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Applied and Environmental Microbiology, October 2007, p. 6159-6165, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.02835-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Indole Derivatives as Self-Growth Inhibitors of Symbiobacterium thermophilum, a Unique Bacterium Whose Growth Depends on Coculture with a Bacillus sp.

Tomo-o Watsuji,¹ Shinya Yamada,² Tomoya Yamabe,¹ Yuka Watanabe,¹ Taira Kato,¹ Takao Saito,² Kenji Ueda,^1* and Teruhiko Beppu¹

Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan,¹ Takasago International Corporation, 1-4-11 Nishi-yawata, Hiratsuka 254-0073, Japan²

Received 6 December 2006/ Accepted 2 August 2007

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Symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a Bacillus sp. Recently, we discovered that CO₂ generated by Bacillus is the major inducer for the growth of S. thermophilum; however, the evidence suggested that an additional element is required for its full growth. Here, we studied the self-growth-inhibitory substances produced by S. thermophilum. We succeeded in purifying two substances from an ether extract of the culture supernatant of S. thermophilum by multiple steps of reverse-phase chromatography. Electron ionization mass spectrometry and nuclear magnetic resonance analyses of the purified preparation identified the substances as 2,2-bis(3'-indolyl)indoxyl (BII) and 1,1-bis(3'-indolyl)ethane (BIE). The pure growth of S. thermophilum was inhibited by authentic BII and BIE with MICs of 12 and 7 µg/ml, respectively; however, its growth in coculture with Bacillus was not inhibited by BII at the saturation concentration and was inhibited by BIE with an MIC of 14 µg/ml. Both BII and BIE inhibited the growth of other microorganisms. Unexpectedly, the accumulation levels of both BII and BIE in the pure culture of S. thermophilum were far lower than the MICs (<0.1 µg/ml) while a marked amount of BIE (6 to 7 µg/ml) equivalent to the MIC had accumulated in the coculture. An exogenous supply of surfactin alleviated the sensitivities of several BIE-sensitive bacteria against BIE. The results suggest that Bacillus benefits S. thermophilum by detoxifying BII and BIE in the coculture. A similar mechanism may underlie mutualistic relationships between different microorganisms.

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Symbiobacterium thermophilum is a thermophilic bacterium that was originally isolated by its ability to produce a thermostable tryptophanase from compost collected at Hiroshima, Japan (19). This bacterium is characterized by a syntrophic property that is reproduced under a laboratory culture condition; although it does not grow or shows impaired growth with a very low cellular yield in pure culture, it effectively propagates, achieving a density of approximately 5 x 10⁸ cells/ml, when it is cocultured with the cognate Bacillus sp. strain S, which was isolated together with S. thermophilum (19). The molecular phylogenetic and genomic sequencing studies (13, 22) uncovered unique genetic and taxonomic features of S. thermophilum. S. thermophilum exhibits a number of genetic features associated with Firmicutes (low-G+C, gram-positive bacteria represented by Bacillus and Clostridium), despite its high G+C content (68.5%). This unusual property has now become a new topic in bacterial systematics (2, 4) and suggests that this kind of bacterium has been left uncharacterized due to its "unculturable nature," despite its wide distribution in the natural environment (20). We are interested in the fundamental details of the syntrophic feature of S. thermophilum and expect that this information will provide us with new knowledge regarding not only the microbial physiology but also the issue of unculturability of environmental microorganisms.

Recently, we discovered that a pure culture of S. thermophilum grew effectively under high atmospheric CO₂ concentrations (24). The lines of evidence suggest that the organism's dependence on CO₂ for growth is based on the requirement for bicarbonate, which is required for the reaction catalyzed by essential enzymes such as phosphoenolpyruvate carboxylase and acetyl-coenzyme A carboxylase. The requirement for exogenous bicarbonate can be attributed to the lack of carbonic anhydrase, which catalyzes the conversion of CO₂ to bicarbonate (17). While bacteria that retain carbonic anhydrase can obtain sufficient bicarbonate for growth by catalytic conversion from environmental CO₂, those lacking this enzyme depend on the high concentration of bicarbonate that naturally occurs in the environment. In fact, knockout mutants for a carbonic anhydrase gene confer a CO₂ dependence phenotype in Ralstonia eutropha (8) and Escherichia coli (10). The result of genomic sequencing revealed that S. thermophilum does not retain the gene for carbonic anhydrase (22). Based on these observations, we speculate that S. thermophilum grows by using the CO₂ that is generated by the cognate Bacillus sp. strain S.

