Guanine Nucleotides Guanosine 5'-Diphosphate 3'-Diphosphate and GTP Co-operatively Regulate the Production of an Antibiotic Bacilysin in Bacillus subtilis*

Takashi InaokaDagger , Kosaku TakahashiDagger , Mayumi Ohnishi-Kameyama§, Mitsuru Yoshida§, and Kozo OchiDagger

From the Dagger  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
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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.

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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.

                              
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Table I
Bacterial strains and plasmids used in this study

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: Delta ywfB-F (5'- aagcttATGATTATATTGGATAATAGCATTCAG-3') and Delta 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-Delta 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.

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.

beta -Galactosidase Assay-- beta -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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.

                              
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Table II
Analysis of culture broths for bacilysin titer and of cell extracts for basilysin synthetase activity


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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).

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.


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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.

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.


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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 beta -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).

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.


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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 beta -galactosidase activity was measured. B, stringent TI112 (squares) and relaxed TI113 (circles) strains were grown as in A.

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).


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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 beta -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.

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.


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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 beta -galactosidase activity was measured. B, strains TI112 (open squares), TI113 (open circles), TI114 (closed squares), and TI115 (closed circles) were grown as in A.


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ABSTRACT
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REFERENCES

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 beta -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-beta -D-galactopyranoside; MOPS, 4-morpholinepropanesulfonic acid; ESI, electrospray ionization; MS, mass spectrometry; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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[Abstract/Free Full Text]
9. Ratnayake-Lecamwasam, M., Serror, P., Wong, K. W., and Sonenshein, A. L. (2001) Genes Dev. 15, 1093-1103[Abstract/Free Full Text]
10. Inaoka, T., and Ochi, K. (2002) J. Bacteriol. 184, 3923-3930[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Free Full Text]
34. Stover, A. G., and Driks, A. (1999) J. Bacteriol. 181, 1664-1672[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
39. Tamehiro, N., Okamoto-Hosoya, Y., Okamoto, S., Ubukata, M., Hamada, M., Naganawa, H., and Ochi, K. (2002) Antimicrob. Agents Chemother. 46, 315-320[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
54. Toulokhonov, I., Shulgina, I., and Hernandez, V. J. (2001) J. Biol. Chem. 276, 1220-1225[Abstract/Free Full Text]
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


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