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Journal of Bacteriology, July 2001, p. 4357-4363, Vol. 183, No. 14
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.14.4357-4363.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Gene Replacement Analysis of the Butyrolactone
Autoregulator Receptor (FarA) Reveals that FarA Acts as a Novel
Regulator in Secondary Metabolism of Streptomyces
lavendulae FRI-5
Shigeru
Kitani,1
Yasuhiro
Yamada,2 and
Takuya
Nihira1,*
Department of Biotechnology, Graduate School
of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka
565-0871,1 and Department of Applied
Biological Science, Faculty of Engineering, Fukuyama University,
Fukuyama, Hiroshima 729-0292,2 Japan
Received 7 December 2000/Accepted 25 April 2001
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ABSTRACT |
IM-2
[(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl
-butanolide] is a -butyrolactone autoregulator which, in
Streptomyces lavendulae FRI-5, switches off the production
of D-cycloserine but switches on the production of a blue
pigment and several nucleoside antibiotics. To clarify the in vivo
function of an IM-2-specific receptor (FarA) in the IM-2 signaling
cascade of S. lavendulae FRI-5, a farA deletion
mutant was constructed by means of homologous recombination. On several
solid media, no significant difference in morphology was observed
between the wild-type strain and the farA mutant (strain
K104), which demonstrated that the IM-2-FarA system does not
participate in the morphological control of S. lavendulae
FRI-5. In liquid media, the farA mutant overproduced nucleoside antibiotics and produced blue pigment earlier than did the
wild-type strain, suggesting that the FarA protein acts primarily as a
negative regulator on the biosynthesis of these compounds in the
absence of IM-2. However, contrary to the IM-2-dependent suppression of
D-cycloserine production in the wild-type strain, overproduction of D-cycloserine was observed in the
farA mutant, indicating for the first time that the
presence of both IM-2 and intact FarA are necessary for the suppression
of D-cycloserine biosynthesis.
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INTRODUCTION |
Members of the filamentous,
gram-positive bacterial genus Streptomyces are versatile
producers of many secondary metabolites, including over two-thirds of
all known antibiotics used in human medicine and in agriculture
(1, 2). These antibiotics are products of complex
biosynthetic pathways, and their production occurs in a growth
phase-dependent manner (4) generally coinciding with
morphological differentiation on solid media. Among factors known to
affect antibiotic production and/or morphological differentiation in
streptomycetes (5, 6, 9, 11, 43), -butyrolactone autoregulators have been shown in several streptomycetes to serve as
extracellular signaling molecules that determine the onset of these two
noteworthy characteristics (13). Among the known -butyrolactone autoregulators, the earliest known was A-factor (7, 15, 16), which is required for both streptomycin
production and sporulation in a streptomycin-negative
(Str
) and sporulation-negative (Spo) mutant
of Streptomyces griseus. Other well-studied
-butyrolactone autoregulators are virginiae butanolides (VBs),
which control virginiamycin production in Streptomyces
virginiae (23, 40), and SCB1, which induces the
precocious production of actinorhodin and undecylprodigiosin in
Streptomyces coelicolor A3(2) (38). The most
unique among the -butyrolactone autoregulators is IM-2 [(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl--butanolide]
of Streptomyces lavendulae FRI-5. In contrast to the solely
positive effects exerted by other members of the autoregulator family, IM-2 is capable not only of switching on the production of a blue pigment (BP) and nucleoside antibiotics (showdomycin and minimycin) but
also of switching off the production of the antituberculosis antibiotic
D-cycloserine (DCS) (8).
In vitro studies of an IM-2-specific receptor protein (FarA) (21,
34, 39) have indicated that FarA is a dimeric DNA binding
protein that, in the absence of IM-2, recognizes and binds to specific
DNA sequences situated in the promoter region of a target gene. IM-2
binding to FarA causes FarA to dissociate from the DNA, which in turn
allows the transcription of the target gene to occur. Similar data have
been obtained in vitro for a VB-specific receptor (BarA) (19,
20) and an A-factor-specific receptor (ArpA) (29,
32), suggesting that all the autoregulator receptors
express a common activity as transcriptional repressors. Yet a common
in vivo trait on the autoregulator-dependent cascade has not been
determined by phenotypic analyses of receptor-deficient mutants. When
an ArpA mutant of S. griseus was created in an
A-factor-negative background, the defect in sporulation was restored
with even earlier initiation than for the wild-type (Str+
Spo+) strain, and the defect in streptomycin production was
restored to a further 10-fold overproduction compared to that of the
wild-type strain (25), indicating that ArpA acts solely as
a repressor of the two processes. However, no morphological difference
was observed between wild-type S. virginiae and a
barA null mutant (26, 27), indicating that the
VB-specific receptor BarA is not involved in morphological
differentiation. Furthermore, in the barA null mutant,
virginiamycin production was suppressed to only 10% that of the
wild-type strain and biosynthesis of VB itself was abolished, which
latter trait is not clear for ArpA. Therefore, it is apparent that
further investigation of another autoregulator-dependent cascade is
needed in order to determine a common trait that will enable us to
predict or manipulate the autoregulator-dependent secondary metabolism
in Streptomyces species.
