Inhibition of cell-cell interactions in Myxococcus xanthus by congo red

The function of molecules associated with the cell surface may be determined by examining the phenotype of cells treated with inhibitors specific to these cell surface molecules. This strategy was used to examine the function of the major Congo red receptor of the myxobacterium Myxococcus xanthus, which has a developmental cycle that involves social interactions among cells. A class of social motility mutations (A+ S-), known as dsp, may inhibit the same subcellular component as Congo red because the phenotype of wild-type cells which had been treated with Congo red resembled in several ways the phenotype of the Dsp mutants. First, Congo red inhibited agglutination of wild-type cells, whereas Dsp cells were incapable of agglutinating, even in the absence of Congo red. Second, Congo red inhibited fruiting body formation by wild-type cells and reduced the yield of myxospores. Untreated Dsp cells were unable to form fruiting bodies and produced few myxospores. Third, Congo red reduced the rate of wild-type gliding motility to a level comparable to that of untreated Dsp cells, but did not inhibit the A motility of Dsp cells. Finally, binding studies showed that Dsp cells lacked the major Congo red receptor. Wild-type cells bound Congo red with an apparent association constant of 2.4 X 10(5) M-1, while Dsp cells bound it with an apparent association constant of 8.5 X 10(3) M-1. Binding of Congo red to wild-type cells was saturated in less than 10 min and was reversible when excess Congo red was removed. These results suggest that the Congo red receptors are controlled by the S motility system and that these receptors are involved in cell cohesion, social motility, and fruiting body formation.     

Mutants of Myxococcus xanthus insensitive to glycerol-induced myxospore formation.

Mutants of Myxococcus xanthus FBt unable to form myxospores in response to 0.5 M glycerol arise spontaneously with a frequency of 1--3 X 10(-5). These mutants are designated glc. Ultraviolet mutagenesis increases the frequency to a maximum of 7% of the survivors. The reversion frequency following ultraviolet irradiation of spontaneous glc mutants is less than 10(-3). Of four glc mutants examined, none form myxospores in response to the alternative inducers, ethylene glycol and dimethyl sulphoxide. One glc mutant is induced by 1.5 M glycerol; strain FBt responds to this glycerol concentration with low efficiency myxospore formation. Strain FBt and glc mutants all produce myxospores with low efficiency in response to phenyl ethanol. Of 117 glc mutants tested, 109 form fruiting bodies containing mature myxospores; thus, mutations to the glc phenotype do not normally block myxospore formation within the fruiting cycle of the organism.     

Isolated fibrils rescue cohesion and development in the Dsp mutant of Myxococcus xanthus.

Extracellular fibrils are involved in cell cohesion and cell development in Myxococcus xanthus. One group of social motility mutants, Dsp, is unable to produce extracellular fibrils; these mutants also lose the abilities to cohere and to develop. Extracellular fibrils isolated from vegetative wild-type cells and added to Dsp cells fully restored the abilities of these cells to cohere and to undergo normal morphological development. The fibrils thus mimic the ability of intact, wild-type cells to carry out the same rescue. Optimal cohesion rescue by fibrils required calcium and magnesium ions, did not require protein synthesis, but was energy dependent, i.e., sodium azide and sodium cyanide blocked rescue. Cohesion rescue was also blocked by the diazo dye Congo red. Cohesion rescue is genus specific, i.e., isolated fibrils did not cause the cohesion of Pseudomonas aeruginosa, Bacillus subtilis, Proteus mirabilis, Escherichia coli, or the related myxobacterium Stigmatella aurantiaca. Developmental rescue of Dsp by isolated fibrils included aggregation, fruiting body formation, and myxospore morphogenesis. Developmental gene expression in the Dsp mutant was only partially rescued by the isolated fibrils.     

The asgE locus is required for cell-cell signalling during Myxococcus xanthus development

In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell-cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor.     

Mutants of Myxococcus xanthus insensitive to glycerol-induced myxospore formation

Mutants of Myxococcus xanthus FB_t unable to form myxospores in response to 0.5 M glycerol arise spontaneously with a frequency of 1–3×10^–5. These mutants are designated glc. Ultraviolet mutagenesis increases the frequency to a maximum of 7% of the survivors. The reversion frequency following ultraviolet irradiation of spontaneous glc mutants is less than 10^–3. Of four glc mutants examined, none form myxospores in response to the alternative inducers, ethylene glycol and dimethyl sulphoxide. One glc mutant is induced by 1.5 M glycerol; strain FB_t responds to this glycerol concentration with low efficiency myxospore formation. Strain FB_t and glc mutants all produce myxospores with low efficiency in response to phenyl ethanol. Of 117 glc mutants tested, 109 form fruiting bodies containing mature myxospores; thus, mutations to the glc phenotype do not normally block myxospore formation within the fruiting cycle of the organism.     

