Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11263–11267, September 1998 Developmental Biology Intercellular signaling in Stigmatella aurantiaca: Purification and characterization of stigmolone, a myxobacterial pheromone (microbial developmentydifferentiationyfruiting bodies) WULF PLAGA†,IRMELA STAMM, AND HANS ULRICH SCHAIRER Zentrum fu¨r Molekulare Biologie der Universita¨tHeidelberg (ZMBH), Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany Communicated by A. Dale Kaiser, Stanford University School of Medicine, Stanford, CA, July 27, 1998 (received for review May 1, 1998) ABSTRACT The myxobacterium Stigmatella aurantiaca named stigmolone, which represents a novel type of prokary- passes through a life cycle that involves formation of a otic signal molecule required for the developmental cycle of S. multicellular fruiting body as the most complex stage. An aurantiaca. The structure of this compound, 2,5,8-trimethyl- early step in this differentiation process depends on a signal 8-hydroxy-nonan-4-one, was determined by NMR and mass factor secreted by the cells when nutrients become limited. spectrometry (5). The formation of a fruiting body from a small cell population can be accelerated by addition of this secreted material. The MATERIALS AND METHODS bioactive compound was found to be steam volatile. It was purified to homogeneity by steam distillation followed by Chemicals. Solvents used for HPLC were: LiChrosolv water reversed-phase and normal-phase HPLC. The pheromone was (Merck), acetonitrile (ultragradient grade, J. T. Baker), named stigmolone, in accordance with the structure 2,5,8- methyl acetate (Fluka), and cyclopentane (Aldrich). trimethyl-8-hydroxy-nonan-4-one, as determined by NMR Bacterial Strains and Growth Conditions. S. aurantpaca and mass spectrometry. Stigmolone represents a structurally DW4y3–1 (6) was grown at pH 7.2 and 32°C in 1% bacto unique and highly bioactive prokaryotic pheromone that is tryptone (Difco), supplemented with 8 mM MgSO4 and 120 y effective in the bioassay at 1 nM concentration. mg liter streptomycin sulfate. Cultures were aerated by gyra- tion (up to 800 ml) or grown in a 10-liter fermenter (ISF 200, p Stigmatella aurantiaca is a myxobacterium, which lives in Infors AG, Bottmingen, Switzerland) at 95% O2 and constant pH. Myxococcus xanthus DK1622 (7) was grown in CTT swarms on solid surfaces. As is typical for myxobacteria, this medium (1% casitone, 8 mM MgSO , 10 mM TriszHCl, 1 mM organism survives periods of starvation by undergoing a 4 potassium phosphate, pH 7.6) (8) at 32°C. developmental process in which the individuals of a swarm Bioassay. The assay of pheromone activity was performed aggregate to form multicellular fruiting bodies. These species- according to Stephens et al. (4). A 200-ml culture of S. specific fruiting bodies harbor the myxospores, which are aurantiaca (4 3 1010 cells) was harvested by centrifugation (15 dormant cells. The morphologically complex fruiting body of min., 4,200 g, 4°C), washed at 4°C with 200 ml of 10 mM CaCl S. aurantiaca has a treelike structure, and spores are packaged 2 in 100 mM HepesyNaOH, pH 7.2, and finally suspended in this in sporangioles located on the top of the structure. When the buffer to a density of 1 3 1010 cellsyml. Samples of 1-ml volume supply of nutrients increases (which also may occur after containing the fractions to be tested were supplemented with dissemination of the sporangioles), the myxospores of a spo- y 1.5% agar (Difco), 7.8 mM CaCl2, 10 mM Hepes NaOH, pH rangiole germinate to build up a new swarm of vegetative cells. 7.2, and placed in wells of a 24-well tissue culture plate Under favorable conditions the growth of the swarm is based (Greiner, Nu¨rtingen, Germany) at 60°C. The solidified agar on transverse binary fission of the cells (1–3). was dried for 20 min in a hood, and the cell suspension was The switch from the socially living single cells to the spotted in portions of 5 ml(53 107 cells) onto the agar. After multicellular fruiting body, which contains a variety of differ- the spots had dried, the covered plates were incubated at 32°C entiated cells, is a prokaryotic paradigm for differentiation in the dark. The appearance of aggregates and fruiting bodies processes and signal transduction. To build up the fruiting was monitored after 10–20 hr. body as an elaborate and spatially complex multicellular The assay conditions for M. xanthus DK1622 were adapted structure, the cells must exchange signals during the entire as follows. Cells of a culture (4 3 107 cellsyml) were harvested process. For the related organism Myxococcus xanthus several and washed as described above. The final suspension was of the genes and developmental