Absolute Chemical Structure of the Myxobacterial Pheromone Of

Home , Myxobacteria, Stigmatella aurantiaca

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

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 fraction 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.

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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 pheromone 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 incubated 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 temperature 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]. to be (M-CH3) , and a peak at m/z 182, which was In the case of japanilure, the sex pheromone of the deduced to be (M-H2O) (Fig. 2B). In addition, all Japanese beetle, only one enantiomer of the phero- of the fragmentation peaks coincided with the pro- mone is active; the opposite enantiomer is inhibi- posed structure of 8-hydroxy-2,5,8-trimethyl-4-nona- tory. In the case of the pheromone of the ambrosia none (1) (Fig. 1) [8]. beetle both enantiomers of the pheromone must be We synthesized racemic (RS)-1, and optically ac- present for optimal activity. In the case of Stigma- tive (R)-1 and (S)-1, to con¢rm the structure of the tella's pheromone each of the enantiomers showed pheromone, and to determine its absolute con¢gura- equal levels of activity, and the combination e¡ect tion. The details of their synthesis will be reported of both enantiomers was neither inhibitory nor syn- elsewhere by K. Mori and M. Takenaka (in prepa- ergistic, but merely additive. We therefore could not ration). determine the absolute con¢guration of the natural We ¢rst compared the results of the GC-MS anal- pheromone from its biological activity. ysis of the natural pheromone and those of the syn- Next, we tried to determine the absolute con¢gu- thesized (RS)-1. The retention time (Fig. 2Ab) and ration of the natural pheromone by GC-MS using

FEMSLE 8258 30-7-98 Y. Morikawa et al. / FEMS Microbiology Letters 165 (1998) 29^34 33

Some bacteria produce their own extracellular signals to communicate and to form multicellular asso- ciations. Butyrolactones in actinomycetes [10] and N- acyl homoserine lactones in Gram-negative bacteria [11] are extracellular signals which have been extensively studied. The pheromone produced by S. aurantiaca has a unique structure and is a new type of communication signal. Further studies on this pheromone will provide us with further insight into the multicellular development of S. aurantiaca and bacterial communication. Downloaded from https://academic.oup.com/femsle/article/165/1/29/623856 by guest on 24 September 2021

Acknowledgments

We thank Dr. Shingo Marumo, Professor Emeri- tus of Nagoya University, for helpful suggestions. This work was supported by a Grant-in-Aid for Ex- Fig. 4. Mass chromatograms of (a) the reduced form of (S)-1; ploratory Research (No. 09876094) from the Minis- (b) the reduced form of (R)-1; and (c) the reduced form of the natural pheromone. try of Education, Science, Sports and Culture of Ja- pan. chiral capillary columns. We attempted to separate the synthetic R- and S-enantiomers directly using References some commercially available chiral columns, but these were unsuccessful (data not shown). We were [1] Dworkin, M. (1996) Recent advances in the social and devel- able to separate the synthetic R- and S-enantiomers opmental biology of the myxobacteria. Microbiol. Rev. 60, by reducing the ketone with sodium borohydride 70^102. (Fig. 4a,b). Both R- and S-enantiomers showed [2] Dworkin, M. and Kaiser, D. (1985) Cell interactions in myx- two peaks, which could be the threo and the erythro obacterial growth and development. Science 230, 18^24. [3] Schairer, H.U. (1993) Stigmatella aurantiaca, an organism for diastereomers generated by the reduction. The mass studying the genetic determination of morphogenesis. chromatogram of the reduced form of the R-enan- In: Myxobacteria II (Dworkin, M. and Kaiser, D., Eds.), tiomer showed peaks at 13.18 min and 13.30 min, pp. 333^346. American Society for Microbiology, Washing- while that of the S-enantiomer showed peaks at ton, DC. 13.20 min and 13.48 min. The reduced form of the [4] Stephens, K., Hegeman, G.C. and White, D. (1982) Phero- mone produced by the myxobacterium Stigmatella aurantiaca. natural pheromone showed three peaks (Fig. 4c) J. Bacteriol. 149, 739^747. which were the same as those of (RS)-racemate [5] Morikawa, Y., Takayama, S., Che, F.S., Fudo, R., Yamana- (data not shown). Those data suggested that the nat- ka, S. and Isogai, A. (1997) Photomorphogenesis of myxobac- ural pheromone exists in an enantiomeric mixture. terium Stigmatella aurantiaca. Abstracts of 5th International We puri¢ed the natural pheromone under mild Congress of Plant Molecular Biology, Singapore, Abstract 500. conditions without using acids or bases in puri¢ca- [6] Stephens, K. and White, D. (1980) Morphogenetic e¡ects of tion solvents. In fact, when we applied the same light and guanine derivatives on the fruiting myxobacterium puri¢cation procedure to each of the synthetic enan- Stigmatella aurantiaca. J. Bacteriol. 144, 322^326. tiomers, the absolute con¢guration of each enan- [7] Fudo, R., Ando, T., Sato, S., Kameyama, T. and Yamanaka, tiomer did not change (data not shown). We, there- S. (1997) Isolation of isoeugenitin as a fruiting body inducer for Stigmatella aurantiaca from a soil fungus Papulaspora sp. fore, concluded that the natural pheromone Biosci. Biotech. Biochem. 61, 183^184. produced in our experiment is truly a mixture of [8] Hull, W.E., Berkessel, A., Stamm, I. and Plaga, W. (1997) both enantiomers. Intercellular signaling in Stigmatella aurantiaca: Proof, puri¢-

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cation and structure of myxobacterial pheromone. Abstracts [10] Horinouchi, S. and Beppu, T. (1992) Autoregulatory factors of the 24th International Conference on the Biology of the and communication in actinomycetes. Annu. Rev. Microbiol. Myxobacteria, New Braunfels, TX, p. 25. 46, 377^398. [9] Mori, K. (1997) Pheromones: synthesis and bioactivity. [11] Gray, K.M. (1997) Intercellular communication and group Chem. Commun. 1153^1158. behavior in bacteria. Trends Microbiol. 5, 184^188. Downloaded from https://academic.oup.com/femsle/article/165/1/29/623856 by guest on 24 September 2021

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