In this study, to confirm that FarA is actually involved in the IM-2
signaling cascade of S. lavendulae FRI-5 and also to identify common traits of autoregulator-dependent phenotypes, a
farA deletion mutant of S. lavendulae was
constructed using homologous recombination, and a phenotypic comparison
between the wild-type strain and a strain with a farA
deletion was reported. Similar to the case of VB biosynthesis in
S. virginiae, FarA was found to regulate IM-2 biosynthesis
in S. lavendulae. Lines of evidence are presented showing
that FarA is involved as a negative regulator in the production of BP
and nucleoside antibiotics. Moreover, it was found that in addition to
the presence of IM-2, intact FarA should be present to suppress DCS
production. This regulation is novel in autoregulator signaling and
suggests that an autoregulator-bound receptor itself, rather than the
unbound receptor, could be an important component in the autoregulator signaling cascade.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, growth conditions, and conjugal
transfer of DNA from Escherichia coli to S. lavendulae FRI-5.
S. lavendulae FRI-5
(MAFF10-06015; National Food Research Institute, Ministry of
Agriculture, Forestry and Fishers, Tsukuba, Japan) was cultured as
described previously at 28°C (8) in medium B (containing
[in grams per liter] yeast extract, 7.5; glycerol, 7.5; NaCl, 1.25 [pH 6.5]) for antibiotic production, in liquid medium containing half
volumes each of YEME medium (12, 17) and Trypticase soy
broth (Oxoid) for preparation of total DNA, and on ISP medium 2 (Difco)
for spore formation. For conjugal transfer of DNA into S. lavendulae FRI-5, the methylation-deficient E. coli
strain ET12567 (dam-13::Tn9 dcm-6 hsdM
hsdS) (24) containing the RP4 derivative pUZ8002
(33) was used as the donor. E. coli K-12 strain
JM101 (Toyobo) was used for routine subcloning. The plasmids used were
pIJ8606 (J. H. Sun and M. J. Bibb, personal communication), a
pIJ2925 (14) derivative containing a thiostrepton resistance gene (tsr), pKC1132 (3), and pSET152
(3). Procedures for standard DNA manipulation in E. coli and Streptomyces were described previously
(reference 35 and references 12 and 17, respectively). All chemicals were of reagent or high-performance liquid
chromatography (HPLC) grade and were purchased from Nacalai Tesque,
Takara Shuzo, or Wako Pure Chemical Industries.
Construction of a farA deletion mutant and a
farA complemented strain.
A 2.8-kb EcoRI
(blunt-ended)-BstPI fragment containing the 5' upstream
sequence plus the 5' 123 bp of farA was isolated from pMW101, a pUC19 derivative containing an 8-kb PstI fragment
(39), and the fragment was cloned into the
EcoRI and SmaI sites of pIJ8606 to give pSG101. A
2.8-kb PvuII-PstI fragment carrying the 3' 30 bp
of farA plus the 3' downstream sequence from pMW101 was
cloned into the PstI and blunt-ended SphI sites
of pSG101, generating pSG102. This reconstructed a contiguous 5.6-kb
segment of the chromosome, except that a 510-bp
BstPI-PvuII fragment internal to farA
was replaced with the 1.1-kb tsr gene. The entire 6.7-kb insert was recovered from pSG102 as an EcoRI-PstI
fragment and cloned into the EcoRI and PstI sites
of pKC1132, yielding pSG103.
E. coli ET12567(pUZ8002) transformed with pSG103 was
conjugated with S. lavendulae FRI-5 K101 as previously
described (22). Exconjugants in which the plasmid pSG103
had presumptively integrated at the farA locus by a single
crossover via homologous recombination were selected with apramycin.
After three rounds of incubation at 28°C on ISP medium 2 containing 5 µg of thiostrepton per ml, putative
farA::tsr deletion mutants formed from
the second crossover were detected by their apramycin sensitivity. One
of the strains was designated strain K104. To complement the
farA deletion mutant (K104), a 2.1-kb SacI
fragment containing the entire farA gene with promoter was
isolated from pMW101 and cloned into SacI-digested and
blunt-ended pSET152, generating pSG104. After conjugal transfer of
pSG104 from E. coli ET12567(pUZ8002) to strain K104,
apramycin resistance exconjugants were obtained and designated strain
K105. The resulting strains were analyzed by Southern hybridization. The probe used was a 902-bp farA internal fragment amplified
by PCR from pMW101 using farAN-3
(5'-CGGGATCCTCATCGGCACACCACGGCCCG-3') and farAC-3
(5'-CGGGATCCTGCACAGGGGAAAGCGGA-3') as primers.
IM-2 binding assay.
Crude extracts for the IM-2 binding
assay were prepared as described previously (34). IM-2
binding activity was routinely assayed using the ammonium sulfate
precipitation method (18) with
[3H]IM-2-C5 (10 pmol; 40 Ci/mmol) in the
presence and absence of non-labeled IM-2-C5 (15 nmol;
1,500-fold molar excess). The radioactivity in the solution was
measured using a liquid scintillation counter (model LS6000; Beckman).
Detection of BP.
At the indicated times, supernatants were
obtained by centrifugation (15,000 × g, 4°C, 10 min)
of culture broths in medium B and were filtrated through
0.2-µm-pore-size filters, and the absorbance at 590 nm was measured.
Analysis of nucleoside antibiotic and DCS production.