Although a supply of CO₂ markedly promoted the growth of S. thermophilum, it was insufficient to fully explain its dependence on coculture with Bacillus for growth. The cellular yield of S. thermophilum obtained in the pure culture under conditions of high atmospheric CO₂ concentrations was approximately 1 x 10⁷ cells/ml, which corresponds to less than 10% of that achieved by coculture with Bacillus strain S (12, 24). It suggested that the Bacillus strain benefits S. thermophilum not only by supplying CO₂ but also by supplying (or eliminating) some other metabolite(s). In this study, based on the observation that dialysis culture promoted the growth of pure S. thermophilum in cultures, we assessed the possibility that the Bacillus strain removes or inactivates a self-growth inhibitor produced by S. thermophilum. We isolated substances from pure cultures of S. thermophilum that inhibited its growth and identified them as indole alkaloids, which have been known as antibacterial metabolites in several gram-negative bacteria. Although the inhibitory activities of these substances do not fully explain the occurrence of self-growth inhibition in S. thermophilum, the evidence strongly suggests that inactivation of the self-growth inhibitor is an important basis of the effective proliferation of S. thermophilum in coculture with Bacillus strain S.

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Microbial strains and culture conditions.
S. thermophilum IAM14863 and Bacillus strain S have been described previously (13, 19). The microbial strains that were used for the MIC assay (Table 1) were obtained from the Japan Collection of Microorganisms (JCM), RIKEN, and the Institute of Fermentation (IFO), Osaka, Japan (NBRC; National Institute of Technology and Evaluation, Japan). The sources of the microbial strains Escherichia coli DH5 (Takara Shuzo), Thermus thermophilus HB27 (M. Nishiyama, University of Tokyo), Bacillus subtilis 168 (Bacillus Genetic Stock Center, OH), Corynebacterium glutamicum AJ1511 (Ajinomoto Co., Inc., Kawasaki, Japan), Rhodococcus strain RHA1 (M. Fukuda, Nagaoka University of Technology), and Saccharomyces cerevisiae X2180-1A (Yeast Genetic Stock Center, University of California) are indicated within parentheses.

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TABLE 1. MICs of BII and BIE in various microorganisms

The conditions for the cultivation (24) and growth measurement (21) of S. thermophilum cells were essentially the same as those described previously. Pure culture of S. thermophilum was performed at 60°C in a Luria-Bertani (LB) broth (1% Bacto tryptone [Difco Laboratories, Detroit, MI], 0.5% yeast extract [Difco], and 0.5% NaCl [Kokusan, Tokyo], pH 7.6) contained in an Erlenmeyer flask (300 ml/500 ml) under anaerobic conditions (N₂/H₂/CO₂ ratio = 80:10:10) without shaking for 3 days. Cocultivation of S. thermophilum with Bacillus strain S was performed under similar conditions, with the exception that the culture was performed under an atmosphere of normal air. Large-scale and small-scale cultures were carried out in a 500-ml Erlenmeyer flask containing 300 ml of medium and a test tube (diameter, 16 mm by 100 mm) containing 4 ml of medium, respectively.

Dialysis culture of S. thermophilum was carried out as follows. A 1-ml volume of LB medium containing S. thermophilum cells was enclosed in a cellulose dialysis tube (molecular weight cutoff, 3,500; Spectrum Laboratories), which was floated in various volumes of sterile LB medium. The whole system was incubated at 60°C in an anaerobic atmosphere for 3 days. To prepare a conditioned medium that contained the culture supernatant of Bacillus strain S, Bacillus strain S was cultured at 60°C for 24 h in LB medium with shaking at 130 rpm. The culture broth was adjusted to a pH of 7.6 with HCl, sterilized by filtration, and added to the same volume of fresh sterile LB medium. The growth of S. thermophilum was measured by the specific quantitative PCR technique as described previously (21).

