Absolute chemical structure of the myxobacterial pheromone of Stigmatella aurantiaca that induces the formation of its fruiting body

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.

Myxobacterium, Stigmatella aurantiaca, Fruiting body, Pheromone, Absolute structure

1 Introduction

Myxobacteria are a class of Gram-negative bacteria which show a social behavior and complex developmental cycle [1, 2]. In response to starvation, they aggregate to form characteristic multicellular fruiting bodies. Among myxobacteria, Stigmatella aurantiaca has the most elaborate fruiting body structure, and requires light for normal development [3]. The fruiting body formation process of S. aurantiaca may be regarded as a model of photomorphogenesis.

In addition to environmental factors, an extracellular signaling molecule, or pheromone, is known to be involved in the developmental cycle of S. aurantiaca[4]. This pheromone is a small lipophilic compound secreted by S. aurantiaca under nutrient-deficient conditions. Production of this pheromone in S. aurantiaca is extensively promoted by light [5]. Furthermore, exogenous addition of this pheromone to a culture of S. aurantiaca in the dark promotes fruiting body formation [4]. Guanosine nucleotides [6], and isoeugenitin, a fungal metabolite [7], have also been reported to induce fruiting body formation in S. aurantiaca in the dark. Studying these exogenous signals will elucidate the signaling cascade in this photomorphogenesis.

W.E. Hull et al. [8] recently isolated the pheromone and determined that its structure is 8-hydroxy-2,5,8-trimethyl-4-nonanone (1) (Fig. 1). However, the absolute configuration of this compound, which has an asymmetric carbon atom at C-5, has not yet been determined. In this study, we have purified the natural pheromone to homogeneity by normal and reversed phase chromatography, and have elucidated some of its chemical properties. We also synthesized both enantiomers of the pheromone stereoselectively. We report the bioactivity of the synthesized enantiomers, as well as the absolute configuration of the natural pheromone.

1

Structure of 8-hydroxy-2,5,8-trimethyl-4-nonanone.

2 Materials and methods

2.1Bacterial strain and growth

S. aurantiaca DW4 (ATCC 33878) were grown in Casitone medium (1% Casitone (Difco Laboratories, Detroit, MI, USA), 8 mM MgSO₄), at 30°C and 140 rpm. Vegetative cells were harvested during the mid-log phase by centrifugation at 3000×g for 10 min.

2.2Assay of pheromone activity

The pheromone activity was assayed as described by R. Fudo et al. [7]. Vegetative cells (1×10⁸/5 μl) were placed on mineral agar medium (3.4 mM CaCl₂, 10 mM KCl, 10 mM NaCl, 1.5% agar) containing samples to be tested. The agar plates were incubated at 28°C for 24 h in the dark. Cells were then observed under a dissection microscope to confirm the fruiting body formation.

2.3Isolation of the pheromone

Vegetative cells were resuspended (2×10⁹ cells ml⁻¹) 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 30°C with shaking at 140 rpm under the light for 24 h. The culture supernatant obtained by the centrifugation (5000×g, 30 min) was applied onto a column of XAD-2 resin (Organo, Tokyo, Japan). The column was washed with water and 20% methanol and then eluted with 80% methanol. After the removal of methanol by evaporation, the pheromone in the residual water layer was extracted with chloroform. The chloroform extract was concentrated by evaporation and applied onto a silica gel column, and the column was eluted with 20% ethyl acetate in chloroform. The active fraction was concentrated by evaporation and further purified by the following four steps of reversed-phase HPLC, using a 626 HPLC system equipped with 996 Diode Array UV Detector (Waters, Milford, MA, USA). The pheromone-containing fraction was sequentially applied onto an ODS column (Capcell Pak C18, UG120, Shiseido, Tokyo, Japan), SymmetryShield RP8 column (Waters), and a CN column (Capcell Pak CN, UG120, Shiseido). Finally, the active fraction was applied to a phenyl column (Capcell Pak Phenyl, UG 120, Shiseido) and eluted with 45% acetonitrile in water. The pheromone was eluted as a symmetrical peak when it was monitored by 195 nm UV absorption.

2.4Structural analysis of the pheromone

Gas chromatography-mass spectrometry (GC-MS) analysis was done on a mass spectrometer (JMS-700 MStation, JEOL, Tokyo, Japan), equipped with a gas chromatograph (5890 Series II, Hewlett-Packard, Waldbronn, Germany), at an ionization energy of 70 eV and a trap current of 300 μA. The natural and synthetic pheromones were analyzed on a DB-5 capillary column (0.32 mm×30 m, J&W Scientific, Folsom, CA, USA). The column temperature was maintained for 2 min at 80°C, then increased at the rate of 4°C min⁻¹ to 160°C. Injections were made in the splitless mode at 180°C.