For
measuring the production of nucleoside antibiotics, culture broth (60 ml of medium B) in a 500-ml baffled flask was collected after 31 h
of cultivation, mycelia were removed by suction filtration, and the
filtrate was adjusted to pH 7.0. The filtrate was applied to an active
charcoal column (5 g) followed by washing with 100 ml of water, and the
absorbed compounds were eluted with 200 ml of methanol. The methanol
eluent was evaporated, dissolved in 5 ml of water, lyophilized, and
redissolved in 1 ml of water for bioassay. The samples thus prepared
contain both showdomycin and minimycin (8), and the
bioassay was performed by measuring clear-zone formation with
Bacillus subtilis PCI219 as a test organism on
glucose-Simmone's agar medium as described by Nishimura et al.
(28) after incubation for 2 to 3 days at 30°C. Authentic showdomycin (a generous gift from Shionogi & Co., Ltd.) was used as the
standard for the bioassay.
For DCS production, samples in medium B were withdrawn at the indicated
times and clarified by filtration through 0.2-µm-pore-size
filters.
Aliquots (50 µl) were separated and quantified by HPLC
(cation-exchange column; Senshu Pak SCX-1251-N; Senshu Scientific
Co.,
Ltd.) with 10 mM ammonium acetate (pH 5.0) as solvent and
detection at
210 nm using authentic DCS (Sigma) as the
standard.
Morphological assessment.
Spores of the wild-type strain, a
farA deletion mutant (strain K104), and a farA
complemented strain (strain K105) were streaked or plated out on ISP
medium 2, oatmeal agar (8), MS agar (mannitol plus soya
flour) (10), minimal medium agar containing 0.5% (wt/vol) mannitol as a carbon source (12, 17), R2 agar (12,
17), and modified SMMS agar (supplemental minimum medium, solid)
as described by Takano et al. (38) and were cultivated at
28°C for 7 days before they were analyzed for morphological differences.
Analysis of IM-2 production.
Spores of each strain (6.6 × 108 spores per 25 ml of medium) were inoculated on ISP
medium 2 agar plates and incubated at 28°C for 19 h. Cultures
from four plates, including agar, were cut into small pieces and kept
frozen at 
80°C for 1 h. Ethanol (60 ml) (adjusted to pH 2.0 with HCl) was added, the supernatant was obtained by centrifugation and
evaporated, and the residue was extracted with ethyl acetate (20 ml).
The ethyl acetate extract was evaporated and dissolved in methanol and
clarified by passage through cotton, and the filtrates were evaporated.
The residue after evaporation was redissolved in
methanol-H2O (4:6) at a concentration of 200 mg/ml and
purified in aliquots of 50 µl by HPLC (C18 reverse-phase column; 10 by 250 mm; Cosmosil C18), with
methanol-H2O (4:6) as a solvent and detection at 210 nm.
Fractions (19.8 to 23.4 ml) corresponding to the elution position of
authentic IM-2-C4 were combined, evaporated, and dissolved
in 3 ml of methanol for the IM-2 bioassay. IM-2 activity in the sample
was assayed by measuring the IM-2-dependent production of BP
(42). One unit of IM-2 activity is the minimum amount
required for the induction of BP production and corresponded to 0.6 ng
(2.97 nM) of IM-2-C5 per ml (36). Authentic
IM-2-C4 was synthesized as described previously
(36).
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RESULTS AND DISCUSSION |
Insertional inactivation of farA in S. lavendulae FRI-5.
To assess the regulatory role(s) of FarA
in the secondary metabolism of S. lavendulae FRI-5, a 510-bp
BstPI-PvuII fragment internal to farA
was replaced with a thiostrepton resistance gene (tsr) by
which 170 amino acids, corresponding to the 42nd to 211th amino acids
of FarA (221 amino acids), were deleted. This deletion includes those
amino acids constituting the second helix of the helix-turn-helix DNA
binding motif (32nd to 51st amino acids) in the N terminus and the
presumed autoregulator binding region in the C-terminal half
(37); thus, the resulting truncated protein should be
devoid of both DNA and IM-2 binding activity. The farA deletion allele together with ca. 2.8 kb of the 5'- and 3'-flanking regions was cloned into pKC1132, a nonreplicating plasmid in
streptomycetes, to generate pSG104. Conjugal transfer from
E. coli ET12567(pUZ8002) harboring pSG104 to
S. lavendulae FRI-5 gave apramycin-resistant exconjugants in
which pSG104 integration by a single crossover event was confirmed by
Southern blot analysis (data not shown). After three rounds of
sporulation of the pSG104-integrated strain in the presence of
thiostrepton, thiostrepton-resistant and apramycin-sensitive colonies were obtained. Southern blot analysis of representative strains, such as strain K104, using PCR-amplified farA
as a probe showed that a 2.1-kb SacI fragment in the
wild-type strain shifted to a 2.6-kb fragment in strain K104 (Fig.
1), confirming that the K104 chromosome
contained only the deleted farA gene by a second crossover.
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FIG. 1.
Construction of a farA deletion mutant and
its complemented strain. (A) Schematic representation of the strategy
used for the disruption of farA and its complementation. The
solid arrow represents the farA gene, the dark gray arrow
represents the farX gene, whose function is unknown, the
light gray arrow represents the thiostrepton resistance gene
(tsr), the open arrow represents the apramycin resistance
gene (apr), the cross-hatched box represents the
oriT sequence, and the black box represents the  C31
attachment site (attB site). Abbreviations: W.T., wild type;
S, SacI; S*, disrupted SacI site. (B) Southern
hybridization analysis of chromosomal DNA from strains K101 (wild-type
strain; lane 1), K104 (a farA deletion mutant; lane 2), and
K105 (a farA complemented strain derived from strain K104;
lane 3) digested with SacI. The probe used was the 0.9-kb
PCR-amplified fragment containing the entire farA gene.