For the MIC assay, certain strains were cultured in LB medium (E. coli, T. thermophilus, B. subtilis, Bacillus strain S, C. glutamicum, and a Rhodococcus sp. strain), nutrient broth (Pseudomonas putida and Janthinobacterium lividum), deMan-Rogosa-Sharpe broth (Lactobacillus paracasei and Lactococcus lactis), or yeast extract-peptone-dextrose medium (Streptomyces griseus, S. cerevisiae, and Aspergillus oryzae). Nutrient broth and deMan-Rogosa-Sharpe broth were purchased from Difco. The yeast extract-peptone-dextrose medium contained 1% Bacto yeast extract (Difco), 2% Bacto peptone (Difco), and 2% glucose (Kokusan). The overnight liquid cultures (10 µl each) were inoculated into 4 ml of fresh medium containing various concentrations (in increments of 1.0 µg/ml, up to 18 µg/ml) of 1,1-bis(3'-indolyl)ethane (BIE) or 2,2-bis(3'-indolyl)indoxyl (BII). E. coli was cultured at 37°C with shaking for 1 day. T. thermophilus was cultured at 70°C with shaking for 1 day. L. paracasei, L. lactis, and S. cerevisiae were cultured at 28°C without shaking for 1 day. Bacillus strain S was cultured at 60°C without shaking for 1 day. A. oryzae and S. griseus were cultured at 28°C with shaking for 3 days. The other strains were cultured at 28°C with shaking for 1 day. Growth was estimated by visual observation. To obtain the effect of surfactin, B. subtilis, C. glutamicum, and J. lividum were inoculated in their corresponding media, to which BIE was added (6 µg/ml for B. subtilis and J. lividum, 10 µg/ml for C. glutamicum) along with various amounts of commercial surfactin (Wako) and cultured at 28°C with shaking. The optical density ( = 600 nm) of each culture broth was measured after an appropriate time period (18 h for B. subtilis, 30 h for C. glutamicum, and 48 h for J. lividum). Shaking was carried out at 110 rpm for all cultures.

Isolation and quantification of growth-inhibitory substances.
In order to isolate the self-growth-inhibitory substance, a large-scale culture of S. thermophilum was performed (see above). After removal of cells by centrifugation, 3,000 ml of the culture supernatant was adjusted to pH 2.5 with HCl and extracted three times with 500 ml of diethyl ether. The resultant ether extract was dried by rotary evaporation, dissolved in 1.5 ml ethanol, and applied onto a reverse-phase column (33 g; ODS-7515-12A; SSC). After being washed with water, the column was developed with a stepwise gradient of acetonitrile in water (water/acetonitrile ratio = 10:0 to 0:10). The activity of the extract was recovered by elution with a water/acetonitrile ratio of 2:8. The fraction was dried by rotary evaporation, dissolved in 1.5 ml ethanol, and applied onto a reverse-phase high-performance liquid chromatography (HPLC) column (diameter, 4.6 by 250 mm; Shodex C18M 4E; Showa Denko). After being washed with water, the column was developed with a 40% to 80% gradient of acetonitrile in water at 1.0 ml·min^–1, and fractions of 1.0 ml were collected while the UV absorption spectra at 214 nm were monitored. According to the results thus obtained, the activity principles were reproducibly fractionated into three separated fractions (no. 31, 38, and 41). After being dried, the fractions were further purified by application onto the same column as that described above and developed by using an isocratic flow (1.0 ml·min^–1) of 3% acetonitrile in water.

