From the Microbial Function Laboratory and
§ Molecular Elucidation Laboratory, National Food Research
Institute, Tsukuba, Ibaraki 305-8642, Japan
Received for publication, August 26, 2002, and in revised form, September 26, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found that a polycistronic operon
(ywfBCDEFG) and a monocistronic gene (ywfH) are
required for the biosynthesis of bacilysin in Bacillus
subtilis. The disruption of these genes by plasmid integration
caused loss of the ability to produce bacilysin, accompanied by a lack
of bacilysin synthetase activity in the crude extract. We investigated
the regulatory mechanism for bacilysin biosynthesis using the
transcriptional lacZ fusion system. The transcription of
these genes was found to be induced at the transition from exponential
to stationary phase. Induction of transcription was accelerated by
depleting a required amino acid, which was done by transferring the
wild-type (rel+) cells to an amino acid-limited
medium. In contrast, no enhancement of the gene expression was detected
in relA mutant cells. In wild-type (rel+) cells, a forced reduction of
intracellular GTP, brought about by addition of decoyinine, which is a
GMP synthetase inhibitor, enhanced the expression of both the
ywfBCDEFG operon and the ywfH gene, resulting
in a 2.5-fold increase in bacilysin production. Disruption of the
codY gene, which regulates stationary phase genes by
detecting the level of GTP, also induced transcription of these genes.
In contrast, the expression of ywfBCDEFG in
relA cells was not activated either by decoyinine addition
or codY disruption, although the expression of
ywfH was induced. Moreover, the codY disruption
resulted in an increase of bacilysin production only in
rel+ cells. These results indicate that
guanosine 5'-diphosphate 3'-diphosphate (ppGpp) plays a crucial role in
transcription of the ywfBCDEFG operon and that the
transcription of these genes are dependent upon the level of
intracellular GTP which is transmitted as a signal via the
CodY-mediated repression system. We propose that, unlike antibiotic
production in Streptomyces spp., bacilysin production in
B. subtilis is controlled by a dual regulation
system composed of the guanine nucleotides ppGpp and GTP.
The stringent response is one of the most important
adaptations, by which bacteria have to survive in a nutrient limited
environment. This response leads to the repression of stable RNA
synthesis (rRNA and tRNA) and gene expression for various translational factors and ribosomal proteins. The stringent response also activates the expression of certain genes, including the amino acids biosynthesis genes. Numerous studies have indicated that the stringent response depends on a transient increase of the hyperphosphorelated guanosine nucleotides, guanosine 5'-diphosphate 3'-diphosphate
(ppGpp),1 in response to the
binding of uncharged tRNA to the ribosomal A site (1). Mutant cells
that are unable to repress stable RNA synthesis under depleted amino
acid conditions have been termed "relaxed." In many cases, these
mutations are found in the relA gene, which encodes the
ppGpp synthetase, or the relC (= rplK) gene,
which codes for the ribosomal protein L11. These relaxed (rel) mutants are unable to initiate ribosome-mediated
synthesis of ppGpp (2-5). Therefore, the RelA and L11 proteins are
thought to be essential mediators, which signal cessation to the
transcriptional machinery.
ppGpp inhibits IMP dehydrogenase, the first enzyme in the GMP synthesis
pathway, resulting in a rapid reduction in the level of GTP (6). Even
under nutrient excess conditions, the accumulation of ppGpp caused by
amino acid depletion results in the reduction of intracellular GTP and
eventually leads to the induction of sporulation and genetic competence
development in the stringent (rel+) but not in
the relaxed (relA) strain (7-10). The deleterious effects
of the relA mutation can be repaired by addition of
decoyinine, a GMP synthetase inhibitor, or by inactivation of CodY,
which regulates the expression of various stationary phase genes by detecting the level of intracellular GTP (9, 10). Thus, a low level of
intracellular GTP can stimulate the transcription of stationary phase
genes, and ppGpp plays a role to accentuate the reduction in the level
of GTP.
Numerous microorganisms, including Bacillus subtilis,
produce a wide variety of secondary metabolites. These secondary
metabolites often possess antibiotic activity but are not essential for
the growth of a producing organism. The secondary metabolism may be taken as one of the adaptive responses to a nutrient limited
environment. Working with the genera Streptomyces and
Bacillus, the correlation between the stringent response and
antibiotic production has been established. In the absence of a
functional relA or rplK gene, antibiotic
production by these organisms is remarkably reduced or lost (11-14).
Therefore, the initiation of antibiotic production is considered to be
positively regulated by the stringent response (15-18), as has
been demonstrated for amino acid biosynthesis (1). B. subtilis provides a feasible system for the study of various biological functions, as denoted by the presence of a transformation system and the availability of genomic information courtesy of the
completed genome project. Thus, we attempted to clarify the regulation
mechanism for antibiotic production in this organism as a model for
bacterial secondary metabolism.
Bacilysin is one of simplest peptide antibiotics produced by B. subtilis (19). It is a dipeptide consisting of an
L-alanine at the N terminus and an unusual amino acid,
L-anticapsin, at the C terminus. Although biosynthesis of
bacilysin has been studied extensively (20-23), little is known about
the regulation mechanism for biosynthesis. In this study, an attempt
was made to identify the genes essential for bacilysin biosynthesis.
Subsequently, we performed an expression analysis of these genes,
mainly in relation to ppGpp and GTP. The results are discussed in the
light of adaptive responses such as sporulation and competence development.
Bacterial Strain, Plasmids, and Culture Conditions--
The
strains of B. subtilis and plasmids used in this study are
listed in Table I. Strains 61884 (rel+) and 61883 (relA1) are isogenic
strains with respect to the relA1 mutation (8). The
relA1 mutation is a leaky mutation (7, 8) and has an amino
acid alteration at position 240 (Gly to Glu) (10). Strains PY79 and
ng79 were provided by G. Özcengiz via A. L. Demain.
