
mutant H 0 2 harbouring pHCYA1, phosphorylation of
most proteins detected in the wild-type strain was
repressed until the end of exponential growth. Because
supplying CAMP exogenously to mutant H 0 2 at a
final concentration of 10 m M also prevented
of p h 0sp h or y 1at ion
phosphor y 1at i on , repression
throughout the exponential growth phase is attributed,
directly o r indirectly, to overproduction of CAMP.
Although little is known about the relationship of
proteins with phosphotyrosines to morphogenesis and
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CAMP and physiology in S. griseus
- ~ ~ - _ _ _ _ _ _
~ _ _ _ _ -
plJ;p
F
pHCYAl
12
24
36
48
60
72
84
Fig. 8. Changes in the profiles of proteins
with phosphotyrosine residues in 5. griseus
mutant H 0 2 harbouring plJ486 or pHCYAl.
Mutant H02 harbouring plJ486 (designated
-) or pHCYAl (designated +) was grown in
YMPD medium, and mycelium taken a t the
indicated times (points A-F) was examined
for proteins with phosphotyrosine residues
by
Western
blotting
with
antiphosphotyrosine antibody. Growth was
measured as wet cell weight. The decision
point apparently corresponds to point C.
Proteins are assigned in the same way as in
Fig. 7.
~
secondary metabolism, it is apparent that in several of
the in cA M P affects ph o sp h ot yr o sin e phosphor y 1at ion.
DISCUSSION
Because only very low A-factor-binding activity and
almost no ArpA were detected in S. griseus mutant
H 0 2 , the mutation(s) in this strain seems to prevent
ArpA production. The additional evidence that introduction of an intact arpA gene does not repress aerial
my ce 1i u ni form at io n or streptomycin prod uc ti on
suggests that a signal relay from ArpA to downstream
genes may also have been interrupted. O u r attempt to
identify the latter mutation in strain H 0 2 led to the
cloning of cyaA encoding a eukaryotic-type adenylate
cyclase. The stiniulatory effect of a low concentration of
CAMP on aerial mycelium formation and possibly also
on secondary metabolism, and the inhibitory effect at
higher concentrations, were observed not only in mutant
H 0 2 but also in a series of K M mutants and in mutant
H 0 1 , all of which contain a mutation in arpA. Although
CAMP provided either as an exogenous supply o r by
disrupting cyaA did not have a very distinct effect in the
wild-type strain, it could be involved in regulating
morphogenesis and secondary metabolism, though not
essential t o the processes. I t might play a role in tuning
~
~-
the physiological conditions in a still unknown way, as
is observed in prokaryotes (Botsford & Harman, 1992)
and eukaryotes (Hunter, 1995; Taylor et al., 1990). The
regulatory roles of cyaA in S. coelicolor A3(2) reported
recently by Susstrunk et al. (1998) suggest that CAMP
does act as a second messenger in Streptornyces spp.
Tata & Menawat (1994) also reported a role for CAMP
in tylosin synthesis in Streptomyces fradiae.
In S. griseus, A-factor is effective only during the short
period when the decision to biosynthesize streptomycin
is established and subsequent production of the antibiotic and differentiation can be influenced (Neumann et
al., 1996). Consistent with this, supplying A-factor to Afactor-deficient mutant strains after the decision point
induces little or no aerial mycelium formation (Ando et
al., 1997b), and providing a large amount at inoculation
severely disturbs aerial mycelium formation, probably
due t o too early an onset of the sequence of metabolic
events (Ando et al., 1997b). A-factor is thus thought to
initiate an ordered sequence of metabolic events required
for morphological and physiological differentiation
during the decision phase. It is noteworthy that the arpA
mutations in mutants KM7 and H 0 1 cause the hosts to
begin aerial mycelium formation and streptomycin
production earlier than in the wild-type strain (Miyake
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1169
~
D.-K. K A N G a n d O T H E R S
et al., 1990; Onaka et al., 1997). We presume that the
absence of ArpA sets off an earlier sequence of some
particular metabolic events that alter the physiological
conditions influencing other metabolic events, but lead
finally to morphological and physiological differentiation. The profile of CAMP accumulation, which shows
a peak exactly at the decision point, suggests a role for
this molecule during the decision phase, as was previously pointed out by Ragan & Vining (1978). T h e
effects of cAMP may be concealed in the wild-type
background, and may become apparent only by their
modulation, directly o r indirectly, of the aberrant
sequence of events induced by the absence of ArpA.