Structural analysis and organic synthesis of BII and BIE.
The chemical structures of the substances corresponding to fraction no. 31 and 38 were determined according to the results of electron ionization mass spectrometry (EI-MS) and nuclear magnetic resonance (NMR) analyses. The EI-MS analysis was carried out with a Hitachi M-80B mass spectrometer (Hitachi, Japan) in a positive mode. NMR spectra were recorded on a 300-MHz, four-nucleus MERCURYplus spectrometer (Varian, Inc.) and a Bruker DRX-500 spectrometer (Bruker Co.). The chemical shifts of CDCl₃ ( 7.26) and (CD₃)₂CO ( 2.04) were used as the internal standards. The spectral data of the active substance corresponding to fraction no. 31 (i.e., BII) were as follows: high-resolution EI-MS (70 eV) m/z 364.1421 [M + H]⁺ (corresponding to C₂₄H₁₈N₃O = 364.1449), 386.1253 [M + Na]⁺; ¹H NMR (500 MHz, acetone-d₆) 10.2 (2H, br, A-1, B-1), 7.53 (1H, dd, J = 1.3, 7.7 Hz, C-4), 7.48 (1H, ddd, J = 1.3, 7.0, 8.1, 10.0 Hz, C-6), 7.43 (2H, dd, J = 8.1, 10.0 Hz, A-4, B-4), 7.35 (2H, dd, J = 8.1, 10.0 Hz, A-7, B-7), 7.22 (2H, d, J = 2.6 Hz, A-2, B-2), 7.10 (1H, br, C-1), 7.04 (2H, ddd, J = 7.0, 8.1, 10.0 Hz, A-6, B-6), 7.02 (1H, dd, J = 0.8, 8.4 Hz, C-7), 6.82 (2H, ddd, J = 7.0, 8.1, 10.0 Hz, A-5, B-5), 6.77 (1H, ddd, J = 0.8, 7.1, 7.7 Hz, C-5); ¹³C NMR (acetone-d₆) 202 (C-3), 162.3 (C-7a), 139.1 (A-7a, B-7a), 138.6 (C-6), 127.7 (A-3a, B-3a, C-4), 125.6 (A-2, B-2), 129.9 (A-6, B-6), 122.5 (A-4, B-4), 120.8 (A-5, B-5), 119.3 (C-5), 116.7 (A-3, B-3), 113.8 (C-7), 113.0 (A-7, B-7), 69.7 (C-2).

Spectral data of the substance corresponding to fraction no. 38 (i.e., BIE) were as follows: High-resolution EI-MS (70 eV) m/z 261.13936 [M + H]⁺ (corresponding to C₁₈H₁₇N₂ = 261.13917); ¹H NMR (300 MHz, CDCl₃) 7.88 (1H, br, A-2, B-2), 7.58 (2H, d, J = 7.8 Hz, A-4, B-4), 7.35 (2H, d, J = 7.8 Hz, A-7, B-7), 7.16 (2H, ddd, J = 0.9, 7.8, 7.8 Hz, A-6, B-6), 7.04 (2H, ddd, J = 0.9, 7.8, 7.8 Hz, A-5, B-5), 6.93 (2H, d, J = 2.1 Hz, A-3, B-3), 4.65 (1H, d, J = 6.9 Hz, 8), 1.81 (3H, d, J = 6.9 Hz, 9). Authentic BII and BIE were synthesized as described previously (7, 16).

Quantification of BII and BIE.
The concentrations of BII and BIE in the S. thermophilum culture broth were measured as follows. After removal of cells by centrifugation, 200 ml of supernatant was adjusted to pH 2.5 with HCl and extracted three times with 70 ml of diethyl ether. After being dried by evaporation and dissolved in 1.0 ml methanol, the ether extract was applied onto a reverse-phase HPLC column (diameter, 4.6 by 250 mm; Shodex C18M 4E, Showa Denko). After being washed with water, the column was developed with a 40% to 80% gradient of acetonitrile in water at 1.0 ml·min^–1 while the UV absorption spectra at 260 nm were monitored. The peaks corresponding to BIE and BII were identified and quantified by comparing them with the peak profile of the authentic samples.