Streptomyces griseoplanus NRRL3507 was a kind gift from Eli
Lilly and Co. (Indianapolis, IN). Staphylococcus aureus 209P
and Salmonella typhimurium SL1102 were used to assay for
bacilysin and anticapsin, respectively.
B. subtilis strains were grown aerobically in S7N medium or
L medium at 37 °C. S7N medium is a modified S7 medium (24)
containing 10 mM ammonium sulfate, 5 mM
potassium phosphate (pH 7.0), 100 mM MOPS (adjusted pH 7.0 by KOH), 2 mM MgCl2, 0.7 mM
CaCl2, 50 µM MnCl2, 5 µM FeCl3, 2 µM thiamin, 1%
glucose, 20 mM glutamate, and 0.1% nutrient broth (Difco).
L medium contained the following (per liter): 10 g of tryptone,
5 g of yeast extract, and 5 g of NaCl. Tryptophan (50 µg/ml) and aspartate (2 mM for L medium or 20 mM for S7N medium) were supplemented as required into the
medium. For selection of B. subtilis transformants, neomycin
(4 µg/ml), and erythromycin (0.5 µg/ml) were used. For selection of
Escherichia coli transformants, ampicillin (100 µg/ml) was used.
Construction of B. subtilis Mutant Strains--
To generate the
TI81 strain, a DNA fragment that contained the open reading frame
for ywfA (580 bp) was amplified by PCR with the primers
ywfA-F (5'-ATGAACAGCAATCAAAACAATG-3') and ywfA-R
(5'-GCATTTCAAGGAGAATCAGC-3'), and cloned into plasmid pCR2.1
resulting in pCR2.1-ywfA. A 1.3-kb SmaI fragment
containing the neo gene was isolated pBEST501 (25) and
inserted into the SspI site of pCR2.1-ywfA. The
resulting plasmid pCR2.1-ywfA::neo was
digested with HindIII, used to transform B. subtilis 61884, and was selected for neomycin resistance on L agar
plates. The resulting B. subtilis strain TI81 was used as a
ywfA disruptant.
For construction of strains TI91, TI92, TI112, and TI113, plasmid
pMutinT3 (26) was integrated into the ywfB gene or
ywfH gene on the chromosome via a single-crossover event as
follows. The DNA fragment containing a partial ywfB gene or
ywfH gene was amplified by PCR with the following specific
primer pairs: Bacilysin Production--
Antibiotic activities were determined
by the paper disk-agar diffusion assay using S. aureus 209P
as a test organism. B. subtilis strains were grown in S7N
medium at 37 °C for 12 h with vigorous shaking. Then, cultures
were diluted 50-fold in fresh S7N medium and further incubated under
the same condition. The supernatants (50 µl) of the culture broth
obtained after centrifugation were applied on paper disk (diameter, 8 mm; Advantec). The paper disks were then placed on a bacilysin assay
plate (20) inoculated with S. aureus 209P. The bacilysin
assay plate contained (per liter): 3.3 g of
Na2HPO4·2H2O, 1 g of
KH2PO4, 1 g of NaCl, 0.7 g of
MgSO4·7H2O, 0.01 g of
FeSO4·7H2O, 0.5 g of trisodium
citrate·2H2O, 2.4 g of sodium
glutamate·H2O, 10 g of glucose, 0.025 g each of the
following amino acids (arginine, cysteine, glycine, histidine, leucine,
methionine, phenylalanine, proline, threonine, tryptophan, tyrosine,
and valine), and 10 g of agar. If necessary, glucosamine hydrochloride was added into the agar plate at the final concentration of 1 mM.
Identification of Bacilysin--
The supernatants of the culture
broths (each 50 ml) for strains 61884 and 61883 obtained after
centrifugation were freeze-dried using a vacuum pump. The residual
material was dissolved in 5 ml of water and put through reverse phase
HPLC (Capcell pack C18 UG120 column, 15 × 250 mm,
Shiseido) using water as a mobile phase at a flow rate of 2.5 ml/min.
The fraction containing antibiotic activity (from the 61884 broth) was
freeze-dried and analyzed by ESI-MS (Thermo Quest LCQ ion trap mass
spectrometer equipped with an ESI source, Thermo Quest). No any
antibiotic activity was detected in other HPLC fractions. The fraction
for 61883 was collected at the same elution time when bacilysin was
detected for 61884.
Detection of Bacilysin Synthetase (Alanine-Anticapsin Ligase)
Activity--
Because anticapsin is not commercially available,
we prepared it from the S. griseoplanus NRRL3507 culture
broth using a method described previously (27). Anticapsin was
purified, and its structure was confirmed by ESI-MS spectrometry
analysis (data not shown). The activity of bacilysin synthetase was
detected using the method described by Sakajoh et al. (20).
Bacilysin activity in cell-free extracts was detected using the
bacilysin assay plate.
Northern Blot Analysis--
Cells were inoculated into S7N
medium, grown at 37 °C with vigorous shaking, and harvested at time
0 (the time point of transition from exponential to stationary phase),
at which time point A650 was ~1.0. The total
cellular RNA was prepared using the Isogen reagent (Nippon Gene). The
resulting RNA sample (15 µg for ywfBCDEFG and 5 µg for
ywfH) was electrophoresed after denaturation with glyoxal
and dimethyl sulfoxide, transferred on a membrane
(Hybond-N+; Amersham Biosciences), and then hybridized with
an RNA probe. RNA probes for ywfB and ywfH were
prepared using T7 RNA polymerase with pCR2.1-ywfB and
pCR2.1-ywfH as the template, respectively. DIG RNA labeling
kit (SP6/T7) was purchased from Roche Diagnostics and used for labeling.
Involvement of the Stringent Response in Bacilysin
Production--
When the B. subtilis wild-type
(rel+) strain 61884 was grown in S7N medium,
cells produced an antibiotic against S. aureus after
8 h of incubation (Table II). On the
other hand, antibiotic production by the isogenic relA
mutant 61883 was significantly lower than that of 61884. Antibiotic
production by 61884 (but not 61883) increased 3-5-fold when cells were
first grown to middle exponential phase with a sufficient amount of
aspartate (20 mM) and then transferred to the same media
except containing a limiting amount of aspartate (2 mM).
