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Absolute Chemical Structure of the Myxobacterial Pheromone Of FEMS Microbiology Letters 165 (1998) 29^34 Absolute chemical structure of the myxobacterial pheromone of Stigmatella aurantiaca that induces the formation of its fruiting body Downloaded from https://academic.oup.com/femsle/article/165/1/29/623856 by guest on 24 September 2021 Yuka Morikawa a, Seiji Takayama a, Ryosuke Fudo b, Shigeru Yamanaka b, Kenji Mori c, Akira Isogai a;* a Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0101, Japan b Central Research Laboratories, Ajinomoto Co. Ltd., Suzuki 1-1, Kawasaki, Kanagawa 210-0801, Japan c Department of Chemistry, Science University of Tokyo, Kagurazaka 1-3, Shinjuku, Tokyo 162-8601, Japan Received 6 May 1998; revised 3 June 1998; accepted 4 June 1998 Abstract Stigmatella aurantiaca, a species of myxobacteria, produces a novel extracellular signaling molecule, 8-hydroxy-2,5,8- trimethyl-4-nonanone, which promotes its developmental cycle. To determine the absolute configuration of this pheromone, which contains one asymmetric carbon, we prepared the R- and S-enantiomers by stereoselective synthesis. The synthesized R- and S-enantiomers each showed nearly the same fruiting body-inducing activities as the natural pheromone. Gas chromatography-mass spectrometry (GC-MS) analysis using a chiral capillary column revealed that the naturally-produced pheromone is a mixture of both enantiomers. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Myxobacterium; Stigmatella aurantiaca; Fruiting body; Pheromone; Absolute structure 1. Introduction ing body formation process of S. aurantiaca may be regarded as a model of photomorphogenesis. Myxobacteria are a class of Gram-negative bacte- In addition to environmental factors, an extracel- ria which show a social behavior and complex devel- lular signaling molecule, or pheromone, is known to opmental cycle [1,2]. In response to starvation, they be involved in the developmental cycle of S. auran- aggregate to form characteristic multicellular fruiting tiaca [4]. This pheromone is a small lipophilic com- bodies. Among myxobacteria, Stigmatella aurantiaca pound secreted by S. aurantiaca under nutrient-de¢- has the most elaborate fruiting body structure, and cient conditions. Production of this pheromone in requires light for normal development [3]. The fruit- S. aurantiaca is extensively promoted by light [5]. Furthermore, exogenous addition of this pheromone to a culture of S. aurantiaca in the dark promotes * Corresponding author. Tel.: +81 (743) 72-5450; Fax: +81 (743) 72-5459; fruiting body formation [4]. Guanosine nucleotides E-mail: [email protected] [6], and isoeugenitin, a fungal metabolite [7], have 0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(98)00252-3 FEMSLE 8258 30-7-98 30 Y. Morikawa et al. / FEMS Microbiology Letters 165 (1998) 29^34 also been reported to induce fruiting body formation under the light for 24 h. The culture supernatant in S. aurantiaca in the dark. Studying these exoge- obtained by the centrifugation (5000Ug, 30 min) nous signals will elucidate the signaling cascade in was applied onto a column of XAD-2 resin (Organo, this photomorphogenesis. Tokyo, Japan). The column was washed with water W.E. Hull et al. [8] recently isolated the phero- and 20% methanol and then eluted with 80% meth- mone and determined that its structure is 8-hy- anol. After the removal of methanol by evaporation, droxy-2,5,8-trimethyl-4-nonanone (1) (Fig. 1). How- the pheromone in the residual water layer was ex- ever, the absolute con¢guration of this compound, tracted with chloroform. The chloroform extract was which has an asymmetric carbon atom at C-5, has concentrated by evaporation and applied onto a sili- not yet been determined. In this study, we have pu- ca gel column, and the column was eluted with 20% ri¢ed the natural pheromone to homogeneity by nor- ethyl acetate in chloroform. The active fraction was mal and reversed phase chromatography, and have concentrated by evaporation and further puri¢ed by Downloaded from https://academic.oup.com/femsle/article/165/1/29/623856 by guest on 24 September 2021 elucidated some of its chemical properties. We also the following four steps of reversed-phase HPLC, synthesized both enantiomers of the pheromone ster- using a 626 HPLC system equipped with 996 Diode eoselectively. We report the bioactivity of the synthe- Array UV Detector (Waters, Milford, MA, USA). sized enantiomers, as well as the absolute con¢gura- The pheromone-containing fraction was sequentially tion of the natural pheromone. applied onto an ODS column (Capcell Pak C18, UG120, Shiseido, Tokyo, Japan), SymmetryShield RP8 column (Waters), and a CN column (Capcell 2. Materials and methods Pak