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Effect of dialysis on the growth of S. thermophilum.
To study the effect of dialysis on the growth of S. thermophilum, its pure culture was dialyzed against various volumes of sterile medium (see Materials and Methods). As shown in Fig. 1A, the cellular yield of S. thermophilum increased according to the increment in the dialysis ratio; by dialysis at a 100-fold dilution ratio, S. thermophilum grew to a density of 9 x 10⁸ cells/ml, which was 100 times higher than the cellular yield achieved in the culture without dialysis (1 x 10⁷ cells/ml). According to this result, we assumed that the growth promotion observed could be due to the elimination of a growth-inhibitory substance(s) that has a low molecular weight (<3,500). In the coculture system, the high cellular yield of S. thermophilum could be due to the elimination (inactivation) or repression of production of the growth inhibitor by some metabolic activity of Bacillus strain S.

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FIG. 1. Growth inhibition of S. thermophilum possibly due to the effect of its own metabolite. (A) Growth promotion by dialysis. S. thermophilum was cultured in a CO2-containing atmosphere with dialysis against various amounts of sterile LB medium, and the cellular concentration was measured after 3 days of incubation at 60°C. Cellular yields (vertical axis) were plotted against the dialysis ratio (horizontal axis). (B) Growth inhibition by the exogenous supply of the ether extract of the culture broth of S. thermophilum. S. thermophilum was cultured in LB medium supplemented with various amounts of the ether extract, and the cellular concentration was measured after 3 days of incubation at 60°C. Cellular yields (vertical axis) were plotted against the relative concentration of the ether extract (horizontal axis); 1.0 U is equivalent to the concentration of the inhibitory substance in the original culture broth used for extraction. Open circle, ether extract of culture supernatant of S. thermophilum; open triangle, ether extract of LB medium without inoculation. The graphs show results representative of a thrice-repeated experiment.

Identification of self-growth inhibitors of S. thermophilum.
Based on the above speculation, we investigated the presence of growth-inhibitory activity in the supernatant of the pure culture of S. thermophilum and detected a marked activity in its ether extract fraction (Fig. 1B). In the culture medium containing 0.5 U of the putative inhibitory substance (1.0 U is equivalent to the concentration of the inhibitory substance in the original culture broth used for extraction), the growth of S. thermophilum was completely inhibited. The addition of an ether extract of a culture medium without bacterial inoculation did not inhibit the growth of S. thermophilum (Fig. 1B). These results indicated that the ether extract of the culture supernatant contained a metabolite(s) of S. thermophilum that inhibited the growth of the organism.

In order to determine the chemical structure of the putative self-growth inhibitor produced by S. thermophilum, the active principle was isolated and purified from the culture supernatant (see Materials and Methods). The activity reproducibly split into different fractions in a step of reverse-phase HPLC (Fig. 2A). In this step, the active principle obtained in the previous reverse-phase column chromatography was fractionated into 60 fractions. The subsequent growth inhibition test showed that fraction no. 31, 38, and 41 each exhibited a growth inhibition activity while none of the other fractions did (data not shown). These inactive fractions, however, showed inhibitory activities when their mixtures were added to culture medium (Fig. 3A). Furthermore, a mixture of all fractions showed much higher inhibitory activity than a mixture of the above-mentioned three active fractions. This suggested that the activity principle contained multiple inhibitors as well as an additional element that promotes the inhibitory activity.

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FIG. 2. Isolation and identification of growth inhibitors. (A) UV ( = 214 nm) elution profile for reverse-phase HPLC. The activity fraction obtained in reverse-phase open column chromatography was applied onto an HPLC column and separated into 60 fractions (see Materials and Methods). The inhibitory activity split into three separated fractions (no. 31, 38, and 41) in this step. Inactive fractions also showed inhibitory activities when their mixture was added to culture medium (refer to text). A result representative of an experiment repeated more than five times is shown. (B) 1H NMR and 13C NMR chemical shift assignments for BII and BIE. BII and BIE were isolated from fraction no. 31 and 38, respectively.