This down-shift condition, which provoked a stringent response, was
accompanied by a temporal increase of ppGpp (from 4 pmol/AM600 to 90 pmol/AM600) and 5-fold
decrease of GTP (8, 13). (AM is the amount of cells that would produce an A650 = 1 if suspended in 1 ml.) B. subtilis produces the dipeptide antibiotic bacilysin, which
interferes with glucosamine synthesis (29). As expected, antibacterial
potency was negated by the addition of glucosamine into the assay plate
(data not shown). Further evidence came from mass-spectrometric
analysis. We isolated the fraction containing antibiotic activity by
reversed phase HPLC (see "Experimental Procedures") and analyzed
this sample by ESI-mass spectrometry (Fig.
1A). The peak at
m/z 271.1, which represents the mass of bacilysin [M + H]+, was detected at a high intensity in the sample
prepared from the 61884 culture extract. ESI-MS/MS of the protonated
molecule of bacilysin, which was used as a precursor ion, revealed
fragment ions derived from cleavage of the amide bond at m/z
200 (corresponding to the protonated molecule of anticapsin) and at
m/z 182 (corresponding to the mass of dehydrated anticapsin)
(Fig. 1B). In contrast, the peak corresponding to [M + H]+ of bacilysin was not detected when a sample from the
relA strain (61883) was analyzed (Fig. 1C). These
results indicate that the antibiotic produced by B. subtilis
61884 is bacilysin and its production is dependent on the stringent
response.
Identification of Genes for Bacilysin Biosynthesis--
The
alanine-anticapsin ligation is thought to be the final step in the
bacilysin biosynthesis pathway. B. subtilis mutant ng79
shows neither a detectable amount of bacilysin in the culture broth nor
alanine-anticapsin ligase (bacilysin synthetase) activity because of a
bac-1 mutation (22). Because the bac-1 mutation in ng79 exists between the ctrA and sacA loci
(22), we utilized this fact to find the gene with bac-1
mutation. We inserted a neomycin-resistant gene (as a selectable
marker) within the ywfA gene, which is located in the
ctrA-sacA region. Disruption of ywfA
gene did not affect bacilysin production (Fig.
2). Strain ng79 was then transformed with
the DNA from the ywfA disruptant strain TI81, and antibiotic
productivity of the resulting transformants were examined. Because 63%
of transformants (19/30) produced antibiotic, we deduced that the
bac-1 mutation is located in the polycistronic operon
ywfBCDEFG (previously designated
ipa80/85d; Ref. 30). We therefore sequenced the
ywfBCDEFG genes in ng79 and compared the data with the
sequence of the parent strain PY79, and found three point mutations,
ywfC118 (C352 to T, Pro-118 to Ser), ywfD178 (C532 to T, Arg-178 to Trp), and ywfE179 (C536 to T, Pro-179
to Leu). Of these mutations, introduction of the ywfE179
mutation with ywfA::neo into 61884 (TI94) resulted in the loss of bacilysin production (Fig. 2), as well
as bacilysin synthetase activity (Table II). It remained unknown
whether ywfC118 and ywfD178 lesions are
responsible for the loss of bacilysin production in ng79. For further
analysis, we constructed ywfB disruption mutants, which
carry an IPTG-dependent ywfCDEFG, and
ywfH interruption mutants, which carry an
IPTG-dependent ywfH. The pMutinT3-derived plasmids used for these constructions allowed the creation of a
transcriptional element fusing the target gene promoter to the lacZ gene. This made it possible to control the expression
of downstream gene(s) that may belong to the same operon via the IPTG-inducible Pspac promoter. In the absence of IPTG, the
constructed mutants TI91 (ywfB::pMutinT3) and
TI112 (ywfH::pMutinT3) did not produce any
antibiotic activity (Fig. 2) or any detectable bacilysin synthetase
activity (Table II). Even in the presence of IPTG, antibiotic
production of these mutants was not re-acquired, although the mutants
did give rise to a detectable amount of bacilysin synthetase activity
(Table II). These results indicate that the YwfB protein is essential
for bacilysin production but not for alanine-anticapsin ligation
because the mutant TI91 has the disrupted ywfB gene. On the
other hand, YwfE and YwfH can be assigned to be involved in the
alanine-anticapsin ligation (Table II). No restoration of antibiotic
production in the ywfH interruption mutant, even in the
presence of IPTG, may indicate that a greater amount of the
ywfH gene product is required for bacilysin production.
Although the induction of antibiotic production and sporulation often
start concomitantly, implying a mechanistic connection of these two
processes, the bacilysin nonproducing mutant sporulated as well as
wild-type strain, producing 3 × 108 spores/ml after
30 h of incubation in S7N medium as examined with strain TI94.
Effect of the Stringent Response on Expression of the Bacilysin
Biosynthesis Genes--
To analyze the expression of bacilysin
biosynthesis gene, we constructed transcriptional lacZ
fusion elements to each promoter. In the rel+
strain, expression of ywfB and ywfH were induced
at the transition between exponential and stationary phase during
growth in S7N medium (Fig. 3,
A and B). However, expression of ywfB
in the relA strain was much less compared with the
rel+ strain (Fig. 3A). On the other
hand, virtually no difference in the expression of ywfH was
observed between the rel+ and relA
strains (Fig. 3B). As demonstrated by lacZ fusion
analysis, the target gene was inactivated upon plasmid integration,
which might influence its own expression. Therefore, we analyzed the level of transcripts directly by Northern blotting using probes for
ywfB and ywfH. The ywfBCDEFG
transcript (6.7 kb) was detected only in rel+
strain 61884 but not in relA strain 61883 (Fig.
3C, left panel). Consistent with the
result from lacZ fusion analysis, there was no significant
difference in the amount of ywfH transcript (0.8 kb) between
the rel+ and relA strains (Fig.
