When glycerol was used to induce myxospore formation in Myxococcus xanthus in the presence of 32Pi, the label was incorporated into a variety of acid-soluble compounds. Incorporation into ribonucleotides was approximately fivefold greater than in vegetative cells or noninducible mutants grown in glycerol. The label was also incorporated into some unknown compounds and material tentatively identified as guanosine tetraphosphate. Marked accumulation into polyphosphates, which were present mainly

more »

... n culture supernatants, occurred relatively late during myxospore formation. The kinetics of accumulation of some of these compounds and their distribution into acid-soluble cell extracts and culture supernatants are described and compared with those in vegetative cells and noninducible mutants. The biology of Myxococcus xanthus, a gramnegative, gliding bacterium, has been the subject of several recent reviews (33, 34). Under starvation conditions on solid medium, the cells aggregate to form raised mounds or fruiting bodies within which the long slender cells undertake morphogenetic change to form spherical myxospores. The asynchronous formation of fruiting bodies is generally complete within 4 to 5 days at 32°C. The study of events concerned with myxospore formation has received impetus from the discovery that glycerol is able to induce spore formation in liquid medium (9). If the medium is made 0.5 M with glycerol, logarithmic-phase cells quantitatively and synchronously differentiate into myxospores in the absence of fruiting body formation. Cells, morphologically indistinguishable from mature myxospores, are formed within 2 h after addition of glycerol (9), although some metabolism continues for an additional 6 h in sporulating cultures (22). During the first 2 h after glycerol addition, numerous metabolic changes take place. The glyoxalate cycle enzymes, isocitratase and malate synthetase, are induced (33), as are the enzymes required for formation and incorporation of N-acetylgalactosamine into spore coats (11) and a variety of other enzymes (34). Changes in synthesis of RNA and DNA also accompany the sporulation process (34). The alterations in biosynthetic events suggest that regulatory mechanisms must be involved. Since the discovery of guanosine tetraphosphate (6), several laboratories have reported the presence of novel phosphorylated compounds in a variety of organisms (12, 17, 19, 23, 24) . The time of appearance of these compounds indicates they may participate in regulatory functions. Originally, this work was intended to look for analogous compounds that may appear during myxospore formation in M. xanthus. Because of the appearance of many compounds, this report represents a survey of the acid-soluble, phosphorylated compounds that appear in cell extracts and supernatant fractions of M. xanthus cultures induced to form myxospores in liquid medium with glycerol. MATERIALS AND METHODS Organism. M. xanthus MD-1 was obtained from M. Dworkin (University of Minnesota, Minneapolis). A tan isolate, competent in fruiting body formation, was used throughout. Five independently isolated spontaneous mutants, unable to sporulate in 0.5 M glycerol, were selected by the method of Burchard and Parish (4) and used in certain experiments. Growth. The growth medium consisted of 1% (wt/vol) Casitone (Difco) containing 10.0 mM MgSO4. The phosphate concentration in this medium, as determined by the method of Ames (1), was 0.5 mM. Cells were routinely grown in 40 ml of the medium in 250-ml Erlenmeyer flasks, which were incubated at 32°C in a gyratory water bath shaker (New Brunswick Scientific Co.) set at 160 rpm. When smaller volumes were required, 5 to 10 ml of the culture was incubated in 50-ml Erlenmeyer flasks under the same conditions except the flasks were tilted at a 300 angle. For myxospore formation, logarithmic-phase cells were centrifuged at 12,000 x g for 10 min at 4°C. The cell pellet was dispersed to the same density in fresh medium and allowed to resume growth at 32°C with shaking. After 60 min, sterile glycerol (6.8 M) was