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Phenotypic characterization of a farA deletion
mutant (K104) in liquid culture.
To confirm the absence of a
functional IM-2 receptor (FarA) in strain K104, IM-2 binding activity
was measured with tritium-labeled IM-2-C5 using crude
extracts prepared from 8.5-h cells of strain K104 and the wild-type
strain (Table 1). The 8.5-h time of
cultivation was carefully selected, because BP production at 10.5 h in the wild-type strain indicates the presence at 8.5 h of
endogenous IM-2, which usually precedes BP production by about 2 h. The presence of endogenous IM-2 not only inhibits the IM-2 binding
assay by competing with labeled IM-2 but also enhances the amount of
FarA in the wild-type strain by derepressing farA
transcription (21, 39). The results of the IM-2 binding
assay demonstrated that strain K104 lost almost all IM-2 binding
activity in comparison with a wild-type strain (strain K101).
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TABLE 1.
IM-2 binding activity and IM-2 production of the S. lavendulae FRI-5 wild-type strain (strain K101), a farA
mutant (strain K104), and a farA complemented strain (strain
K105)a
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To examine whether the
farA mutation affects growth
characteristics in liquid culture, the growth of strains K101 and K104
was measured with or without exogenous addition of IM-2 at 5 h
of
cultivation (Fig.
2). While the growth of
the wild-type strain
was repressed by the addition of synthetic
IM-2-C
5 at a final
concentration of 100 nM, strain K104
continued to grow irrespective
of IM-2 addition, indicating that strain
K104 became insensitive
to the presence of IM-2.
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FIG. 2.
Growth curves in liquid culture of the wild-type strain
(strain K101) (A), a farA deletion mutant (strain K104) (B),
and a farA complemented strain (strain K105) (C). Each
strain was grown in medium B at 28°C without IM-2 addition (open
triangles) or with IM-2 addition (final concentration, 100 nM) at
5 h of cultivation (solid squares). Growth was monitored by
measuring the optical density at 600 nm (OD600). Arrows
indicate the timing of the IM-2 addition.
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To evaluate how the
farA deletion affects IM-2 signaling, BP
production in strain K104 was initially monitored by measuring
the
absorbance at 590 nm (Fig.
3A). In the
wild-type strain (strain
K101) without exogenous IM-2 addition, BP
production was observed
after 10.5 h of cultivation, while the
addition of IM-2 at 5 h
of cultivation induced BP production from
7 h of cultivation.
In the
farA mutant strain K104,
however, regardless of IM-2 addition,
BP production was observed after
8 h of cultivation, with much
reduced levels of BP (about 4% that
of the wild-type strain with
external IM-2). This phenomenon in strain
K104 resembled that
of the VB receptor disruptants of
S. virginiae in which virginiamycin
production began much earlier, if
not occurring constitutively,
than in the parental strain but at
an amount of only 10% that
of the parental strain (
26,
27).
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FIG. 3.
Time courses of BP production (A) and DCS production (B)
in a farA deletion mutant and a wild-type strain. The
amounts of BP and DCS at the indicated times were measured as described
in Materials and Methods. Strains K101 (wild-type strain; solid
diamonds), K104 (farA mutant; open circles), and K105
(complemented strain; solid triangles) were grown at 28°C in medium
B. The designation (+) IM-2 indicates that exogenous IM-2 (final
concentration of 100 nM) was added to the culture at 5 h of
cultivation, and cultivation was continued further, and () IM-2
indicates that there was no exogenous IM-2 addition. Arrows indicate
the timing of the IM-2 addition.
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Similar to BP production, the production of nucleoside antibiotics
(showdomycin and minimycin) is induced by IM-2 in a wild-type
strain
(
8). However, without the addition of IM-2, the production
was small or negligible, but external IM-2 clearly caused production
(
8). This suggests that the concentration of endogenously
produced
IM-2 (about 27 nM) was not high enough to fully activate the
biosynthesis
of nucleoside antibiotics and that full activation
requires that
much higher concentrations of IM-2 be generated by
external IM-2
addition. In the
farA mutant strain K104,
however, the production
of nucleoside antibiotics was higher even
without IM-2 addition
than was that of the wild-type with the external
addition of IM-2
(Fig.
4), indicating
that the
farA deletion resulted in overproduction
of
nucleoside antibiotics. This phenomenon is similar to the case
of an
A-factor receptor (ArpA)-deficient mutant of
S. griseus in
which a 10-fold final overproduction of streptomycin was observed,
with
a 1-day earlier initiation of production than in the wild-type
strain
(
25), suggesting that
farA is, as proposed for
arpA in
the streptomycin biosynthesis of
S. griseus, the primary negative
regulatory gene for the biosynthesis
of nucleoside antibiotics
in
S. lavendulae FRI-5.
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FIG. 4.
Comparison of nucleoside antibiotic production between
strain K101 and strain K104 with a bioassay using B. subtilis PCI219. Supernatants from culture broth were applied onto
active charcoal columns, and the absorbed compounds were eluted with
methanol, concentrated, and used for bioassay. Each strain was
cultivated for 31 h without () or with (+) IM-2 addition at
5 h of cultivation.