3C, right panel). These Northern
analysis results confirm the transcriptional fusion analysis results.
Therefore, we used the transcriptional lacZ fusion system
for further analysis.
To confirm the significance of the stringent response in expression of
the bacilysin biosynthesis genes, we monitored the expression of
ywfBCDEFG and ywfH genes under conditions of
amino acid deprivation, which provokes a typical stringent response in
rel+ cells (6, 13). Strains 61884 (rel+) and 61883 (relA1) were grown
to mid-exponential phase in S7N medium containing a sufficient (20 mM) amount of aspartate, and then cells were transferred to
the same medium containing a limiting (2 mM) amount of
aspartate. The expression of ywfBCDEFG increased in
rel+ cells immediately after aspartate
starvation compared with nonstarved control culture (Fig.
4A). There was, however, no
increase in expression when the isogenic relA strain 61883 was subjected to similar starvation. The expression of ywfH
was enhanced in rel+ cells by aspartate
starvation, despite the fact that ywfH gene expression is
activated at late growth phase in both rel+ and
relA strains (see Fig. 3B). It was also an
unexpected result that the expression of ywfH was not
enhanced but rather repressed (3-fold drop) in relA cells.
The ywfH transcription activation (in
rel+ cells) and repression (in relA
cells) observed under the down-shift conditions may be a result from a
secondary stringent response effect. Thus, transcription of both
ywfBCDEFG and ywfH was enhanced directly or
indirectly under the conditions that elicit stringent response.
Decrease in the Level of Intracellular GTP Stimulates Transcription
of the Bacilysin Biosynthesis Genes--
Previous studies demonstrated
that the RelA protein is involved in the rapid reduction of
intracellular GTP by synthesizing ppGpp, which inhibits IMP
dehydrogenase, leading to the initiation of sporulation and competence
development (6-8, 10). Therefore, we investigated whether or not a
forced reduction in the level of GTP leads to activation of the
bacilysin biosynthesis genes. We added decoyinine (a GMP synthetase
inhibitor) into the culture at time 0 (the time point of transition
from exponential to stationary phase). As shown in Fig.
5A, decoyinine (2 mM) activated the transcription of the ywfBCDEFG
operon in rel+ cells, displaying a 4-fold
increase compared with non-treated cells. In contrast, relA
cells failed to increase expression when treated with decoyinine. The
level of intracellular GTP decreased 30-50% of the initial level 30 min after decoyinine was added for both rel+ and
relA cells, and the low level of GTP was maintained at least for 2 h as actually measured by HPLC (data not shown). These
results suggest that sustaining a certain ppGpp level is necessary to elicit induction of ywfBCDEFG expression by reducing the
level of GTP. Addition of decoyinine did not induce ppGpp accumulation in rel+ (and also relA) cells (data
not shown). In contrast, expression of ywfH could be induced
by decoyinine even in relA cells (Fig. 5B). Thus,
expression of ywfH gene is dependent upon the level of
intracellular GTP rather than ppGpp. Treatment of
rel+ cells with decoyinine actually gave rise to
a 2.5-fold increase in bacilysin production (Table II), indicating a
5-fold increase in terms of productivity per unit cell (data not
shown).
GTP-regulated Bacilysin Biosynthesis Is Mediated by the CodY
Protein--
The GTP-binding protein CodY regulates the transcription
of various stationary phase genes in B. subtilis (9,
31-33). Recently, the CodY protein was demonstrated to play a key role
in the initiation of sporulation and genetic competence by detecting
intracellular GTP (9, 10). These results taken together with our
present results with decoyinine suggest that the bacilysin biosynthesis genes may also belong to this regulon. To assess this possibility, we
analyzed the effect of a codY disruption on expression of
the ywfBCDEFG and ywfH genes. Disruption of the
codY gene gave rise to a 2-3-fold increase in the
expression of the ywfBCDEFG gene in
rel+ but not in relA cells (Fig.
6A). Bacilysin production was
actually enhanced 2-fold by disruption of codY in
rel+ cells (TI70), but there was substantially
no (or only slight) effect in relA cells (TI71) (Table II).
In agreement with results shown in the decoyinine addition experiment,
disruption of codY showed a positive effect on
ywfH transcription even in relA cells (Fig.
6B). These results were also confirmed by Northern dot-blot analysis (data not shown). Thus, ppGpp and GTP both play a critical role in bacilysin production by B. subtilis.
B. subtilis strains are reported to produce three
ribosomal antibiotics (TasA (Ref. 34), subtilosin (Ref. 35), and
sublancin (Ref. 36)) and three nonribosomal antibiotics (surfactin
(Ref. 37), bacilysin (Ref. 19), and plipastatin (Ref. 38)). Recently in
our laboratory, the novel phospholipid antibiotic bacilysocin (39) was
discovered in strain 168 (the standard strain of B. subtilis Marburg). More recently, we also found that a certain mutant derived from B. subtilis 168 possesses the ability to
produce another antibiotic,
3,3'-neotrehalosadiamine,2
which had been previously isolated from Bacillus pumilus
(40). Our principal findings in the present study are (i)
identification of genes for bacilysin biosynthesis and (ii) a novel
regulation system for those genes under the conditions that elicit the
stringent response. We successfully identified ywfBCDEFG
operon and ywfH gene as the genes that operate for bacilysin
synthesis. Of these genes, the direct evidence to involvement in
bacilysin synthesis was displayed for ywfBE and
ywfH (but not for ywfCDFG) by gene disruption
experiments or by missense allele analysis. The amino acid sequence for
the ywfB gene deduced from the gene sequence demonstrated a
28% homology (in amino acid identity) to the prephenate dehydratase in
Streptomyces coelicolor (accession no. T36068). Likewise,
the ywfG gene product demonstrated similarity to aspartate aminotransferase found in various organisms (30). Anticapsin, a
component of bacilysin, is shown to be derived from prephenate, an
intermediate in the aromatic amino acid biosynthesis pathway (21), and
thus the biosynthesis of anticapsin is considered to proceed in analogy
with the biosynthesis of phenylalanine that utilizes prephenate and
amino donor such as aspartate. Therefore, it is highly likely that the
gene products of ywfB and ywfG are enzymes that
participate in the anticapsin biosynthesis pathway. The ywfF
gene product had a similarity to a multidrug-efflux transporter protein
from Pyrococcus abyssi (26% amino acid identity in 175 residues), indicating that the YwfF protein may be involved in the
self-resistance to bacilysin. Participation of ywfBFG in
bacilysin synthesis thus can be warranted by putative functions of
these gene products. The ywfD gene product is similar to
glucose dehydrogenase found in various organisms (30). Although the
functions of YwfC and YwfD protein in bacilysin synthesis remained
unknown, these proteins may be involved in anticapsin biosynthesis
because ywfC and ywfD are both located in a
single operon ywfBCDEFG. When these results are taken
together, the ywfBCDEFG operon and ywfH gene are
both thought to be the structural genes for bacilysin biosynthesis.
