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.     

Starvation-independent sporulation in Myxococcus xanthus involves the pathway for β-lactamase induction and provides a mechanism for competitive cell survival

Myxococcus xanthus is a Gram-negative, soil-dwelling bacterium with a complex life cycle which includes fruiting body formation and sporulation in response to starvation. This developmental process is slow, requiring a minimum of 24–48 h, and requires cells to be at high cell density on a solid surface. It is known that, in the absence of starvation, vegetatively growing cell suspensions can form 'glycerol spores' when exposed to high levels of glycerol, usually 0.5 M. The cells differentiate from rods to resistant spheres rapidly (2–4 h) and synchronously. We have found that the chromosomally encoded β-lactamase of M. xanthus can be induced by numerous β-lactam antibiotics as well as by non-specific inducers including glycine and many D -amino acids. In addition, D -cycloserine, phosphomycin, and hen egg-white lysozyme also induce β-lactamase in this bacterium. Unexpectedly, agents which induce β-lactamase can induce 'glycerol spores'; all of the agents tested which induce glycerol spores (glycerol, DMSO, ethylene glycol) also induce β-lactamase. During the induction of sporulation, β-lactamase activity increases, reaching a peak during the morphological transition from rod-shaped cells to spherical spores. These spores are viable and resistant to many treatments which disrupt vegetatively growing rods but are not as resistant as fruiting body spores. The concomitant induction of β-lactamase and starvation-independent sporulation suggests that these processes share a common signal-transduction pathway. These results also suggest that starvation-independent sporulation may be an adaptation of cells in order to resist agents that damage peptidoglycan structure and therefore threaten cell survival.     

Branched-chain fatty acids: the case for a novel form of cell-cell signalling during Myxococcus xanthus development

The esg locus is required for the formation of muiti-cellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus Studies have suggested that esg mutants are defective in the production of an essential signal (E-signal) used in cell-cell communication and that E-signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched-chain keto acid dehydrogenase. a multienzyme complex involved in branched-chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus. this enzyme complex appears to participate in the production of the branched-chain fatty acids found in this organism. M. xanthus fatty acids (including the branched-chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include; (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development-defective mutants. These and other results suggest a model in which the branched-chain fatty acids. Synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting-body formation. This model proposes that one or more of the branched-chain fatty acids that are released constitutes the E-signal which must be transmitted between cells to complete M. xanthus development.     

Changes in cell surface hydrophobicity of Myxococcus xanthus are correlated with sporulation-related events in the developmental program.

Cell surface hydrophobicity was measured in the bacterium Myxococcus xanthus during vegetative growth, fruiting body formation, and glycerol-induced spore formation by the method of Rosenberg et al. (FEMS Microbiol. Lett. 9:29-33, 1980). A significant decrease in cell surface hydrophobicity was observed 12 to 36 h after fruiting body formation and 60 to 120 min after glycerol-induced sporulation. The hydrophilic shift was correlated with the ability of the cells to sporulate but not with their ability to aggregate. Sucrose gradient purification removed the hydrophilic substance from the fruiting body spores but not from the glycerol-induced spores. The change in cell surface hydrophobicity in M. xanthus should be a useful developmental marker.     

A common step for changing cell shape in fruiting body and starvation-independent sporulation of Myxococcus xanthus

Myxococcus xanthus can sporulate in either of two ways: at the end of the program of fruiting body development or after exposure of growing cells to certain reagents such as concentrated glycerol. Fruiting body sporulation requires starvation, while glycerol sporulation requires rapid growth, and since the two types of spores are structurally somewhat different, it has generally been assumed that the two processes are different. However, a Tn5 Lac insertion mutation, Omega7536, has been isolated which simultaneously blocks the development of fruiting body spores as well as glycerol-induced spores. Both sporulation pathways are blocked in the mutant within the process that converts a rod-shaped cell into a spherical spore. The Omega7536 locus is expressed at the time of cell shape change appropriate to each process, early after glycerol induction and late after starvation induction. On the C-signal response pathway, it is possible to identify positions for the normal function of the Omega7536 locus and for the inducing stimulus from glycerol that are unique and consistent with the observations. Although the two sporulation pathways differ in certain respects, it is shown that they share at least one step for changing a rod-shaped cell into a spherical spore.     

Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus

We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.     

Phosphorylation and methylation of proteins during Myxococcus xanthus spore formation

Post-translational modification of proteins was examined during the life cycle of Myxococcus xanthus. A specific pattern of protein phosphorylation was observed in vegetative cells. When spore formation was induced by glycerol, significant changes in the pattern of protein phosphorylation were observed, including the phosphorylation of two membrane proteins. In in vitro experiments, the same membrane proteins were phosphorylated by ATP when the membrane preparation from cells treated with glycerol was used. Changes in the pattern of protein methylation were also observed during spore formation induced by glycerol or fruiting body formation. These results suggest that post-translational protein modification may be required for spore formation or fruiting body formation.     

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.     

Patterns of Protein Production in Myxococcus xanthus During Spore Formation Induced by Glycerol, Dimethyl Sulfoxide, and Phenethyl Alcohol

Spore formation of Myxococcus xanthus can occur not only on agar plates during fruiting body formation, but also in a liquid culture by simply adding glycerol, dimethyl sulfoxide, or phenethyl alcohol to the culture. This chemically-induced spore formation occurs synchronously and much faster than that occurring during fruiting body formation. Dramatic changes in patterns of protein synthesis were observed during chemically-induced spore formation, as had previously been observed during fruiting body formation (Inouye et al., Dev. Biol. 68:579-591, 1979). However, the production of protein S, one of the major development-specific proteins during fruiting body formation, was not detected at all, although protein U, another development-specific protein, was produced in a late stage of spore formation as in the case of fruiting body formation. This indicates that the control of the gene expression during chemically-induced spore formation is significantly different from that during fruiting body formation. It was also found that during spore formation, every cell seems to have a potential to form a spore regardless of its age, since smaller cells as well as larger cells separated by sucrose density gradient centrifugation could equally form spores upon the addition of glycerol. Patterns of protein synthesis were almost identical for all the three chemicals. However, the final yield of spores was significantly different depending upon the chemicals used. When phenethyl alcohol was added with glycerol or dimethyl sulfoxide, the final yields were determined by the multiple effect of the two chemicals added. This suggests that although these chemicals are able to induce the gene functions required for spore formation, they may have inhibitory effects on some of the gene functions or the processes of spore formation.     

Purification and characterization of SP21, a development-specific protein of the myxobacterium Stigmatella aurantiaca.

Stigmatella aurantiaca is a gram-negative bacterium with a complex life cycle, including cellular aggregation resulting in the formation of a characteristic three-dimensional structure, the so-called fruiting body. During fruiting and upon chemical induction of sporulation, a major development-specific protein, SP21, is synthesized. SP21 was purified to homogeneity from the membranous fraction of chemically induced spores. Expression of SP21 was studied with an antiserum raised against the purified protein.     

Ribonucleic acid synthesis during fruiting body formation in Myxococcus xanthus.