For stereochemical analysis, the natural pheromone and the synthetic pheromones were reduced, and then analyzed by GC-MS. One hundred nanograms of the pheromones were dissolved in 5 μl 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 CB column (0.25 mm×25 m, Chrompack, Middelburg, The Netherlands). The chiral column temperature was maintained for 2 min at 120°C, then increased at the rate of 4°C min⁻¹ to 180°C. Injections were made in the splitless mode at 180°C.

3 Results and discussion

The pheromone of S. aurantiaca was purified to homogeneity by subjecting the culture medium to the XAD-2 column, chloroform extraction, silica gel column, and a series of reversed-phase HPLC. The overall yield of the pheromone was approximately 1 μg from 10 l of starvation medium. The UV spectrum of the pheromone showed an end-absorption below 200 nm and a weak absorption around 280 nm, which suggests the presence of a ketone. The capillary GC-MS analysis of the purified natural pheromone revealed a total ion mass chromatogram having a single peak (Fig. 2 Aa), whose mass spectrum showed a peak at m/z 185, which was deduced to be (M-CH₃)⁺, and a peak at m/z 182, which was deduced to be (M-H₂O)⁺ (Fig. 2B). In addition, all of the fragmentation peaks coincided with the proposed structure of 8-hydroxy-2,5,8-trimethyl-4-nonanone (1) (Fig. 1) [8].

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.

We synthesized racemic (RS)-1, and optically active (R)-1 and (S)-1, to confirm the structure of the pheromone, and to determine its absolute configuration. The details of their synthesis will be reported elsewhere by K. Mori and M. Takenaka (in preparation).

We first compared the results of the GC-MS analysis of the natural pheromone and those of the synthesized (RS)-1. The retention time (Fig. 2 Ab) and mass fragmentation pattern (data not shown) of the synthesized (RS)-1 were indistinguishable from those of the natural pheromone. Therefore, the structure of the pheromone was confirmed as being 1.

To determine the absolute structure of the pheromone, we first compared the fruiting body inducing activity of (RS)-1, (R)-1, (S)-1, and the natural pheromone. The dose-response curves of each pheromone are shown in Fig. 3. The natural pheromone and each of the synthetic enantiomers, (R)-1, and (S)-1, induce fruiting body formation at nearly equal levels. Racemic (RS)-1 also showed nearly the same activity as either enantiomer.

3

The dose-activity curves of (RS)-1, (R)-1, (S)-1, and the natural pheromone. Natural pheromone (triangle), (R)-1 (open circle), (S)-1 (close circle) and (RS)-1 (square). Error bars represent S.E. (n= 4).

The relationship between the stereochemistry and bioactivity of the pheromones of insects is diverse [9]. In the case of japanilure, the sex pheromone of the Japanese beetle, only one enantiomer of the pheromone is active; the opposite enantiomer is inhibitory. In the case of the pheromone of the ambrosia beetle both enantiomers of the pheromone must be present for optimal activity. In the case of Stigmatella's pheromone each of the enantiomers showed equal levels of activity, and the combination effect of both enantiomers was neither inhibitory nor synergistic, but merely additive. We therefore could not determine the absolute configuration of the natural pheromone from its biological activity.

Next, we tried to determine the absolute configuration of the natural pheromone by GC-MS using chiral capillary columns. We attempted to separate the synthetic R- and S-enantiomers directly using some commercially available chiral columns, but these were unsuccessful (data not shown). We were able to separate the synthetic R- and S-enantiomers by reducing the ketone with sodium borohydride (Fig. 4a,b). Both R- and S-enantiomers showed two peaks, which could be the threo and the erythro diastereomers generated by the reduction. The mass chromatogram of the reduced form of the R-enantiomer showed peaks at 13.18 min and 13.30 min, while that of the S-enantiomer showed peaks at 13.20 min and 13.48 min. The reduced form of the natural pheromone showed three peaks (Fig. 4c) which were the same as those of (RS)-racemate (data not shown). Those data suggested that the natural pheromone exists in an enantiomeric mixture.

4

Mass chromatograms of (a) the reduced form of (S)-1; (b) the reduced form of (R)-1; and (c) the reduced form of the natural pheromone.

We purified the natural pheromone under mild conditions without using acids or bases in purification solvents. In fact, when we applied the same purification procedure to each of the synthetic enantiomers, the absolute configuration of each enantiomer did not change (data not shown). We, therefore, concluded that the natural pheromone produced in our experiment is truly a mixture of both enantiomers.

Some bacteria produce their own extracellular signals to communicate and to form multicellular associations. 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.

Acknowledgements

We thank Dr. Shingo Marumo, Professor Emeritus of Nagoya University, for helpful suggestions. This work was supported by a Grant-in-Aid for Exploratory Research (No. 09876094) from the Ministry of Education, Science, Sports and Culture of Japan.