One of the most intriguing challenges in biology is to elucidate
mechanisms by which cells detect and respond to extracellular nutritional conditions. Among prokaryotes, B. subtilis and
Streptomyces spp. offer a feasible system for studying such
mechanisms because they display a wide range of adaptations to nutrient
limitations, including production and secretion of antibiotics and
enzymes, and formation of aerial mycerium (in Streptomyces
spp.) and endospores (in B. subtilis) as an extreme response
to nutrient limitation (33). The stringent response, a general and
ubiquitous response to nutritional environmental stress in prokaryotic
microorganisms, is mediated by the unique nucleotide ppGpp (1). By
analyzing mutants with an impaired ability to elicit the stringent
response, Ochi proposed that ppGpp plays a role in triggering the onset of antibiotic production in Streptomyces spp., whereas
morphological differentiation is triggered by detecting a reduced
amount of GTP (5, 16, 41, 42). The most interesting observation in the
present study is that GTP, in addition to ppGpp, is the key factor for
initiating antibiotic production in B. subtilis as
demonstrated by the addition of decoyinine and by CodY disruption experiments (Figs. 5 and 6). Namely, GTP acts as a signal molecule for
both sporulation and antibiotic production in B. subtilis. This report is the first to demonstrate that GTP
plays a critical role in initiating the onset of bacterial antibiotic
production. However, it should be noted that the GTP effect can be
elicited only in rel+ (wild-type) but not
relA (mutant type) cells. This fact strongly indicates that
ppGpp plays a pivotal role as a positive regulator even in B. subtilis and that GTP functions as a negative regulator, thus
producing a synergistic effect on antibiotic production when its level
declined. In the framework of this notion, a GTP-binding protein CodY
should be highlighted. The CodY protein mediates the inhibitory effects
of glucose and amino acids on stationary phase gene expression (31) and
is considered to be a GTP-detecting transcriptional regulator
containing a predicted GTP binding pocket (9) as demonstrated in
Sonenshein's laboratory. Recently, Inaoka and Ochi (10) demonstrated
that the RelA protein plays an essential role in the induction of
competence development (at least under certain physiological
conditions) by reducing the level of intracellular GTP and overcoming
CodY-mediated regulation. Likewise, bacilysin production in
B. subtilis is apparently controlled by CodY activity, because codY disruption increased the level of expression
for genes involved in bacilysin biosynthesis (Fig. 6). GTP-bound CodY is thought to be the active form of this protein because reduction of
the level of GTP by decoyinine addition released the repression of
genes necessary for bacilysin biosynthesis (Fig. 5). Genome sequence
analysis has revealed the existence of a widespread distribution of
CodY homologues in low G + C Gram-positive bacteria but not in
Streptomyces spp. This may be a reason for the fact that,
unlike B. subtilis, antibiotic production by
Streptomyces spp. never increased (instead always decreased)
upon treatment with decoyinine as examined with various
Streptomyces spp., although morphological differentiation
was enhanced (42, 43).3 Thus,
it is thought that the extent of secondary metabolism in organisms
containing the CodY homologue may be activated by modulating the level
of GTP. In Streptomyces spp. the GTP-binding protein Obg was
presented as a candidate protein for detecting the level of GTP for
initiating morphological development of this genus (44, 45).
Introduction of multiple copies of obg into wild-type S. coelicolor suppresses not only aerial mycerium but also
antibiotic (actinorhodin) production (45).
Involvement of the quorum sensing system in biological events is a
current topic in microbiology (46). Recently, Yazgan et al.
(47) reported that bacilysin production in B. subtilis is
under the control of quorum sensing via the OppA (oligopeptide permease)-imported peptide pheromone PhrC, which is necessary for
efficient sporulation and competence development. Our preliminary results indicated that expression of the oppA-lacZ gene in a
relA mutant was only 5-10% compared with the
rel+ strain in the culture conditions used
here.2 Because decoyinine treatment or
codY disruption fully induces sporulation and competence
development even in the relA strain (10), the transcription
of oppA is probably under the control of CodY-GTP rather
than ppGpp as is the case for the dpp gene (31). Because
neither decoyinine treatment nor codY disruption activated
transcription of the ywfBCDEFG operon in relA
cells (Figs. 5 and 6), a basal level of ppGpp (~4
pmol/AM650) appears to be primarily essential for
transcription of the ywfBCDEFG operon. Because decoyinine
does not induce ppGpp accumulation even in the stringent
(rel+) cells, expression of the operon must
sense the basal levels of ppGpp that differ between
rel+ and relA cells. In fact, the
basal level of ppGpp (0.5-1 pmol/AM600) in relA
cells is 4-fold less than that in rel+
cells.3 Supporting is the fact that a relC
mutant 61953 having ppGpp less than a detectable level (<0.5
pmol/AM600) is also not capable of producing bacilysin
(13). B. subtilis relA null mutant requires valine,
isoleucine, leucine, and methionine for growth in synthetic medium
(48). The anticapsin biosynthesis pathway mediated by the
ywfBCDEFG operon may belong to a similar regulon that
controls the biosynthesis of those amino acids. Thus, ppGpp is
essential for the transcription of the ywfBCDEFG operon and
GTP regulates the transcription of both ywfBCDEFG and
ywfH genes via the CodY-mediated regulation system. We
therefore propose that bacilysin production in B. subtilis
is controlled by a dual regulation system composed of the guanine
nucleotides, ppGpp and GTP.