Sporulation timing in Myxococcus xanthus is controlled by the espAB locus

The fruiting body development of Myxococcus xanthus consists of two separate but interacting pathways: one for aggregation of many cells to form raised mounds and the other for sporulation of individual cells into myxospores. Sporulation of individual cells normally occurs after mound formation, and is delayed at least 30 h after starvation under our laboratory conditions. This suggests that M. xanthus has a mechanism that monitors progress towards aggregation prior to triggering sporulation. A null mutation in a newly identified gene, espA (early sporulation), causes sporulation to occur much earlier compared with the wild type (16 h earlier). In contrast, a null mutation in an adjacent gene, espB, delays sporulation by about 16 h compared with the wild type. Interestingly, it appears that the espA mutant does not require raised mounds for sporulation. Many mutant cells sporulate outside the fruiting bodies. In addition, the mutant can sporulate, without aggregation into raised mounds, under some conditions in which cells normally do not form fruiting bodies. Based on these observations, it is hypothesized that EspA functions as an inhibitor of sporulation during early fruiting body development while cells are aggregating into raised mounds. The aggregation-independent sporulation of the espA mutant still requires starvation and high cell density. The espA and espB genes are expressed as an operon and their translations appear to be coupled. Expression occurs only under developmental conditions and does not occur during vegetative growth or during glycerol-induced sporulation. Sequence analysis of EspA indicates that it is a histidine protein kinase with a fork head-associated (FHA) domain at the N-terminus and a receiver domain at the C-terminus. This suggests that EspA is part of a two-component signal transduction system that regulates the timing of sporulation initiation.     

Isolation of Physarum polycephalum plasmodial mutants altered in sporulation by chemical mutagenesis of flagellates

Plasmodia are giant, multinucleate single cells which develop from mononucleate amoebae during the developmental cycle of Physarum polycephalum. In visible light, starving plasmodia lose their unlimited replicative potential and terminally differentiate into fruiting bodies (sporulation). Aiming at genetic dissection of the circuits controlling commitment and differentiation, we worked out a standardized procedure for the generation and screening of plasmodial mutants altered in sporulation by mutagenesis with ethylnitrosourea. To obtain a homogeneous population of cells of those strains which cannot grow axenically, we describe a protocol for preparing a suspension of flagellates to be used as starting material for mutagenesis. Flagellates can transform into plasmodia via the amoebal stage. Pilot phenotypic screening yielded plasmodial mutants altered in the photocontrol of sporulation or with disturbed developmental program. The existence of mutants with a disturbed developmental program indicates that the sequence and synchrony of morphogenetic steps of fruiting body formation can be uncoupled through mutation. Complementation testing by plasmodial fusion identified three complementation groups of non-sporulating mutants. The work described provides an experimental basis for performing mass screens for Physarum mutants altered in sporulation.     

The Myxococcus xanthus lipopolysaccharide O-antigen is required for social motility and multicellular development

The gliding bacterium Myxococcus xanthus aggregates to form spore-filled fruiting bodies when nutrients are limiting. Defective fruiting-body formation and sporulation result from mutations in the sasA locus, which encodes the wzm wzt wbgA (formerly rfbABC ) lipopolysaccharide (LPS) O-antigen biosynthesis genes. Mutants carrying these same sasA mutations are defective in social motility and form small glossy colonies. We report here that the developmental and motility phenotypes of four mutants each containing different Tn 5 insertions in LPS O-antigen biosynthesis genes are similar to those of the original sasA locus mutants. All of the LPS O-antigen mutants tested exhibited defective developmental aggregation and sporulated at only 0.02–15% of the wild-type level. In addition, all of the LPS O-antigen mutants were determined by genetic analyses to be wild type for adventurous motility and defective in social motility, indicating that the LPS O-antigen is necessary for normal development and social motility. The two previously identified cell-surface components required for social motility, type IV pili and the protein-associated polysaccharide material termed fibrils, were detected on the surfaces of all of the LPS O-antigen mutants. This indicates that LPS O-antigen is a third cell-surface component required for social motility.     