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FIG. 3. Inhibitory activities against the growth of S. thermophilum. (A) Inhibitory activity exhibited by mixtures of fractions obtained by reverse-phase HPLC. Mixtures of all fractions (closed circle), fractions that showed inhibitory activities (no. 31, 38, and 41) (open square), and fractions that did not show inhibitory activity (all except no. 31, 38, and 41) (closed triangle) were added to culture medium at various concentrations; 1.0 U is equivalent to the concentration of the inhibitory substance in the original culture broth used for extraction. (B) Inhibitory activity of BII and BIE. Pure culture of S. thermophilum (–Bacillus) and coculture with Bacillus strain S (+Bacillus) in the medium supplied with various amounts of authentic BII (upper panel) and BIE (lower panel) are seen. Pure growth of S. thermophilum in a conditioned medium containing culture supernatant of Bacillus strain S (±sup) is also shown. The cells were quantified after 3 days of growth at 60°C by the quantitative PCR technique (21). All graphs show results representative of thrice-repeated experiments.

From 102 liters of culture supernatant, approximately 10 mg (fraction no. 31), 3 mg (fraction no. 38), and 1 mg (fraction no. 41) of the purified active substances were obtained. From the spectral data obtained from EI-MS and NMR spectroscopy, the active substances corresponding to fraction no. 31 and 38 were identified as BII and BIE, respectively (Fig. 2B). The identities of these substances were confirmed by comparing the retention time on HPLC and the spectral data obtained for the active substance with those for the authentic samples, which were synthesized by the method described above (see Materials and Methods). We could not identify the substance corresponding to fraction no. 41, due to the relatively low quality and quantity of the preparation. The indole derivatives BII and BIE have been isolated from culture broths of several gram-negative bacteria, such as Pseudomonas aureofaciens (5), Vibrio parahaemolyticus (1), and Haemophilus influenzae (18); moreover, they are known for their growth-inhibitory activities against Staphylococcus aureus and B. subtilis (1).

Growth-inhibitory activities of BII and BIE.
Figure 3B shows the growth-inhibitory effects of the exogenous supplies of authentic BII and BIE against S. thermophilum. The saturation concentrations of both the substances in water were 18 µg/ml. In the pure-culture system, the supplies of BII and BIE at or more than 12 and 6 µg/ml, respectively, inhibited the growth of S. thermophilum. Meanwhile, in the coculture with Bacillus strain S, BII did not cause any marked inhibition against the growth of S. thermophilum (or Bacillus strain S) up to a concentration of 18 µg/ml. The minimal concentration of BIE required to inhibit the growth of S. thermophilum in the coculture was 14 µg/ml, twofold that required in the pure culture. At this concentration, BIE also inhibited the growth of Bacillus strain S (see below). Therefore, there is a possibility that the inhibition observed here is not due to the direct effect of BIE on S. thermophilum.

Table 1 shows the MICs of BII and BIE studied for various microbial strains (see Materials and Methods). BII did not inhibit the growth of most of the organisms studied, including Bacillus strain S. Only two actinobacteria, S. griseus and the Rhodococcus sp., were sensitive to BII. On the other hand, BIE showed a broad antimicrobial spectrum; it effectively inhibited the growth of microbes belonging to gram-positive bacteria, gram-negative bacteria, the genus Thermus, and even fungi. The MIC of BIE for Bacillus strain S was 14 µg/ml, which was the same value as that obtained for S. thermophilum in the coculture with Bacillus strain S.

Production of BII and BIE by S. thermophilum.
Figure 4 shows the accumulation profiles of BII and BIE in the culture of S. thermophilum as well as the corresponding growth curves. In the pure culture, BII started to accumulate in the early stationary phase and reached the maximum level (0.37 µg/ml) in 60 h, while the other substance, i.e., BIE, gradually accumulated throughout the late stationary phase and reached the maximum level (0.9 µg/ml) in 108 h. The concentrations of these substances that accumulated in the pure culture were lower than the inhibitory concentrations determined as described above.