A method has been devised that allowed us, for the first time, to pulse-label M. xanthus cells with precursors for ribonucleic acid biosynthesis while they were undergoing fruiting body formation. Using this method, we examined patterns of ribonucleic acid (RNA) accumulation throughout the process of fruiting body formation. As development proceeded, the rate of RNA accumulation increased at two periods of the developmental cycle: once just before aggregation and once late in the cycle, when sporulation was essentially completed. In contrast to vegetatively growing cells, in which only stable RNA species are labeled during a 30-min pulse, the majority of radioactivity found in RNA from 30-min pulse-labeled developing cells was found in an unstable heterodisperse fraction that migrated to the 5S to 16S region of sucrose density gradients and sodium dodecyl sulfate-polyacrylamide gels. This pattern of incorporation could not be induced (i) by a shift down of vegetatively growing cells to a nutritionally poor medium, in which the generation time was increased to that of developing cells during the growth phase, or (ii) by plating of vegetative cells onto the same solid-surface environment as that of developing cells, but which surface supported vegetative growth rather than fruiting body formation. Thus, the RNA synthesis pattern observed appeared to be related to development per se rather than to nutritional depletion or growth on a solid surface alone. The radioactivity incorporated into the unstable 5S to 16S RNA fraction accumulated as the pulse length was increased from 10 to 30 min; in contrast, an analogous unstable fraction from vegetative cells decreased as pulse length was increased. This suggested that developmental 5S to 16S RNA was more stable than vegetative cell 5S to 16S RNA (presumptive messenger RNA). However, during a 45-min chase period, radioactivity in 30-min-pulse-labeled developmental 5S to 16S RNA decayed to an extent twice that of developmental RNA located in 16S and 23S regions of sucrose density gradients and was considerably less stable than the 5S, 16S, and 23S RNA species labeled during a 30-min pulse of vegetative cells.     

Induction of beta-lactamase influences the course of development in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus.     

Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus.

One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and germination.     

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis.

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.     

Release of a cell surface protein during development of Myxococcus xanthus.

VGP is a major cell-surface glycoprotein present in vegetative cells of Myxococcus xanthus. Serological assays indicated that this protein was released from cells and accumulated in the medium during development, i.e., aggregation, fruiting body formation, and myxosporulation. Cells induced to form spores in the absence of aggregation retained VGP, indicating that loss of VGP was associated with developmental aggregation rather than myxosporulation. Anti-VGP antibodies inhibited vegetative cell gliding, suggesting the protein may also be required for motility.     

Glycolaldehyde Dehydrogenase,Its Involvement in Vitamin B6 Biosynthetic Pathway of Escherichia coli B

The deoxyribonucleic acid (DNA) polymerases were partially purified from spores and vegetative cells of Bacillus subtilis. Some biochemical properties of the enzymes from the spores were studied in comparison with those from the vegetative cells. The spores and vegetative cells had at least three species of DNA polymerases (DNA polymerase I, II and III). These DNA polymerases in spores could not be distinguished from those in vegetative cells, respectively, with regard to the reresponses to ionic strength, the sensitivity to thiol-blocking agents, the template specificity, pH and temperature optima in assay, and the sedimentation behavior. It is inferred that DNA polymerases from spores was essentially identical to those from vegetative cells.

The DNA polymerase activity decreased rapidly in the course of sporulation, and only about 20% is recovered in the spores, suggesting that an extentive inactivation mechanism of the enzymes would be involved during sporulation.     

Small acid-soluble proteins with intrinsic disorder are required for UV resistance in Myxococcus xanthus spores

Bacterial sporulation in Gram-positive bacteria results in small acid-soluble proteins called SASPs that bind to DNA and prevent the damaging effects of UV radiation. Orthologs of Bacillus subtilis genes encoding SASPs can be found in many sporulating and nonsporulating bacteria, but they are noticeably absent from spore-forming, Gram-negative Myxococcus xanthus. This is despite the fact that M. xanthus can form UV-resistant spores. Here we report evidence that M. xanthus produces its own unique group of low-molecular-weight, acid-soluble proteins that facilitate UV resistance in spores. These M. xanthus-specific SASPs vary depending upon whether spore formation is induced by starvation inside cell aggregations of fruiting bodies or is induced artificially by glycerol induction. Molecular predictions indicate that M. xanthus SASPs may have some association with the cell walls of M. xanthus spores, which may signify a different mechanism of UV protection than that seen in Gram-positive spores.     

Sporulation in distilled water

Spores are formed when vegetative cells of sporing aerobes are shaken with distilled water at 37°. These spores are derived from the small number of cells which survive lysis. The sporulation process involves increase and concentration of solid material in the cell, and is achieved at the expense of the products of lysis of 80 to 90 per cent of the resuspended cells.     

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.