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Although phenotypically positive effects on antibiotic production such
as those by IM-2 on nucleoside antibiotics and BP are
common among
autoregulators, phenotypically negative effects on
antibiotic
production are unusual among
-butyrolactone autoregulators
and are
seen only as the termination of DCS by IM-2 (
8). Because
the repressor function seems common to all the autoregulator receptors
(
19,
20,
21,
32), we hypothesized that FarA should repress
a negative regulator of DCS production and expected that DCS production
would be abolished in the
farA mutant. However, strain K104
not
only continued to produce DCS but also overproduced it (Fig.
3B),
suggesting the presence of a completely different mode of regulation
by
FarA.
Phenotypic characterization of a farA deletion mutant
on solid medium.
Recently, the involvement of an A-factor
receptor, ArpA, in the morphological control of S. griseus
was clearly demonstrated, in which ArpA indirectly represses
transcription of the adsA gene encoding an extracytoplasmic
sigma factor necessary for morphological differentiation via repressing
the adpA gene encoding an activator of adsA
(41). To clarify whether farA is involved in
the morphological control of S. lavendulae, morphological
characteristics of strains K101 and K104 were carefully compared on a
range of different solid media. Because no differences in morphology
were detected between strains K101 and K104 (data not shown), the
IM-2-FarA cascade was concluded to play no role in the morphological
differentiation of S. lavendulae FRI-5.
The wild-type strain with a confluent lawn of growth produced a BP-like
pigment on ISP medium 2 and R2 agar medium which was
lacking or too
small to be visible for strain K104 (Fig.
5), indicating
that the phenomenon is
FarA dependent. Because this phenomenon
can be considered to be an
IM-2-triggered onset of secondary metabolism
on solid media, we
assessed the concentration of IM-2 in the solid
media (Table
1). As
expected, the wild-type strain (K101) produced
6.48 nM IM-2, which is
2.2-fold higher than the minimum effective
concentration of IM-2 (2.97 nM). Surprisingly, strain K104 produced
only 11% of the amount of IM-2
produced by the wild-type strain,
indicating that FarA should be
necessary for IM-2 biosynthesis.
The similar requirement of an intact
autoregulator receptor for
the production of a corresponding
autoregulator has been observed
in the BarA null mutant
(
26) of
S. virginiae and the SCBI receptor
(ScbR) mutant of
S. coelicolor A3(2) (E. Takano and M. J. Bibb,
personal communication). Thus, an autoregulator receptor should
generally be involved in the regulation of autoregulator biosynthesis.
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FIG. 5.
BP production by strains K101, K104, and K105 on ISP
medium 2. Spores of each strain were plated out on ISP medium 2, and
plates were incubated for 19 h at 28°C.
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trans complementation of a farA deletion
mutant.
To confirm that the phenotypic differences observed
between the wild-type strain and the farA mutant (strain
K104) were due to the lack of functional FarA protein, an intact
farA gene was transconjugated and integrated into an
attB site of strain K104 via pSG105, a derivative of a
Streptomyces integration plasmid, pSET152, containing a
C31 attP site and int. The integration of
intact farA in apramycin-resistant exconjugants was
confirmed by Southern hybridization (Fig. 1), and they were designated
strain K105. All the characteristics of strain K105 (the IM-2 binding activity [Table 1], growth in the presence of IM-2 [Fig. 2], BP
production [Fig. 3A], DCS production [Fig. 3B], nucleoside antibiotic production [data not shown], and IM-2 production [Table 1]) were restored to the wild-type phenotypes. Furthermore, pigment production on solid media (ISP medium 2 and R2 agar) was also restored
in strain K105 (Fig. 5), suggesting that pigment production on solid
media is FarA dependent, similar to the FarA-dependent BP production in
liquid media. These lines of evidence clearly demonstrated that the
phenotypic changes in strain K104 were due solely to the
loss-of-function mutation of farA.
All the known
-butyrolactone autoregulator receptors (FarA as an
IM-2 receptor [
34], BarA as a VB receptor
[
30], ArpA
as an A-factor receptor [
31],
and ScbR as an SCBI receptor [Takano
and Bibb, personal
communication]) are highly conserved in the
DNA binding motif present
in their N-terminal portions and have
been proposed to play roles as
transcriptional regulators in antibiotic
production (
19,
20,
21,
29,
32). Previously, our in
vitro analysis of an IM-2-specific
receptor, FarA, revealed that
one of the target genes of FarA is the
farA gene itself (
21).
The FarA protein in the
absence of IM-2 represses
farA transcription
by binding to
specific FarA binding sequences in the
farA promoter
region.
In the presence of IM-2, transcription of
farA is
derepressed
by the dissociation of IM-2-bound FarA from the FarA
binding sequences,
forming an autoregulatory circuit between IM-2 and
FarA.
Phenotypic analyses of a
farA deletion mutant revealed that
FarA is involved in the IM-2-dependent growth suppression in liquid
media, the production of BP and nucleoside antibiotics, and the
termination of DCS production, confirming that FarA is a mediator
of
IM-2 signaling in
S. lavendulae. Furthermore, it became
clear
that FarA is somehow participating in the regulation of IM-2
biosynthesis.
However, contrary to the case for the A-factor-ArpA
system of
S. griseus, no regulatory role in regard to
morphology was observed
for the IM-2-FarA system, which agreed well
with phenomena observed
in two other autoregulator systems, namely, the
VB-BarA system
of
S. virginiae and the recently found
SCB1-ScbR system of
S. coelicolor A3(2) (Takano and Bibb,
personal communication). Therefore,
a lack of a regulatory role as
regards morphology seems to be
common, rather than an exception, to
-butyrolactone autoregulators
of
streptomycetes.