One of the metabolic events, actually affected independently of A-factor by CAMP, is synthesis of the
stringent response factor ppGpp. This event not only
reflects nutrient conditions, such as nitrogen limitation
and amino acid starvation, but is important for initiating
both physiological and morphological differentiation
(Ochi, 1986,1987a7 1988; Chakraburtty & Bibb, 1997).
S. griseus mutant H 0 2 , like the wild-type strain, began
to accumulate ppGpp at the onset of aerial mycelium
formation. In S. griseus I F 0 13189, ppGpp was shown
to be a strong inhibitor of IMP dehydrogenase, which
eventually led to a decrease in the intracellular G T P pool
size (Ochi, 1987b). We therefore assume that the
inability to form aerial mycelium of mutant H 0 2
carrying a high-copy number of c y a A is due to a slower
decrease in its GTP pool size. T h e greatly reduced
accumulation of ppGpp when c y a A was introduced on
the high-copy-number plasmid accounts for the
observed repression of aerial mycelium and streptomycin production in these transformants. S. griseus
mutant H 0 2 will offer a feasible system to examine the
possible effect of cAMP on the expression of relA
encoding ppGpp synthetase (Martinez-Costa et al.,
1996; Chakraburtty & Bibb, 1997). Such an effect may
not be readily detected in the wild-type strain, which
sporulates and produces streptomycin almost normally
in the presence of a high concentration of CAMP.
We have recently found that an AfsK/AfsR homologous
system controls aerial mycelium formation in response
to glucose in S. griseus (T.Umeyame, P.-C. Lee, K. Ueda
& S. Horinouchi, unpublished results). In S. coelicolor
A3(2), a protein serine/threonine kinase, AfsK, and its
target protein AfsR control secondary metabolism
(Matsumoto et al., 1994). In the present study, we found
that profiles of protein tyrosine phosphorylation
changed dramatically after the decision point (Fig. 7).
Similar changes in protein tyrosine phosphorylation
have been reported in Streptom yces h ygroscopicus,
Streptornyces lividans and Streptomyces lavendulae
(Waters et al., 1994). In mutant H 0 2 , the tyrosine
phosphorylation pattern is almost the reverse of that in
the wild-type strain ; most proteins phosphorylated
before or a t the decision point in the wild-type strain are
not phosphorylated in mutant H 0 2 until after the
decision point. Although the role of tyrosine
phosphorylation in physiological and morphological
differentiation is not yet clear, this finding suggests
1170
that mutant H 0 2 differentiates physiologically and
morphologically in an aberrant protein tyrosine
phosphorylation background. Since the tyrosine
phosphorylation pattern in the A-factor-deficient mutant strain HH1 containing the intact arpA gene is
similar to that in the wild-type strain (data not shown),
the change in the phosphorylation pattern is ascribed to
ArpA. Higher concentrations of cAMP either directly o r
indirectly inhibit tyrosine phosphorylation after the
decision point in mutant H 0 2 , and this may result in
repression of physiological and morphological differentiation. Further studies are necessary to fully elucidate
the role of tyrosine phosphorylation and to relate it to
CAMP. Modulatory activities of cAMP are observed
exclusively with protein serine/threonine kinases in
eukaryotes (Taylor et al., 1990; Hunter, 1995), but
activation of a protein tyrosine kinase in Acinetobacter
calcoaceticus belonging to the Neisseriaceae family has
been reported (Grangeasse et al., 1997).
In conclusion, our data, together with those of Susstrunk
et al. (1998) and Tata & Menawat (1994), show a
pleiotropic regulatory role for cAMP in Streptomyces.
One effect is on ppGpp synthesis, reflecting the nutrient
conditions closely associated with morphological
and physiological differentiation. Protein tyrosine
phosphorylation, also suggested to be important for
differentiation in Streptomyces (Hong et al., 1993), is
also affected by cAMP in conjunction with ArpA, one of
the important proteins in the A-factor regulatory cascade. These findings suggest that a complex cAMP
regulatory network participates in a coordinated and
concerted way with other regulatory networks to control
physiological functions.
This work was supported, in part, by the Nissan Science
Foundation, by the ‘Research for the Future’ Program of the
Japan Society for Promotion of Sciences (JSPS), and by the Bio
Design Program of the Ministry of Agriculture, Forestry, and
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Received 20 November 1998; revised 13 January 1999; accepted
20 January 1999.
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