Defects in motility and development of Myxococcus xanthus lipopolysaccharide mutants.

Five transposon Tn5 mutants of the procaryote Myxococcus xanthus had been shown previously to be defective in lipopolysaccharide biosynthesis (J. M. Fink,-M. Kalos, and J. F. Zissler, J. Bacteriol. 171:2033-2041, 1989). These mutants were studied for possible defects in gliding motility and multicellular development. Wild-type M. xanthus cells glide both as single cells and as groups of cells. We found that the Tn5 lipopolysaccharide O-antigen mutants were defective in single-cell motility but were unaltered in group motility. These mutant strains were slow to develop but eventually gave rise to normal, spore-filled fruiting bodies. We also had shown previously that 56 (ethyl methanesulfonate-induced and spontaneous) phage-resistant mutants were defective in lipopolysaccharide biosynthesis. We found that many of these lipopolysaccharide O-antigen mutants were defective in single-cell motility but were unaltered in group motility. These mutants also gave rise to normal, spore-filled fruiting bodies. We also studied several phage-resistant mutants which were lacking a side-chain carbohydrate on the lipopolysaccharide core. These mutants possessed both single-cell motility and group motility but were altered in the magnitude of gliding. These mutants were blocked early in development and could not form multicellular fruiting bodies. Several of the mutations in the developmentally aberrant strains were mapped to a single locus by using a collection of genetically linked transposons as genetic markers.     

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB^A116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB^A116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB^A116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.     

Mutants of Myxococcus xanthus dsp defective in fibril binding.

The dsp mutant of Myxococcus xanthus lacks extracellular fibrils and as a result is unable to undergo cohesion, group motility, or development (J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5765-5770, 1983; J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5771-5777, 1983; R. M. Behmlander and M. Dworkin, J. Bacteriol. 173:7810-7821, 1991; L. J. Shimkets, J. Bacteriol. 166:837-841, 1986; L. J. Shimkets, J. Bacteriol. 166:842-848, 1986). However, cohesion and development can be phenotypically restored by the addition of isolated fibrils (R. M. Behmlander, Ph.D. thesis, University of Minnesota, Minneapolis, 1994; B.-Y. Chang and M. Dworkin, J. Bacteriol. 176:7190-7196, 1994). As part of our attempts to examine the interaction of fibrils and cells of M. xanthus, we have isolated a series of secondary mutants of M. xanthus dsp in which cohesion, unlike that of the parent strain, could not be rescued by the addition of isolated fibrils. Cells of M. xanthus dsp were mutagenized either by ethyl methanesulfonate or by Tn5 insertions. Mutagenized cultures were enriched by selection of those cells that could not be rescued, i.e., that failed to cohere in the presence of isolated fibrils. Seven mutants of M. xanthus dsp, designated fbd mutants, were isolated from 6,983 colonies; these represent putative fibril receptor-minus mutants. The fbd mutants, like the parent dsp mutant, still lacked fibrils, but displayed a number of unexpected properties. They regained group motility and the ability to aggregate but not the ability to form mature fruiting bodies. In addition, they partially regained the ability to form myxospores. The fbd mutant was backcrossed into the dsp mutant by Mx4 transduction. Three independently isolated transconjugants showed essentially the same properties as the fbd mutants--loss of fibril rescue of cohesion, partial restoration of myxospore morphogenesis, and restoration of group motility. These results suggest that the physical presence of fibrils is not necessary for group motility, myxospore formation, or the early aggregative stage of development. We propose, however, that the perception of fibril binding is required for normal social behavior and development. The dsp fbd mutants (from here on referred to as fbd mutants) open the possibility of isolating and characterizing a putative fibril receptor gene.     

Synergism between morphogenetic mutants of Myxococcus xanthus.

Myxococcus xanthus, a social procaryotic microorganism, forms fruiting bodies and myxospores. We have isolated a collection of mutants of M. xanthus that are defective in fruiting morphogenesis and have studied synergistic interaction in pairwise mixtures of these mutants. Certain pairs of these fruiting-defective mutants can fruit when mixed together. Similarly, certain mutants that cannot sporulate under standard fruiting conditions can form myxospores in the presence of wildtype or other nonsporulating mutants. The pattern of synergism between pairs of conditional nonsporulating mutants defines at least three and probably four groups of mutants, such that members of a group cannot synergize with each other but can synergize with members of other groups.     

Cell surface properties correlated with cohesion in Myxococcus xanthus.