signals involved in fruiting adjusted to 8 3 108 cellsyml and spotted in portions of 5 ml onto body formation have been characterized, as summarized in a the agar containing up to 0.2 mM Stigmatella pheromone. The review (1). appearance of aggregates and fruiting bodies was monitored In 1982 Stephens et al. (4) demonstrated a pheromone after 12–24 hr. activity in an aqueous eluate obtained from filter paper on Dialysis Experiment. A dialysis membrane (from a Visking which S. aurantiaca had formed fruiting bodies. This activity dialysis tube, exclusion limit 8,000–15,000 dalton) was also was found in chloroform extracts of the eluate, but mounted in a microdialysis apparatus (model 1200MD, Be- purification of active substances was not performed. In this thesda Research Laboratories) with 6.8 mM CaCl2 in buffer paper we demonstrate that specific signal(s) trigger the aggre- wells on the lower side of the membrane and air on the upper gation of cells, a step that is essential for the initiation of the side. Aliquots of 3 3 108 cells (5 ml) per well were placed on differentiation process for S. aurantiaca. We describe the the membrane and dried. Intensive dialysis was achieved by purification of a hydroxy ketone with the formula C12H24O2, pumping the calcium chloride solution through the buffer wells; the content of the wells was mixed by permanent stirring. The publication costs of this article were defrayed in part by page charge Dialysis was performed at 32°C under fluorescent light (ca. 500 payment. This article must therefore be hereby marked ‘‘advertisement’’ in lx). The possible influence of light intensity was not investi- accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9511263-5$2.00y0 †To whom reprint requests should be addressed. e-mail: wplaga@ PNAS is available online at www.pnas.org. sun0.urz.uni-heidelberg.de. 11263 Downloaded by guest on September 28, 2021 11264 Developmental Biology: Plaga et al. Proc. Natl. Acad. Sci. USA 95 (1998) gated in this study, but see ref. 9 for effects of light on tial role of secreted extracellular factor(s) for aggregation and Stigmatella development. the differentiation process under starvation conditions. S. Large-Scale Production of the Pheromone. Cells of a 10-liter aurantiaca cells were placed on a dialysis membrane (Fig. 1), 3 12 culture (2 10 cells) were harvested by tangential filtration and during dialysis against 6.8 mM CaCl2 (starvation), no using a Pellicon 0.45-mm cassette (Millipore) and centrifuga- aggregation took place. If dialysis was stopped after 24 hr, tion (4,200 g, 4°C). The cells were washed at 4°C with 700 ml aggregates began to form. In this case the time required for y of 10 mM CaCl2 in 100 mM Hepes NaOH, pH 7.2, and finally aggregation was shorter than in a control experiment per- suspended in ca. 50 ml buffer to a density of 4 3 1010 cells per formed without dialysis pretreatment. This difference is most ml. This suspension was spotted in 5-ml portions on 7.5 3 30 likely caused by the time lag observed for controls between the cm filter paper strips (Whatman 3MM) moistened with 6.8 mM initiation of starvation conditions and the beginning of pher- y 2 CaCl2 at a density of about 1.8 spots cm . The paper strips omone secretion (see below and Fig. 2). were eluted in a chamber for descending paper chromatogra- Large-Scale Production of the Pheromone. Several attempts phy under white fluorescent light (ca. 500 lx) using 6.8 mM to obtain pheromone produced in suspension cultures of S. CaCl2 as descending solvent. The eluate from the strips was aurantiaca cells were undertaken, but all failed. The secretion collected over a period of 130 hr (about 3 liters from 2 3 1012 of the pheromone may depend on the contact of the cells with cells) and stored at 220°C. a solid surface or with each other. Therefore, we took advan- Steam Distillation. The paper eluate (1.7 liters) was heated tage of the fact that S. aurantiaca cells stick tightly to filter in a two-liter flask, and steam from an external generator was paper. In a conventional paper chromatography chamber cells blown through the sample. The condensed distillate (1.7 l) was on strips of filter paper remained in place even with a 2 stored at 20°C. Up to 22 liters of such distillates were pumped descending flow of 6.8 mM CaCl2 through the strips while all through a glass column (2.5-cm diameter) containing 10 g of soluble, secreted molecules were eluted continuously from the C18 solid-phase extraction resin (Chromabond C18, Macherey paper. The time course for the appearance of pheromone & Nagel). The bound pheromone was recovered in 14 ml by activity in the eluate is given in Fig. 2. During the first 15 hr elution with 60% CH3CN.