Insight into the mechanism by which ppGpp functions in initiating
antibiotic production came from studying rifampicin-resistant mutants,
which provided several clues for understanding the ppGpp-RNA polymerase
interrelationship (49, 50). In E. coli, ppGpp is known to
have both positive and negative effects on transcriptional regulation,
and one of the most prominent effects of ppGpp accumulation is a
reduction in the rate of gene transcription for rRNA and tRNA, which
eventually leads to a cessation of protein synthesis (1). Recent
genetic and biochemical studies, performed solely in E. coli, have revealed that ppGpp binds directly to RNA polymerase, modulating its function perhaps in an allosteric manner (51-54). Indeed, as demonstrated in E. coli, the mutant RNA
polymerase may show altered promoter selectivity (55). Recently we have found that introduction of certain mutations that confer resistance to
rifampicin activates antibiotic production in S. coelicolor and Streptomyces lividans (49, 50). These mutations were
found in the so-called rif domain within the RNA polymerase
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bacterial strains and plasmids used in this study
ywfB-F (5'-
aagcttATGATTATATTGGATAATAGCATTCAG-3') and
ywfB-R (5'-
ggatccTAGGTGAAGGATGTGAAACAAT-3') (for ywfB), and
ywfH-F (5'-aagcttCATTTTTTTCAAAGGGGTGC-3') and ywfH-R (5'-
ggatccTTATCAAATGTGTCAGGCGC-3') (for ywfH).
These primers have the HindIII or BamHI site as
indicated by the lowercase boldface letters. The fragments were cloned
into pCR2.1 to generate pCR2.1-ywfB and
pCR2.1-ywfH, respectively. HindIII-BamHI fragments were fully sequenced to
confirm correct sequence and then ligated into the corresponding
restriction sites for pMutinT3, resulting in
pMutinT3-
ywfB and pMutinT3-ywfH, respectively. These plasmids were used to transform B. subtilis 61884 and
were selected for erythromycin resistance on L agar plates, thus
generating strains TI91 and TI112. To generate strains TI92 and TI113,
strain 61883 was transformed with the chromosomal DNAs derived from
TI91 and TI112, respectively. The correct constructions were confirmed by PCR analysis using the chromosomal DNA from each mutant.
-Galactosidase Assay--
-Galactosidase assay was carried
out as described by Miller (28). The specific activity was expressed as
[absorbance at 420 nm (A420) per minute per
milliliter of culture per A650] × 1000.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Analysis of culture broths for bacilysin titer and of cell extracts for
basilysin synthetase activity
View larger version (30K):
[in a new window]
Fig. 1.
ESI-mass spectra of the culture broths of
61884 and 61883. The lyophilized culture supernatants was
subjected to reverse phase HPLC. Then, the fraction containing
antibiotic activity was analyzed by ESI-MS. Strain 61884 showed the
peak corresponding to [M + H]+ of bacilysin at
m/z 271 (A), which was not observed in
strain 61883 (C). The product ions of the peak were found at
m/z 200 and m/z 182 (B).
View larger version (23K):
[in a new window]
Fig. 2.
Location and organization of the bacilysin
biosynthesis genes. Shown are the mutant constructions and the
corresponding bacilysin phenotype. Genes are shown as thick
arrows. Bacilysin activity in the culture broths were
determined as in Table II. Pspac and stem-loop structure
indicate IPTG-dependent spac promoter and the
transcriptional terminator, respectively. lacI encodes a
repressor of Pspac. neo and erm
represent neomycin-resistant and erythromycin-resistant genes,
respectively.
View larger version (49K):
[in a new window]
Fig. 3.
Expression analysis of the
ywfBCDEFG operon and the ywfH
gene. The transcription of ywfBCDEFG and
ywfH were analyzed using transcriptional lacZ
fusion constructs with ywfB (A) and
ywfH (B) or by Northern blotting (C).
A, stringent TI91 (squares) and relaxed TI92
(circles) strains were grown in S7N medium supplemented with
required amino acids (50 µg/ml Trp and 20 mM Asp).
Culture samples were withdrawn at the indicated times and culture
density (A650, open
symbols), and the -galactosidase activity
(closed symbols) was measured. B,
stringent TI112 (squares) and relaxed TI113
(circles) strains were grown as in A. C, stringent 61884 (lanes 1 and
3) and relaxed 61883 (lanes 2 and
4) strains were grown as in A, and harvested at
time 0. The total cellular RNA of each strain was prepared using the
Isogen reagent. The resulting RNA samples were subjected to
electrophoresis, transferred to a membrane, and then hybridized with
probe RNA for ywfB (left panel) or
ywfH (right panel).
View larger version (25K):
[in a new window]
Fig. 4.
Effect of amino acid deprivation on
expression of the bacilysin biosynthesis genes, ywfB
and ywfH. A, stringent TI91
(squares) and relaxed TI92 (circles) strains were
grown in S7N medium supplemented with sufficient amount of required
amino acids (50 µg/ml Trp and 20 mM Asp) until
A650 reached 0.5. Cells were harvested by
centrifugation (5 min at 25 °C) and immediately resuspended in the
same volume of the fresh medium containing a sufficient (20 mM, open symbols) or a limiting (2 mM, closed symbols) amount of
aspartate. Culture samples were withdrawn at the indicated times, and
-galactosidase activity was measured. B, stringent TI112
(squares) and relaxed TI113 (circles) strains
were grown as in A.
View larger version (27K):
[in a new window]
Fig. 5.