The gliding behavior of Myxococcus xanthus cells is controlled by two multigene systems, A and S, which encode information for adventurous and social behaviors, respectively. The S system can be genetically disrupted through mutation, such as a dsp mutation, or phenotypically disrupted by treating cells with the diazo dye Congo red (Arnold and Shimkets, J. Bacteriol. 170:5765-5770, 1988). One of the functions controlled by the S system is cell agglutination. Immediately after the induction of agglutination, wild-type cells begin to form aggregates, and within 30 min the cells are packed side-to-side in clumps containing thousands of cells. Changes in the cohesive properties of S+ cells are correlated with changes in the topology of the cell surface observed by electron microscopy. Two types of cell-associated appendages were observed on wild-type cells: thin filaments (ca. 5 nm in diameter), which have been called fimbriae or pili, at one cell pole, and thick, flaccid filaments (ca. 50 nm in diameter), referred to as fibrils, at both the sides and tips of cells. Cohesion was correlated with the secretion of the thick fibrils, which coat the cell surface and form an extracellular matrix in which the cells are interconnected. Several lines of evidence suggest that these thick fibrils are involved in cohesion. First, Dsp cells were unable to agglutinate or secrete this extracellular material. Second, wild-type cells which were treated with Congo red neither agglutinated nor secreted the extracellular fibrils. Finally, removal of the Congo red from wild-type cells restored cohesion and also restored production of the thick fibrils. Attempts to estimate the efficiency with which two cells cohered following collision suggested that under optimal conditions, one in three collisions resulted in stable contact. The collision efficiency decreased linearly as the cell density increased, suggesting a cell density-dependent regulation of cohesion. Some aspects of gliding behavior can be explained in terms of an inducer and an inhibitor of S motility.     

Cell-cell interactions in developmental lysis of Myxococcus xanthus

The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death. Two phenotypically complementable strains of M. xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants. Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented. We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated. The effect of the cell-free extract could be replaced by exogenously supplied glucosamine or mannosamine.     

dsg, a gene required for cell-cell interaction early in Myxococcus development.

dsg mutants of Myxococcus xanthus are conditionally defective in fruiting body development, including sporulation. Unable to develop on their own, these mutants can assemble fruiting bodies with spores if they are mixed with wild-type cells. To elucidate the developmental defect in dsg mutants by close comparison with wild type, such mutants have been backcrossed by transduction, using a closely linked insertion of transposon Tn5 for selection. Backcrossed dsg mutants form aggregates that are larger, less compact, and less symmetrical than dsg+ fruiting bodies. Also, the starvation-induced sporulation in dsg aggregates is delayed and reduced. However, dsg mutants can be induced by glycerol or dimethyl sulfoxide to sporulate at levels approaching those of wild type. dsg mutants may thus have a primary defect early in development which diminishes their capacity to aggregate and which indirectly decreases the number of fruiting body spores. The linked insertion of Tn5 also facilitated cloning the dsg gene. The cloned dsg+ allele was shown to be dominant to both the dsg-429 and dsg-439 alleles, and both mutant alleles were shown to belong to the same genetic complementation group. Subcloning of restriction fragments, deletions, and insertions of transposon Tn5 agree in locating the dsg gene to an 850-base-pair segment of the cloned region.     

Genes required for developmental signalling in Myxococcus xanthus: three asg loci.

asg-carrying strains of Myxococcus xanthus arose in a selection for mutants defective in cell-cell signalling during fruiting body development. All 15 asg mutations examined were found to lie in one of three genetic loci, asgA, asgB, or asgC. The loci were defined by linkage to different insertions of transposon Tn5 and molecular cloning of asgA. asg mutants of all three types were deficient in the aggregation of cells into mounds of the sort that normally give rise to fruiting bodies. asg mutants were also deficient in spore formation; sporulation is normally one of the last steps in fruiting body development. Consistent with a requirement for cell-to-cell signalling, at 1 to 2 h asg+-carrying cells release a material called A-factor that can rescue development of asg mutants. asgA, asgB, and asgC mutants released 5% or less of the asg+ level of A-factor, as measured by bioassay. The experimental results are consistent with the hypothesis that a deficiency in A-factor production or release is the primary developmental defect in asg mutants and that aggregation and sporulation depend on A-factor. asg mutations at all three loci also changed the color and morphology of growing colonies, and failure to release A-factor may itself arise from a defect in growing cells.     

