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.     

Effects of deletion of the gene for the development-specific protein S on differentiation in Myxococcus xanthus

A deletion mutation of the gene for protein S (tps), a development-specific protein of Myxococcus xanthus, was constructed. No significant differences in the process of fruiting body formation or the yield of myxospores were observed between mutant and wild-type cells. On the other hand, when the tps gene was deleted together with a 2.0-kilobase sequence including the ops gene immediately upstream of the tps gene, fruiting body formation was substantially delayed, and the yield of myxospores was reduced. These results indicate that protein S is not essential for differentiation of M. xanthus, whereas a gene product(s) coded from the sequence upstream of the tps gene appears to be required for normal fruiting body formation.     

Role of cell cohesion in Myxococcus xanthus fruiting body formation.

Dsp mutants of Myxococcus xanthus have a complex phenotype with abnormal cell cohesion, social motility, and development. All three defects are the result of a single mutation in the dsp locus, a region of DNA about 14 kilobases long. Cohesion appears to play a central role in social motility, since nonsocial mutants exhibit weak agglutination or, in the case of Dsp cells, no agglutination (L. J. Shimkets, J. Bacteriol. 166:837-841, 1986). However, Dsp cells can be agglutinated by cohesive strains of M. xanthus. This provided the opportunity to examine the role of cohesion during development by comparing the developmental phenotype of Dsp cells with that of Dsp cells mixed with cohesive strains. Dsp mutants were unable to complete any of the developmental behaviors: aggregation, fruiting body formation, developmental autolysis, and sporulation. Contact with cohesive strains seemed to restore some developmental characteristics to the Dsp cells. When allowed to develop with wild-type cells, Dsp cells accumulated in fruiting bodies and underwent developmental autolysis, but did not form a significant portion of the spore population. Igl mutants, which may be similar to the previously described frizzy mutants, are cohesive strains that are unable to form fruiting bodies. Mixing Igl cells with Dsp cells under developmental conditions resulted in fruiting body formation, although the Dsp cells were unable to form significant levels of myxospores. In spite of their inability to sporulate under developmental conditions, Dsp mutants did not appear to be defective in the sporulation process. In fact, they formed normal levels of myxospores in response to the chemical inducer glycerol.     

Adenylate energy charge during fruiting body formation by Myxococcus xanthus.

The adenylate energy charge of developing Myxococcus xanthus cells was measured. The energy charge of vegetative cells (0.81) does not change significantly during the course of fruiting body formation. Furthermore, myxospores, which are resistant, resting cells present in the fruiting body, have a relatively high energy charge (0.73).     

Effects of heat shock upon the expression of developmentally regulated genes in Myxococcus xanthus

Abstract The effects of heat shock upon the expression of several developmentally regulated genes of Myxococcus xanthus were examined. No effects were observed on levels or timing of developmentally regulated β-galactosidase expression in eight randomly selected Tn5lac insertion mutants. However, heat shock significantly affected the fruiting behavior of temperature-sensitive aggregation ( tag ) mutants of M. xanthus . The tag mutant phenotype exhibits the normal aggregation of cells to form fruiting bodies at temperatures < 34°C, but cells fail to aggregate at temperatures ⩾ 34°C. Heat shock administered to tag mutant strains prior to starvation prohibited fruiting body formation at permissive temperatures. Additionally, tag mutant strains were found to be extremely sensitive to killing at 40°C. Heat shock was also found to increase tagA and tagE expression by 22 and 47%, respectively. Mutations in tagA blocked heat shock induced expression of tagE .     

DNA replication during aggregation phase is essential for Myxococcus xanthus development

Previous studies have demonstrated that fruiting body-derived Myxococcus xanthus myxospores contain two fully replicated copies of its genome, implying developmental control of chromosome replication and septation. In this study, we employ DNA replication inhibitors to determine if chromosome replication is essential to development and the exact time frame in which chromosome replication occurs within the developmental cycle. Our results show that DNA replication during the aggregation phase is essential for developmental progression, implying the existence of a checkpoint that monitors chromosome integrity at the end of the aggregation phase.     

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.     

Identification and characterization of the Myxococcus xanthus bsgA gene product.

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.     

Morphogenesis in Myxococcus xanthus and Myxococcus virescens (Myxobacterales)

1. Myxococcus xanthus B and M. virescens V2 were compared with a view to establishing the control of their morphogenetic cycles. Both organisms are typical myxococci and on solid media with low concentrations of nutrient they form fruiting bodies, within which vegetative cells convert to myxospores. Ultrathin sections of vegetative M. virescens resembled those of M. xanthus and contained prominent heavily stained bodies, presumed to be polyphosphate granules. Shadowed preparations showed fimbriae associated with M. xanthus but not with M. virescens. 2. M. xanthus B converted to myxospores in liquid medium in response to certain alcohols. M. virescens V2 produced phase-refractile spheres, which were not viable and had an unusual ultrastructure. 3. The distributions of fruiting bodies on solid media containing 0.02% Casitone were recorded for the two species and were compared with a Poisson distribution. Cells responded to differences in cell density in a manner suggestive of a response to a chemotactic attractant. Cells growing vegetatively and also cells forming fruiting bodies produced 3',5'-cyclic adenosine monophosphate (cAMP) as measured by the incorporation of exogeneous [3H] adenosine into cAMP. 4. The significance of these findings for theories of fruiting body formation are discussed.     