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FIG. 4. Production of BII and BIE by S. thermophilum. (Upper) Cellular growth of S. thermophilum in the pure culture (closed circles) and coculture with Bacillus strain S (open circles). (Lower) Amounts of BII and BIE accumulated in the above-mentioned pure culture (closed squares, BII; closed triangles, BIE) and coculture (open squares, BII; open triangles, BIE). Results representative of a twice-repeated experiment are shown.

In contrast to those in the pure culture, the accumulation levels of the two substances were markedly high in the coculture (Fig. 4); they began to accumulate in the late logarithmic phase and reached the maximum levels (BII, 6.3 µg/ml; BIE, 6.7 µg/ml) in 36 h. The concentration of BII that accumulated in the coculture corresponded to that which caused a 10-fold reduction in the growth of the pure culture of S. thermophilum (Fig. 3B). Similarly, the concentration of BIE that accumulated in the coculture corresponded to that which inhibited the growth of S. thermophilum in the pure culture. BII and BIE were not detected in the pure culture of Bacillus strain S.

The above observation suggested that coculture with Bacillus confers resistance against BII and BIE to S. thermophilum. Hence, we examined the effect of the addition of culture supernatant of Bacillus strain S on the sensitivity of S. thermophilum to BII and BIE. As shown in the graphs presented in Fig. 3B, the addition of culture supernatant of Bacillus strain S reduced the sensitivity of S. thermophilum to some extent. The minimal concentrations of BII and BIE required to inhibit the growth of S. thermophilum in the conditioned medium were 18 and 11 µg/ml, respectively.

Detoxification of BIE by surfactin.
Based on the above-mentioned results, we thought it possible that the toxic effect of BIE is alleviated to a certain extent by some metabolite of Bacillus strain S. Figure 5 shows the effect of an exogenous supply of surfactin, a surfactant produced by Bacillus spp., on the growth of the three representative BIE-sensitive organisms: B. subtilis, C. glutamicum, and J. lividum. While the growth of these bacteria was completely inhibited in the presence of BIE (6 µg/ml for B. subtilis and J. lividum and 10 µg/ml for C. glutamicum), it was restored by the addition of surfactin in a dose-dependent manner. This result suggests that sequestration by a surfactant is one of the mechanisms responsible for the tolerance of some bacteria to the indole derivative compound. We could not study the effect of surfactin on the BIE-dependent growth inhibition of S. thermophilum, since surfactin was toxic to this organism.

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FIG. 5. Effects of the exogenous supply of surfactin on the inhibitory effect of BIE against the three BIE-sensitive bacteria. Each organism was cultured in an appropriate medium supplied with BIE and various amounts of commercial surfactin (see Materials and Methods). Growth was quantified by measuring optical density at 600 nm (OD600). Diamonds, B. subtilis; squares, C. glutamicum; triangles, J. lividum. Results representative of a twice-repeated experiment are shown.

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It has been known that dialysis culture enables the high-cell-density cultivation of some bacteria, including E. coli and lactobacilli, as well as mammalian cells (reviewed by Pörtner and Märkl) (15). It is predicted that the high cellular yield achieved by dialysis culture is due to the removal of a growth inhibitor(s) that is generated during the growth of the corresponding organism. To date, some biochemical information regarding such growth inhibitors is available; for example, the precise characterization of the high-cell-density culture by Nakano et al. (11) revealed that acetic acid serves as a self-growth inhibitor for E. coli. Inoue et al. (6) found that the growth of Legionella pneumophila was enhanced by the addition of Diaion resin to the culture, and sulfur was identified as the self-growth inhibitor of this bacterium. It is predicted that the elimination of lactic acid and ammonia is responsible for the growth enhancement of mammalian cells in dialysis culture (9, 14).