CN, UG120, Shiseido). Finally, the active frac- tion was applied to a phenyl column (Capcell Pak 2.1. Bacterial strain and growth Phenyl, UG 120, Shiseido) and eluted with 45% ace- tonitrile in water. The pheromone was eluted as a S. aurantiaca DW4 (ATCC 33878) were grown in symmetrical peak when it was monitored by 195 Casitone medium (1% Casitone (Difco Laboratories, nm UV absorption. Detroit, MI, USA), 8 mM MgSO4), at 30³C and 140 rpm. Vegetative cells were harvested during the 2.4. Structural analysis of the pheromone mid-log phase by centrifugation at 3000Ug for 10 min. Gas chromatography-mass spectrometry (GC-MS) analysis was done on a mass spectrometer (JMS-700 2.2. Assay of pheromone activity MStation, JEOL, Tokyo, Japan), equipped with a gas chromatograph (5890 Series II, Hewlett-Packard, The pheromone activity was assayed as described Waldbronn, Germany), at an ionization energy of by R. Fudo et al. [7]. Vegetative cells (1U108/5 Wl) 70 eV and a trap current of 300 WA. The natural were placed on mineral agar medium (3.4 mM and synthetic pheromones were analyzed on a DB- CaCl2, 10 mM KCl, 10 mM NaCl, 1.5% agar) con- 5 capillary column (0.32 mmU30 m, JpW Scienti¢c, taining samples to be tested. The agar plates were Folsom, CA, USA). The column temperature was incubated at 28³C for 24 h in the dark. Cells were then observed under a dissection microscope to con- ¢rm the fruiting body formation. 2.3. Isolation of the pheromone Vegetative cells were resuspended (2U109 cells ml31) in starvation medium (10 mM Tris-HCl, 1 mM K2HPO4, 8 mM MgSO4, 0.2% monosodium glutamate, 0.4% glucose, and 0.1% CaCl2, pH 7.6), and incubated at 30³C with shaking at 140 rpm Fig. 1. Structure of 8-hydroxy-2,5,8-trimethyl-4-nonanone. FEMSLE 8258 30-7-98 Y. Morikawa et al. / FEMS Microbiology Letters 165 (1998) 29^34 31 Downloaded from https://academic.oup.com/femsle/article/165/1/29/623856 by guest on 24 September 2021 Fig. 2. GC-MS analysis of the natural pheromone of Stigmatella aurantiaca, and the synthetic (RS)-1. A: Total ion mass chromatograms of (a) the natural pheromone; and (b) the synthetic (RS)-1. B: Mass spectrum of the natural pheromone. FEMSLE 8258 30-7-98 32 Y. Morikawa et al. / FEMS Microbiology Letters 165 (1998) 29^34 maintained for 2 min at 80³C, then increased at the rate of 4³C min31 to 160³C. Injections were made in the splitless mode at 180³C. For stereochemical analysis, the natural phero- mone and the synthetic pheromones were reduced, and then analyzed by GC-MS. One hundred nano- grams of the pheromones were dissolved in 5 Wl of 25 nM sodium borohydride in ethanol, and incu- bated at room temperature for 30 min. The reaction mixtures were diluted with water and then extracted with diethyl ether. The obtained organic layers were applied to GC-MS analysis using a Chirasil-DEX Downloaded from https://academic.oup.com/femsle/article/165/1/29/623856 by guest on 24 September 2021 CB column (0.25 mmU25 m, Chrompack, Middel- burg, The Netherlands). The chiral column temper- ature was maintained for 2 min at 120³C, then in- Fig. 3. The dose-activity curves of (RS)-1,(R)-1,(S)-1, and the natural pheromone. Natural pheromone (triangle), (R)-1 (open 31 creased at the rate of 4³C min to 180³C. Injections circle), (S)-1 (close circle) and (RS)-1 (square). Error bars repre- were made in the splitless mode at 180³C. sent S.E. (n = 4). 3. Results and discussion mass fragmentation pattern (data not shown) of the synthesized (RS)-1 were indistinguishable from those The pheromone of S. aurantiaca was puri¢ed to of the natural pheromone. Therefore, the structure homogeneity by subjecting the culture medium to the of the pheromone was con¢rmed as being 1. XAD-2 column, chloroform extraction, silica gel col- To determine the absolute structure of the phero- umn, and a series of reversed-phase HPLC. The mone, we ¢rst compared the fruiting body inducing overall yield of the pheromone was approximately activity of (RS)-1,(R)-1,(S)-1, and the natural pher- 1 Wg from 10 l of starvation medium. The UV spec- omone. The dose-response curves of each pheromone trum of the pheromone showed an end-absorption are shown in Fig. 3. The natural pheromone and below 200 nm and a weak absorption around each of the synthetic enantiomers, (R)-1, and (S)-1, 280 nm, which suggests the presence of a ketone. induce fruiting body formation at nearly equal levels. The capillary GC-MS analysis of the puri¢ed natural Racemic (RS)-1 also showed nearly the same activity pheromone revealed a total ion mass chromatogram as either enantiomer. having a single peak (Fig. 2Aa), whose mass spec- The relationship between the stereochemistry and trum showed a peak at m/z 185, which was deduced bioactivity of the pheromones of insects is diverse [9].
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