Fruiting-body formation and myxospore differentiation and germination in Mxyococcus xanthus viewed by scanning electron microscopy.

Streaming cells, fruiting bodies, and single cells undergoing myxospore differentiation and germination were examined in the FB strain of Myxococcus xanthus by scanning electron microscopy. Myxospores differentiated in fruiting bodies differed in size, in kinetics of germination, in the fate of the myxospore capsule, and in the external structure of the walls of newly emerged cells when compared with myxospores differentiated in liquid medium after glycerol induction. Vegetative cells outgrowing from glycerol-induced myxospores were regularly pleomorphic, a condition that persisted through the first cell division.     

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.     

EspC is involved in controlling the timing of development in Myxococcus xanthus

The espC null mutation caused accelerated aggregation and formation of tiny fruiting bodies surrounded by spores, which were also observed in the espA mutant and in CsgA-overproducing cells in Myxococcus xanthus. In addition, the espC mutant appeared to produce larger amounts of the complementary C-signal than the wild-type strain. These findings suggest that EspC is involved in controlling the timing of fruiting body development in M. xanthus.     

Myxospore induction in a nondispersed growing mutant of Myxococcus xanthus.

Myxococcus xanthus RB5, a rough-colony-forming, nondispersed growing mutant of strain FBt, forms macroscopic, multicellular masses of radially oriented cells in shake cultures. The cells appear to be held together by slime fibrils. Physical and enzymatic methods to disrupt the spheres were unsuccessful as were attempts to isolate dispersed growing mutants. During incubation of the spheres in starvation medium, the cells within convert to myxospores, indistinguishable from those formed in fruiting bodies. Myxospores were also induced in artifically constructed, dense masses of cells of a nonmotile strain.     

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.     

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.     

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.     

A new putative sigma factor of Myxococcus xanthus.

A third putative sigma factor gene, sigC, has been isolated from Myxococcus xanthus by using the sigA gene (formerly rpoD of M. xanthus) as a probe. The nucleotide sequence of sigC has been determined, and an open reading frame of 295 residues (M(r) = 33,430) has been identified. The deduced amino acid sequence of sigC exhibits the features which are characteristic of other bacterial sigma factors. The characterization of a sigC-lacZ strain has demonstrated that sigC expression is induced immediately after cells enter into the developmental cycle and is dramatically reduced at the onset of sporulation. A deletion mutant of sigC grows normally in vegetative culture and is able to develop normally. However, in contrast to the wild-type cells, the sigC deletion mutant cells became capable of forming fruiting bodies and myxospores on semirich agar plates. This suggests that sigC may play a role in expression of genes involved in negatively regulating the initiation of fruiting body formation.     

Purine-containing compounds, including cyclic adenosine 3'',5''-monophosphate, induce fruiting of Myxococcus xanthus by nutritional imbalance.

Induction of Myxococcus xanthus fruiting by a number of different purine-containing compounds, including cyclic adenosine 3',5'-monophosphate, is defective in a mutant resistant to 2,6-diaminopurine. Furthermore, the purine-induced fruiting of wild-type cultures is uniquely blocked by a low concentration of added glycine. These results imply that different purine-containing compounds induce fruiting through a single mechanism involving nutritional imbalance.     

Methionine inhibits developmental aggregation of Myxococcus xanthus by blocking the biosynthesis of S-adenosyl methionine.

Previous studies showed that high concentrations of methionine (> 1 mM) inhibited aggregation and fruiting body formation in Myxococcus xanthus (E. Rosenberg, D. Filer, D. Zafriti, and S. H. Kindler, J. Bacteriol. 115: 29-34, 1973, and J. M. Campos and D. R. Zusman, Proc. Natl. Acad. Sci. USA 72:518-522, 1975). However, the mechanism for the inhibition was unclear. In this study, we found that high levels of methionine inhibited the biosynthesis of S-adenosylmethionine (SAM) and that reduced intracellular levels of SAM are correlated with defective chemotactic movements and reduced developmental gene expression. In addition, we found that methionine analogs and high concentrations of amino acids which are known to affect SAM synthesis in other bacteria, such as threonine, lysine, and isoleucine, also caused reduced cellular levels of SAM and blocked fruiting body formation in M. xanthus. These results indicate that SAM is required for development of M. xanthus and the inhibitory effect of methionine on development results, at least in part, from its blocking of the biosynthesis of SAM.     

Rifampin-resistant mutants of Myxococcus xanthus defective in development.

Rifampin, an antibiotic which is known to bind to and inhibit RNA polymerase, was used to probe the molecular regulation of development in Myxococcus xanthus. Rifampin-resistant mutants were screened for defects in fruiting-body formation. About 20% of the isolates in the initial screenings showed major defects in developmental aggregation or sporulation. Eleven independent mutants with wild-type growth rates and stable phenotypes were analyzed by transduction. In these strains, the rifampin-resistant and nonfruiting phenotypes showed cotransduction frequencies equal to or greater than 99.0 to 99.9%. The RNA polymerase activities were resistant to rifampin in vitro, indicating that the RNA polymerase is altered in these strains. Although their fruiting phenotypes are heterogeneous, these strains can be divided into two classes based on the level of aggregation. The results suggest that RNA polymerase plays a significant role in the regulation of development in M. xanthus since mutations which cause no apparent changes in vegetative growth result in striking defects in fruiting-body formation.