Effect of decoyinine on expression of the
bacilysin biosynthesis genes, ywfB and
ywfH. A, stringent TI91
(squares) and relaxed TI92 (circles) strains were
grown in S7N medium supplemented with a sufficient amount of required
amino acids as in Fig. 3. Decoyinine (2 mM to final
concentration, closed symbols) was added into the
culture at time 0 as indicated by the arrows. Culture
samples were withdrawn at the indicated times, and -galactosidase
activity was measured. Untreated culture samples (open
symbols) were also examined in parallel as a reference.
B, stringent TI112 (squares) and relaxed TI113
(circles) strains were grown and treated with decoyinine as
in A.
View larger version (26K):
[in a new window]
Fig. 6.
Effect of a codY disruption
on expression of the bacilysin biosynthesis genes, ywfB
and ywfH. A, strains TI91
(open squares), TI92 (open
circles), TI95 (closed squares), and
TI100 (closed circles) were grown in S7N medium
supplemented with a sufficient amount of required amino acids as in
Fig. 3. Culture samples were withdrawn at the indicated times, and
-galactosidase activity was measured. B, strains TI112
(open squares), TI113 (open
circles), TI114 (closed squares), and
TI115 (closed circles) were grown as in
A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit. Because these rif mutations activate antibiotic
production even in cells with the genetic background of relA
and relC, it is conceivable that ppGpp can modulate the
function of RNA polymerase, eventually allowing expression of genes for
antibiotic biosynthesis. These results may be helpful in clarifying the
intrinsic mechanism by which ppGpp activates gene expression for the
secondary metabolism in B. subtilis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Chie Kobayashi for technical assistance in several experiments, M. Sakajoh for advice in bacilysin assay, and A. L. Demain (Massachusetts Institute of Technology, Cambridge, MA) and G. Özcengiz (Middle East Technical University, Ankara, Turkey) for providing the B. subtilis strains.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Organized Research Combination System of the Science and Technology Agency of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: National Food Research Inst., 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Tel.: 81-298-38-8125; Fax: 81-298-38-7996; E-mail: kochi@affrc.go.jp.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M208722200
2 T. Inaoka, J. Yasuda, K. Takahashi, H. Yada, M. Yoshida, and K. Ochi, unpublished results.
3 K. Ochi, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ppGpp, guanosine
5'-diphosphate 3'-diphosphate;
IPTG, isopropyl-1-thio--D-galactopyranoside;
MOPS, 4-morpholinepropanesulfonic acid;
ESI, electrospray ionization;
MS, mass spectrometry;
HPLC, high performance liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Cashel, M., Gentry, D. R., Hernandez, V. J., and Vinella, D. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C. , Curtiss, R., III , Ingraham, J. L. , Lin, E. C. C. , Low, K. B. , Magasanik, B. , Reznikoff, W. S. , Riley, M. , Schaechter, M. , and Umbarger, E., eds), 2nd Ed., Vol. 1 , pp. 1458-1496, American Society for Microbiology, Washington, D. C. |
2. | Swanton, M., and Edlin, G. (1972) Biochem. Biophys. Res. Commun. 46, 583-588[Medline] [Order article via Infotrieve] |
3. | Smith, I., Paress, P., and Pestka, S. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 5993-5997[Abstract] |
4. | Ochi, K. (1990) J. Gen. Microbiol. 136, 2405-2412[Medline] [Order article via Infotrieve] |
5. | Ochi, K. (1990) J. Bacteriol. 172, 4008-4016[Medline] [Order article via Infotrieve] |
6. | Ochi, K., Kandala, J., and Freese, E. (1982) J. Bacteriol. 151, 1062-1065[Medline] [Order article via Infotrieve] |
7. | Lopez, J. M., Dromerick, A., and Freese, E. (1981) J. Bacteriol. 146, 605-613[Medline] [Order article via Infotrieve] |
8. |
Ochi, K.,
Kandala, J. C.,
and Freese, E.
(1981)
J. Biol. Chem.
256,
6866-6875 |
9. |
Ratnayake-Lecamwasam, M.,
Serror, P.,
Wong, K. W.,
and Sonenshein, A. L.
(2001)
Genes Dev.
15,
1093-1103 |
10. |
Inaoka, T.,
and Ochi, K.
(2002)
J. Bacteriol.
184,
3923-3930 |
11. | Chakraburtty, R., and Bibb, M. (1997) J. Bacteriol. 179, 5854-5861[Abstract] |
12. |
Martinez-Costa, O. H.,
Arias, P.,
Romero, N. M.,
Parro, V.,
Mellado, R. P.,
and Malpartida, F.
(1996)
J. Biol. Chem.
271,
10627-10634 |
13. | Ochi, K., and Ohsawa, S. (1984) J. Gen. Microbiol. 130, 2473-2482[Medline] [Order article via Infotrieve] |
14. | Shima, J., Hesketh, A., Okamoto, S., Kawamoto, S., and Ochi, K. (1996) J. Bacteriol. 178, 7276-7284[Abstract] |
15. | Kawamoto, S., Zhang, D., and Ochi, K. (1997) Mol. Gen. Genet. 255, 549-560[CrossRef][Medline] [Order article via Infotrieve] |
16. | Ochi, K., Zhang, D., Kawamoto, S., and Hesketh, A. (1997) Mol. Gen. Genet. 256, 488-498[CrossRef][Medline] [Order article via Infotrieve] |
17. | Hesketh, A., Sun, J., and Bibb, M. (2001) Mol. Microbiol. 39, 136-144[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Sun, J.,
Hesketh, A.,
and Bibb, M.