Genes required for both gliding motility and development in Myxococcus xanthus

Myxococous xanthus cells can glide both as individual cells, dependent on A dventurous motility (A motility), and as groups of cells, dependent upon S ocial motility (S motility), Tn5-lac mutagenesis was used to generate 16 new A^- and nine new S^- mutations. In contrast with previous results, we find that subsets of A^- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S^- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S^- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S^- mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S^- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S^- insertion resembles the frz^- sglA1^- mutants upon development, suggesting that this S^- gene defines a new chemotaxis component in M. xanthus.     

Developmental bypass suppression of Myxococcus xanthus csgA mutations.

The csgA mutations of Myxococcus xanthus (formerly known as spoC) inhibit sporulation as well as rippling, which involves ridges of cells moving in waves. Sporulating revertants of CsgA cells were isolated by direct selection, since spores are much more resistant to heat and ultrasonic treatment than are vegetative cells. The revertants fell into seven groups on the basis of phenotype and the chromosomal location of the suppressor alleles. Group 1 contained one allele that was a back mutation of the original csgA mutation. Group 2 contained two linked alleles that were unlinked to the csgA locus and restored fruiting-body formation, sporulation, and rippling. Group 3 revertants regained the ability to sporulate in fruiting bodies but not the ability to ripple. Revertants in groups 4 to 7 were able to sporulate but unable to form fruiting bodies or ripples. The suppressors were all found to be bypass suppressors even though they were not selected as such in most cases. The csgA mutation prevented expression of several developmentally regulated promoters, each fused to a lacZ reporter gene and assayed by beta-galactosidase production. In four of five suppressor groups (groups 4 to 7), expression of each of these csgA-dependent fusions was restored, which suggests that bypass suppression restores developmental gene expression near the point at which expression is disrupted in CsgA mutants. Bypass suppression did not restore production of C factor, and morphological manifestations of development such as rippling and fruiting-body formation were usually abnormal. One interpretation of these results is that C factor has multiple functions and few suppressors can compensate for all of them.     

Effects of glucosamine on lysis, glycerol formation, and sporulation in Myxococcus xanthus.

Glucosamine (GlcN), which has previously been shown to rescue fruiting body formation, lysis, and sporulation in a developmental mutant (G. Janssen and M. Dworkin, Dev. Biol. 112:194-202, 1985), induced lysis in vegetative and developing wild-type cells and inhibited fruiting body formation. It also resulted in a transient, intracellular increase in the concentration of glycerol, a known sporulation inducer, and sporulation of the surviving cells. Phospholipase activity, which was shown to be normally developmentally regulated, increased 7.6-fold after treatment of vegetative cells with 50 mM GlcN. Likewise, autocidal activity, which normally increased 18 to 24 h after the initiation of development, increased 20% when vegetative or developing cells were exposed to GlcN. Two mutants resistant to GlcN-induced lysis (MD1021 and MD1022) were isolated and showed neither an increase in autocide production nor an increase in phospholipase activity in response to added GlcN. MD1021 was developmentally deficient, and GlcN rescued fruiting body formation as well as phospholipase activity and autocide production. We propose that GlcN exerts its lytic effect by regulating the activity of phospholipase enzymes that release autocides, compounds that are believed to be responsible for developmental autolysis. GlcN-induced sporulation was found to depend on several factors: the initial cell density, the amount of lysis induced by GlcN, and the presence of tan-phase variants. An initial cell density of greater than 2 x 10(5) cells per ml was required to support GlcN-induced sporulation, and sporulation did not occur unless 50 to 75% of these cells had lysed. Mutants that were resistant to GlcN-induced lysis also did not sporulate in the presence of GlcN. The effects of GlcN on developing cells depended on the concentration of GlcN added; the addition of low concentrations of GlcN resulted in enhancement of sporulation, while higher concentrations resulted in the inhibition of sporulation. The ultrastructure of GlcN-induced spores resembled that of spores induced by the exogenous addition of glycerol, in contrast to spores isolated from mature fruiting bodies. A model by which GlcN may regulate both lysis and sporulation is presented.     

Sporulation of Myxococcus xanthus in liquid shake flask cultures.

When suspended in a liquid starvation medium, exponentially growing Myxococcus xanthus sporulated within 3 days. These myxospores were similar to spores developed within fruiting bodies, as determined by electron microscopy and the production of spore-specific protein S. This liquid sporulation system may be useful as a means of preparing large quantities of myxospores and extracellular fluid for biochemical studies, including isolation of chemical signals produced during the sporulation process.