We propose the following model for the IM-2-mediated signaling cascade
for the secondary metabolism of
S. lavendulae FRI-5
(Fig.
6). Initially, FarA acts as a
transcriptional repressor
on
farA itself, forming an
autoregulatory circuit which should
serve to sense and maintain
intracellular free IM-2 concentrations
under some threshold level in
the cells. This autoregulatory circuit
has also been observed in the
VB-BarA system of
S. virginiae (
19,
20) and in
the recently found SCB1-ScbR system of
S. coelicolor A3(2);
thus, it seems to be common to
-butyrolactone autoregulator-receptor
systems of streptomycetes. Next, FarA regulates the biosynthesis
of
IM-2 itself, as shown by the observation that IM-2 production
was
dramatically decreased in the
farA disruptant. Thirdly, the
biosyntheses of both BP and nucleoside antibiotics are negatively
controlled by FarA. However, there exists a difference in the
degrees
of regulation. FarA seems to be a primary dominant-negative
regulator
of the biosynthesis of nucleoside antibiotics, since
its disruption
resulted in the overproduction of nucleoside antibiotics,
a phenomenon
identical to ArpA-streptomycin production in
S. griseus (
25). On the other hand, FarA does not seem to be the
dominant
regulator of the biosynthesis of BP in that its disruption
resulted
in earlier initiation of BP production but with a much reduced
amount compared to the wild-type strain.
S. lavendulae
should
have an additional regulatory mechanism which terminates the
premature
initiation of BP, a phenomenon similar to BarA-virginiamycin
production
in
S. virginiae (
26,
27). Finally,
but most striking for the
farA mutant, was the
overproduction of DCS, which was completely
opposite to the expected
lack of DCS production. Supposing that
IM-2-unbound FarA is either
repressing a repressor or activating
an activator of DCS biosynthesis,
the loss-of-function mutation
of
farA should result in the
loss of DCS production. Therefore,
if unbound FarA is the only
functional form, this overproduction
phenomenon in the
farA
mutant is unexplainable and indicates that
both the intact FarA and the
presence of IM-2 are necessary for
the termination of DCS biosynthesis,
suggesting as the simplest
model that the IM-2-FarA complex may act as
a regulator. Because
genes under the control of the IM-2-FarA complex
are predicted
to be bound by the IM-2-FarA complex, searching for DNA
fragments
bound by FarA in the presence of IM-2 is under way and will
give
us clues on the signaling mechanism in
S. lavendulae in
particular
and, in general, on the
-butyrolactone-dependent
regulatory cascade
in streptomycetes.
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|
FIG. 6.
Proposed model of the IM-2 regulatory cascade leading to
the production of secondary metabolites in the absence (A) or presence
(B) of IM-2. Solid lines and dashed lines show direct or indirect
regulation of expression of subordinate proteins, respectively. Arrows
and horizontal lines show activation and repression of the regulation,
respectively.
|
|
| |
ACKNOWLEDGMENTS |
This work was supported in part by a grant from the Research for
the Future Program of the Japan Society for the Promotion of Science (JSPS).
We are grateful to M. J. Bibb (John Innes Centre, United Kingdom)
for helpful discussions regarding the manuscript and to E. Takano (John
Innes Centre) for helpful comments on the manuscript. We also thank
J. H. Sun (John Innes Centre) for the gift of pIJ8606 and Shionogi
& Co., Ltd., for the gift of showdomycin.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7433. Fax:
81-6-6879-7432. E-mail:
nihira{at}bio.eng.osaka-u.ac.jp.
| |
REFERENCES |
| 1.
|
Alderson, G.,
D. A. Ritchie,
C. Cappellano,
R. H. Cool,
N. M. Ivanova,
A. S. Huddleston,
C. S. Flaxman,
V. Kristufek, and A. Lounes.
1993.
Physiology and genetics of antibiotic production and resistance.
Res. Microbiol.
144:665-672[Medline].
|
| 2.
|
Berdy, J.
1995.
Are actinomycetes exhausted as a source of secondary metabolites?, p. 13-14.
In
V. Debabov, Y. Dudnik, and V. Danlienko (ed.), Proceedings of the 9th International Symposium on the Biology of Actinomycetes. All-Russia Scientific Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
|
| 3.
|
Bierman, M.,
R. Logan,
K. O'Brien,
E. T. Seno,
R. N. Rao, and B. E. Schoner.
1992.
Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.
Gene
116:43-49[CrossRef][Medline].
|
| 4.
|
Chater, K. F., and M. J. Bibb.
1997.
Regulation of bacterial antibiotic production, p. 57-105.
In
H. Kleinkauf, and H. V. Dohren (ed.), Biotechnology, vol. 7. Products of secondary metabolism. VCH Press, Weinheim, Germany.
|
| 5.
|
Demain, A. L.
1992.
Microbial secondary metabolism: a new theoretical frontier for academia, a new opportunity for industry.
Ciba Found. Symp.
171:3-16[Medline].
|
| 6.
|
Demain, A. L., and A. Fang.
1995.
Emerging concepts of secondary metabolism in actinomycetes.
Actinomycetologica
9:98-117.
|
| 7.
|
Hara, O., and T. Beppu.
1982.
Mutants blocked in streptomycin production in Streptomyces griseus the role of A-factor.
J. Antibiot.
35:349-358[Medline].
|
| 8.
|
Hashimoto, K.,
T. Nihira,
S. Sakuda, and Y. Yamada.
1992.