(2001)
J. Bacteriol.
183,
3488-3498 |
19. | Walker, J. E., and Abraham, E. P. (1970) Biochem. J. 118, 563-570[Medline] [Order article via Infotrieve] |
20. | Sakajoh, M., Solomon, N. A., and Demain, A. L. (1987) J. Ind. Microbiol. 2, 201-208 |
21. | Hilton, M. D., Alaeddinoglu, N. G., and Demain, A. L. (1988) J. Bacteriol. 170, 482-484[Medline] [Order article via Infotrieve] |
22. | Hilton, M. D., Alaeddinoglu, N. G., and Demain, A. L. (1988) J. Bacteriol. 170, 1018-1020[Medline] [Order article via Infotrieve] |
23. | Özcengiz, G., Alaeddinoglu, N. G., and Demain, A. L. (1990) J. Ind. Microbiol. 6, 91-100[Medline] [Order article via Infotrieve] |
24. | Freese, E. B., Vasantha, N., and Freese, E. (1979) Mol. Gen. Genet. 170, 67-74[Medline] [Order article via Infotrieve] |
25. | Itaya, M., Kondo, K., and Tanaka, T. (1989) Nucleic Acids Res. 17, 4410[Medline] [Order article via Infotrieve] |
26. | Moriya, S., Tsujikawa, E., Hassan, A. K., Asai, K., Kodama, T., and Ogasawara, N. (1998) Mol. Microbiol. 29, 179-187[CrossRef][Medline] [Order article via Infotrieve] |
27. | Shah, R., Neuss, N., Gorman, M., and Boeck, L. D. (1970) J. Antibiot. 23, 613-619[Medline] [Order article via Infotrieve] |
28. | Miller, J. H. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
29. | Kenig, M., and Abraham, E. P. (1976) J. Gen. Microbiol. 94, 37-45[Medline] [Order article via Infotrieve] |
30. | Glaser, P., Kunst, F., Arnaud, M., Coudart, M. P., Gonzales, W., Hullo, M. F., Ionescu, M., Lubochinsky, B., Marcelino, L., Moszer, I., Presecan, E., Santana, M., Schneider, E., Schweizer, J., Vertes, A., Rapoport, G., and Danchin, A. (1993) Mol. Microbiol. 10, 371-384[Medline] [Order article via Infotrieve] |
31. | Slack, F. J., Serror, P., Joyce, E., and Sonenshein, A. L. (1995) Mol. Microbiol. 15, 689-702[Medline] [Order article via Infotrieve] |
32. | Serror, P., and Sonenshein, A. L. (1996) J. Bacteriol. 178, 5910-5915[Abstract] |
33. |
Dworkin, J.,
and Losick, R.
(2001)
Genes Dev.
15,
1051-1054 |
34. |
Stover, A. G.,
and Driks, A.
(1999)
J. Bacteriol.
181,
1664-1672 |
35. | Babasaki, K., Takao, T., Shimonishi, Y., and Kurahashi, K. (1985) J. Biochem. 98, 585-603[Abstract] |
36. |
Paik, S. H.,
Chakicherla, A.,
and Hansen, J. N.
(1998)
J. Biol. Chem.
273,
23134-23142 |
37. | Nakano, M. M., Marahiel, M. A., and Zuber, P. (1988) J. Bacteriol. 170, 5662-5668[Medline] [Order article via Infotrieve] |
38. |
Tsuge, K.,
Ano, T.,
Hirai, M.,
Nakamura, Y.,
and Shoda, M.
(1999)
Antimicrob. Agents Chemother.
43,
2183-2192 |
39. |
Tamehiro, N.,
Okamoto-Hosoya, Y.,
Okamoto, S.,
Ubukata, M.,
Hamada, M.,
Naganawa, H.,
and Ochi, K.
(2002)
Antimicrob. Agents Chemother.
46,
315-320 |
40. | Tsuno, T., Ikeda, C., Numata, K., Tomita, K., Konishi, M., and Kawaguchi, H. (1986) J. Antibiot. 39, 1001-1003[Medline] [Order article via Infotrieve] |
41. | Ochi, K. (1986) J. Gen. Microbiol. 132, 2621-2631[Medline] [Order article via Infotrieve] |
42. | Ochi, K. (1987) J. Bacteriol. 169, 3608-3616[Medline] [Order article via Infotrieve] |
43. | Ochi, K. (1986) J. Gen. Microbiol. 132, 299-305[Medline] [Order article via Infotrieve] |
44. | Okamoto, S., Itoh, M., and Ochi, K. (1997) J. Bacteriol. 179, 170-179[Abstract] |
45. | Okamoto, S., and Ochi, K. (1998) Mol. Microbiol. 30, 107-119[CrossRef][Medline] [Order article via Infotrieve] |
46. | Lazazzera, B. A. (2000) Curr. Opin. Microbiol. 3, 177-182[CrossRef][Medline] [Order article via Infotrieve] |
47. | Yazgan, A., Ozcengiz, G., and Marahiel, M. A. (2001) Biochim. Biophys. Acta 1518, 87-94[Medline] [Order article via Infotrieve] |
48. | Wendrich, T. M., Beckering, C. L., and Marahiel, M. A. (2000) FEMS Microbiol. Lett. 190, 195-201[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Hu, H.,
Zhang, Q.,
and Ochi, K.
(2002)
J. Bacteriol.
184,
3984-3991 |
50. | Xu, J., Tozawa, Y., Lai, C., Hayashi, H., and Ochi, K. (2002) Mol. Genet. Genomics 268, 179-189[CrossRef][Medline] [Order article via Infotrieve] |
51. | Glass, R. E., Jones, S. T., and Ishihama, A. (1986) Mol. Gen. Genet. 203, 265-268[Medline] [Order article via Infotrieve] |
52. | Reddy, P. S., Raghavan, A., and Chatterji, D. (1995) Mol. Microbiol. 15, 255-265[Medline] [Order article via Infotrieve] |
53. |
Chatterji, D.,
Fujita, N.,
and Ishihama, A.
(1998)
Genes Cells
3,
279-287 |
54. |
Toulokhonov, I.,
Shulgina, I.,
and Hernandez, V. J.
(2001)
J. Biol. Chem.
276,
1220-1225 |
55. | Ishihama, A., Fujita, N., Igarashi, K., and Ueshima, R. (1990) in Structure and Function of Nucleic Acids and Proteins (Felicia, Y., Wu, H. , and Cheng, W. W., eds) , pp. 153-159, Raven Press, New York |