BII and BIE, the compounds identified as self-growth inhibitors of S. thermophilum in this study, have been known to occur in culture broths of several gram-negative bacteria. V. parahaemolyticus generates various types of indole derivatives, including BII and BIE (1, 23). Bell et al. (1) observed that BII and BIE demonstrated antimicrobial activities against S. aureus and B. subtilis. On the other hand, Stull et al. (18) discovered that the biosynthesis of BII was induced by the presence of nitrate in H. influenzae and suggested that the synthesis of this compound was a result of nitrate respiration using indole as an electron donor. Although several studies have reported that indole derivatives are formed even by the nonbiological oxidation of indole (3), the lines of evidence, including those obtained in this study, indicate that the formation of these compounds is carried out or is markedly facilitated by the specific metabolic activities of certain bacteria. Currently, no information regarding the mechanism of biosynthesis and the mode of action of BII and BIE is available. It appears most likely that indoles generated by the activity of tryptophanase produced by S. thermophilum serve as a precursor of these substances. We speculate that the substance present in fraction no. 41 is also an indolyl metabolite based on its ¹H NMR spectrum (our unpublished observation).

The accumulation levels of both BII and BIE in the pure culture of S. thermophilum were distinctively lower than the inhibitory concentrations. This raises a possibility that the inhibitory effects of these substances are not directly responsible for the growth yield of S. thermophilum being lower in the pure culture under CO₂ supply than in the coculture with Bacillus strain S (typical growth curves are shown in Fig. 4, upper). However, the marked growth inhibition caused by the mixture of fractions obtained in reverse-phase HPLC (Fig. 3A) suggests that the ether extract contained multiple inhibitors as well as some effector, which itself does not cause growth inhibition but enhances the activities of inhibitors. Hence, we currently speculate that BII and BIE are the major self-growth inhibitors of S. thermophilum and that their effect is enhanced in the presence of unidentified metabolites, although we do not know the mechanism of enhancement.

A significant discovery was that the sensitivity of S. thermophilum to the two substances was ameliorated by its coculture with Bacillus strain S (Fig. 3B). Furthermore, the quantitative analysis demonstrated that the amounts of the two compounds accumulated in the culture broth were markedly increased in the coculture. We consider it most likely that these substances were generated by the metabolic activity of S. thermophilum since Bacillus strain S did not produce these substances in the pure culture; however, we cannot exclude the possibility that the Bacillus strain also contributed to the formation of these substances by utilizing the indole produced by S. thermophilum. Recently, we discovered that the expression of tryptophanase and a related S. thermophilum gene was induced by coculture with Bacillus strain S and that S. thermophilum performs energy metabolism upon degradation of tryptophan (unpublished data). Hence, currently we assume that the marked accumulation of BII and BIE is a result of a unique metabolism that depends on syntrophism with Bacillus.

A notable point was that the amounts of BII and BIE produced in coculture exceeded the concentrations that effectively repressed the growth of S. thermophilum in its pure culture. These results imply that a certain activity of the Bacillus strain S alleviates the toxicity of BII and BIE against S. thermophilum. Currently, the mechanism of detoxification is not clear; however, the effect of the addition of the culture supernatant of Bacillus strain S, which increased the MICs in the pure culture of S. thermophilum (Fig. 3B), strongly suggests that some substance produced by Bacillus strain S has an activity for reducing the inhibitory activities of BII and BIE. It is possible that the substance responsible for the detoxification is a surfactant similar to surfactin, although we have not yet been successful in identifying the substance.

The marked effect of the exogenous supply of surfactin on the growth of the BIE-sensitive organisms leads us to assume that the surfactant produced by Bacillus and other related bacteria might benefit the other organisms by protecting them from BIE and its related substances that are generated by certain types of bacteria. Similar ideas could be applied to various situations of microbial commensalism in the natural environment. We assume that the supply of nutritional elements, along with the elimination or inactivation of inhibitory substances, serves as a basic mechanism for microbial-community structuring.

 
We thank Shoichi Amano for valuable technical assistance.

This study was supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan. T.W. was supported by a fellowship of the COE program.

 
* Corresponding author. Mailing address: Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan. Phone: 81-466-84-3937. Fax: 81-466-84-3935. E-mail: ueda{at}brs.nihon-u.ac.jp

Published ahead of print on 10 August 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, October 2007, p. 6159-6165, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.02835-06
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