IM-2, a butyrolactone autoregulator, induces production of several antibiotics in Streptomyces sp. FRI-5.
J. Ferment. Bioeng.
73:449-455[CrossRef].
|
| 9.
|
Hobbs, G.,
A. I. C. Obanye,
J. Petty,
J. C. Mason,
E. Barratt,
D. C. J. Gardner,
F. Flett,
C. P. Smith,
P. Broda, and S. G. Oliver.
1992.
An integrated approach to studying regulation of production of the antibiotic methylenomycin by Streptomyces coelicolor A3(2).
J. Bacteriol.
174:1487-1494[Abstract/Free Full Text].
|
| 10.
|
Hobbs, G.,
C. Frazer,
D. C. J. Gardner,
J. Cullum, and S. G. Oliver.
1989.
Dispersed growth of Streptomyces in liquid culture.
Appl. Microbiol. Biotechnol.
31:272-277.
|
| 11.
|
Hood, D. W.,
R. Heidstra,
U. K. Swoboda, and D. A. Hodgson.
1992.
Molecular genetic analysis of proline and tryptophan biosynthesis in Streptomyces coelicolor A3(2): interaction between primary and secondary metabolisma review.
Gene
115:5-12[CrossRef][Medline].
|
| 12.
|
Hopwood, D. A.,
M. J. Bibb,
K. F. Chater,
T. Kieser,
C. J. Bruton,
H. M. Kieser,
D. J. Lydiate,
C. P. Smith,
J. M. Ward, and H. Schrempf.
1985.
Genetic manipulation of Streptomyces: a laboratory manual.
The John Innes Foundation, Norwich, England.
|
| 13.
|
Horinouchi, S., and T. Beppu.
1992.
Autoregulatory factors and communication in actinomycetes.
Annu. Rev. Microbiol.
46:377-398[CrossRef][Medline].
|
| 14.
|
Janssen, G. R., and M. J. Bibb.
1993.
Derivatives of pUC18 that have BglII sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of Escherichia coli colonies.
Gene
124:133-134[CrossRef][Medline].
|
| 15.
|
Khokhlov, A. S.
1988.
Results and perspectives of actinomycete autoregulator studies, p. 338-345.
In
Y. Okami, T. Beppu, and H. Ogawara (ed.), Biology of actinomycetes '88. Japan Scientific Societies Press, Tokyo, Japan.
|
| 16.
|
Khokhlov, A. S.,
I. I. Tovarova,
L. N. Borisova,
S. A. Pliner,
L. A. Schevshenko,
E. Y. Kornitskaya,
N. S. Ivkina, and I. A. Rapoport.
1967.
A-factor responsible for the biosynthesis of streptomycin by a mutant strain of Actinomyces streptomycini.
Dokl. Akad. Nauk SSSR
177:232-235[Medline].
|
| 17.
|
Kieser, T.,
M. J. Bibb,
M. J. Buttner,
K. F. Chater, and D. A. Hopwood.
2000.
Practical Streptomyces genetics.
The John Innes Foundation, Norwich, United Kingdom.
|
| 18.
|
Kim, H. S.,
T. Nihira,
H. Tada,
M. Yanagimoto, and Y. Yamada.
1989.
Identification of binding protein of virginiae butanolide C, an autoregulator in virginiamycin production, from Streptomyces virginiae.
J. Antibiot.
43:692-706.
|
| 19.
|
Kinoshita, H.,
H. Ipposhi,
S. Okamoto,
H. Nakano,
T. Nihira, and Y. Yamada.
1997.
Butyrolactone autoregulator receptor protein (BarA) as a transcriptional regulator in Streptomyces virginiae.
J. Bacteriol.
179:6986-6993[Abstract/Free Full Text].
|
| 20.
|
Kinoshita, H.,
T. Tsuji,
H. Ipposhi,
T. Nihira, and Y. Yamada.
1999.
Characterization of binding sequences for butyrolactone autoregulator receptors in streptomycetes.
J. Bacteriol.
181:5075-5080[Abstract/Free Full Text].
|
| 21.
|
Kitani, S.,
H. Kinoshita,
T. Nihira, and Y. Yamada.
1999.
In vitro analysis of the butyrolactone autoregulator receptor protein (FarA) of Streptomyces lavendulae FRI-5 reveals that FarA acts as a DNA-binding transcriptional regulator that controls its own synthesis.
J. Bacteriol.
181:5081-5084[Abstract/Free Full Text].
|
| 22.
|
Kitani, S.,
M. J. Bibb,
T. Nihira, and Y. Yamada.
2000.
Conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces lavendulae FRI-5.
J. Microbiol. Biotechnol.
10:535-538.
|
| 23.
|
Kondo, K.,
Y. Higuchi,
S. Sakuda,
T. Nihira, and Y. Yamada.
1989.
New virginiae butanolides from Streptomyces virginiae.
J. Antibiot.
42:1873-1876[Medline].
|
| 24.
|
MacNeil, D. J.,
J. L. Occi,
K. M. Gewain,
T. MacNeil,
P. H. Gibbons,
C. L. Ruby, and S. J. Danis.
1992.
Complex organization of the Streptomyces avermetilis genes encoding the avermectin polyketide synthase.
Gene
115:119-125[CrossRef][Medline].
|
| 25.
|
Miyake, K.,
T. Kuzuyama,
S. Horinouchi, and T. Beppu.
1990.
The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.
J. Bacteriol.
172:3003-3008[Abstract/Free Full Text].
|
| 26.
|
Nakano, H.,
C. K. Lee,
T. Nihira, and Y. Yamada.
2000.
A null mutant of the Streptomyces virginiae barA gene encoding a butyrolactone autoregulator receptor and its phenotypic and transcriptional analysis.
J. Biosci. Bioeng.
90:204-207.
|
| 27.
|
Nakano, H.,
E. Takehara,
T. Nihira, and Y. Yamada.
1998.
Gene replacement analysis of the Streptomyces virginiae barA gene encoding the butyrolactone autoregulator receptor reveals that BarA acts as a repressor in virginiamycin biosynthesis.
J. Bacteriol.
180:3317-3322[Abstract/Free Full Text].
|
| 28.
|
Nishimura, H.,
M. Mayama,
Y. Komatsu,
H. Kato,
N. Shimaoka, and Y. Tanaka.
1964.
Showdomycin, a new antibiotic from a Streptomyces sp.
J. Antibiot.
17:148-155[Medline].
|
| 29.
|
Ohnishi, Y.,
S. Kameyama,
H. Onaka, and S. Horinouchi.
1999.
The A-factor regulatory cascade leading to streptomycin biosynthesis in Streptomyces griseus: identification of a target gene of the A-factor receptor.
Mol. Microbiol.
34:102-111[CrossRef][Medline].
|
| 30.
|
Okamoto, S.,
K. Nakamura,
T. Nihira, and Y. Yamada.
1995.
Virginiae butanolide binding protein from Streptomyces virginiae: evidence that VbrA is not the virginiae butanolide binding protein and reidentification of the true binding protein.
J. Biol. Chem.
270:12319-12326[Abstract/Free Full Text].
|
| 31.
|
Onaka, H.,
N. Ando,
T. Nihira,
Y. Yamada,
T. Beppu, and S. Horinouchi.
1995.
Cloning and characterization of the A-factor receptor gene from Streptomyces griseus.
J. Bacteriol.
177:6083-6092[Abstract/Free Full Text].
|
| 32.
|
Onaka, H., and S. Horinouchi.
1997.
DNA-binding activity of the A-factor receptor protein and its recognition DNA sequences.
Mol. Microbiol.
24:991-1000[CrossRef][Medline].
|
| 33.
|
Paget, M. S. B.,
L. Chamberlin,
A. Atrih,
S. J. Foster, and M. J. Buttner.
1999.
Evidence that the extracytoplasmic function sigma factor  E is required for normal cell wall structure in Streptomyces coelicolor A3(2).
J. Bacteriol.
181:204-211[Abstract/Free Full Text].
|
| 34.
|
Ruengjitchatchawalya, M.,
T. Nihira, and Y. Yamada.
1995.
Purification and characterization of the IM-2-binding protein from Streptomyces sp. strain FRI-5.
J. Bacteriol.
177:551-557[Abstract/Free Full Text].
|
| 35.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 36.
|
Sato, K.,
T. Nihira,
S. Sakuda,
M. Yanagimoto, and Y. Yamada.
1989.
Isolation and structure of a new butyrolactone autoregulator from Streptomyces sp. FRI-5.
J. Ferment. Bioeng.
68:170-173[CrossRef].
|
| 37.
|
Sugiyama, M.,
H. Onaka,
T. Nakagawa, and S. Horinouchi.
1998.
Site-directed mutagenesis of the A-factor receptor protein: Val-41 important for DNA-binding and Trp-119 important for ligand-binding.
Gene
222:133-144[CrossRef][Medline].
|
| 38.
|
Takano, E.,
T. Nihira,
Y. Hara,
J. J. Jones,
C. J. L. Gershater,
Y. Yamada, and M. J. Bibb.
2000.
Purification and structural determination of SCBI, a -butyrolactone that elicits antibiotic production in Streptomyces coelicolor A3(2).
J. Biol. Chem.
275:11010-11016[Abstract/Free Full Text].
|
| 39.
|
Waki, M.,
T. Nihira, and Y. Yamada.
1997.
Cloning and characterization of the gene (farA) encoding the receptor for an extracellular regulatory factor (IM-2) from Streptomyces sp. strain FRI-5.
J. Bacteriol.
179:5131-5137[Abstract/Free Full Text].
|
| 40.
|
Yamada, Y.,
K. Sugamura,
K. Kondo,
M. Yanagimoto, and H. Okada.
1987.
The structure of inducing factors for virginiamycin production in Streptomyces virginiae.
J. Antibiot.
40:496-504[Medline].
|
| 41.
|
Yamazaki, H.,
Y. Ohnishi, and S. Horinouchi.
2000.
An A-factor-dependent extracytoplasmic function sigma factor (AdsA) that is essential for morphological development in Streptomyces griseus.
J. Bacteriol.
182:4596-4605[Abstract/Free Full Text].
|
| 42.
|
Yanagimoto, M., and T. Enatsu.
1983.
Regulation of a blue pigment production by -nonalactone in Streptomyces sp.
J. Ferment. Technol.
61:545-550.
|
| 43.
|
Yang, K.,
L. Han, and L. C. Vining.
1995.
Regulation of jadomycin B production in Streptomyces venezuelae ISP5230: involvement of a repressor gene, jadR2.
J. Bacteriol.
177:6111-6117[Abstract/Free Full Text].
|
Journal of Bacteriology, July 2001, p. 4357-4363, Vol. 183, No. 14
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.14.4357-4363.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
