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.     

Behavior of peripheral rods and their role in the life cycle of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium with a complex life cycle including a developmental phase in which cells aggregate and sporulate in response to starvation. In previous papers, we have described a heretofore unsuspected layer of complexity in the development of M. xanthus: vegetatively growing cells differentiate into two cell types during development. In addition to the differentiation of spores within fruiting bodies, a second cell type, peripheral rods, arises outside fruiting bodies. The pattern of expression of proteins in peripheral rods is different from that of either vegetatively growing cells or spores, and peripheral rods express a number of recognized developmental markers. In this report, we examine four aspects of the biology of peripheral rods: (i) the influence of nutrients on the proportion of peripheral rods in a population of developing cells, (ii) the capacity of peripheral rods to recapitulate development, (iii) the development of peripheral rods on conditioned medium, and (iv) the ability of peripheral rods to resume growth on low amounts of exogenously added nutrients. The results of these studies suggest that peripheral rods play a significant role in the life cycle of M. xanthus by allowing the exploitation of low amounts or transient influxes of nutrients without the investment of energy in spore germination. The differentiation of vegetatively growing cells into two cell types that differ significantly in biology, shape, and localization within the population has been incorporated into a model of the life cycle of M. xanthus.     

Lipid body formation plays a central role in cell fate determination during developmental differentiation of Myxococcus xanthus

Cell differentiation is widespread during the development of multicellular organisms, but rarely observed in prokaryotes. One example of prokaryotic differentiation is the Gram-negative bacterium Myxococcus xanthus . In response to starvation, this gliding bacterium initiates a complex developmental programme that results in the formation of spore-filled fruiting bodies. How the cells metabolically support the necessary complex cellular differentiation from rod-shaped vegetative cells into spherical spores is unknown. Here, we present evidence that intracellular lipid bodies provide the necessary metabolic fuel for the development of spores. Formed at the onset of starvation, these lipid bodies gradually disappear until they are completely used up by the time the cells have become mature spores. Moreover, it appears that lipid body formation in M. xanthus is an important initial step indicating cell fate during differentiation. Upon starvation, two subpopulations of cells occur: cells that form lipid bodies invariably develop into spores, while cells that do not form lipid bodies end up becoming peripheral rods, which are cells that lack signs of morphological differentiation and stay in a vegetative-like state. These data indicate that lipid bodies not only fuel cellular differentiation but that their formation represents the first known morphological sign indicating cell fate during differentiation.     

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.     

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.     

Multicellular development in Myxococcus xanthus is stimulated by predator-prey interactions

Myxococcus xanthus is a predatory bacterium that exhibits complex social behavior. The most pronounced behavior is the aggregation of cells into raised fruiting body structures in which cells differentiate into stress-resistant spores. In the laboratory, monocultures of M. xanthus at a very high density will reproducibly induce hundreds of randomly localized fruiting bodies when exposed to low nutrient availability and a solid surface. In this report, we analyze how M. xanthus fruiting body development proceeds in a coculture with suitable prey. Our analysis indicates that when prey bacteria are provided as a nutrient source, fruiting body aggregation is more organized, such that fruiting bodies form specifically after a step-down or loss of prey availability, whereas a step-up in prey availability inhibits fruiting body formation. This localization of aggregates occurs independently of the basal nutrient levels tested, indicating that starvation is not required for this process. Analysis of early developmental signaling relA and asgD mutants indicates that they are capable of forming fruiting body aggregates in the presence of prey, demonstrating that the stringent response and A-signal production are surprisingly not required for the initiation of fruiting behavior. However, these strains are still defective in differentiating to spores. We conclude that fruiting body formation does not occur exclusively in response to starvation and propose an alternative model in which multicellular development is driven by the interactions between M. xanthus cells and their cognate prey.     

Isolation and phenotypic characterization of Myxococcus xanthus mutants which are defective in sensing negative stimuli.

Myxococcus xanthus is a gram-negative gliding bacterium that exhibits a complex life cycle. Exposure of M. xanthus to chemicals like dimethyl sulfoxide (DMSO) at nondeleterious concentrations or the depletion of nutrients caused several negative responses by the cells. DMSO (> 0.1 M) or nutrient depletion triggered a repellent response: cell swarming was inhibited and FrzCD (a methyl-accepting chemotaxis protein) was demethylated; higher concentrations of DMSO (> 0.3 M) or prolonged starvation induced an additional response which involved cellular morphogenesis: DMSO caused cells to convert from rod-shaped vegetative cells to spherical, environmentally resistant "DMSO spores," and starvation induced myxospore formation in the fruiting bodies. In order to investigate the nature of these responses, we isolated a number of mutants defective in negative chemotaxis and/or sporulation. Characterization of these mutants indicated that negative chemotaxis plays an important role in colony swarming and in developmental aggregation. In addition, the results revealed some of the major interrelationships between the signal transduction pathways which respond to negative stimuli: (i) DMSO exposure and starvation were initially sensed by different systems, the neg system for DMSO and the stv system for starvation; (ii) the repellent response signals triggered by DMSO or starvation were then relayed by the frz signal transduction system; mutants defective in these responses showed altered FrzCD methylation patterns; and (iii) the morphogenesis signals in response to DMSO or starvation utilize a group of genes involved in sporulation (spo).     

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.     

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.     

Murein components rescue developmental sporulation of Myxococcus xanthus

Murein (peptidoglycan) components are able to rescue sporulation in certain sporulation-defective mutants of Myxococcus xanthus. N-Acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, and D-alanine each increase the number of spores produced by SpoC mutants. When all four components are included they have a synergistic effect, raising the number of spores produced by SpoC mutants to the wild-type level. Murein-rescued spores are resistant to heat and sonic oscillation and germinate when plated on a nutrient-rich medium. They appear to be identical to fruiting body spores in their ultrastructure, in their protein composition, and in their resistance to boiling sodium dodecyl sulfate. Murein rescue of sporulation, like fruiting body sporulation, requires high cell density, a low nutrient level, and a solid surface.     

Differential expression of the three multicopper oxidases from Myxococcus xanthus

Myxococcus xanthus is a soil bacterium that undergoes a unique life cycle among the prokaryotes upon starvation, which includes the formation of macroscopic structures, the fruiting bodies, and the differentiation of vegetative rods into coccoid myxospores. This peculiarity offers the opportunity to study the copper response in this bacterium in two different stages. In fact, M. xanthus vegetative rods exhibit 15-fold-greater resistance against copper than developing cells. However, cells pre-adapted to this metal reach the same levels of resistance during both stages. Analysis of the M. xanthus genome reveals that many of the genes involved in copper resistance are redundant, three of which encode proteins of the multicopper oxidase family (MCO). Each MCO gene exhibits a different expression profile in response to external copper addition. Promoters of cuoA and cuoB respond to Cu(II) ions during growth and development; however, they show a 10-fold-increased copper sensitivity during development. The promoter of cuoC shows copper-independent induction upon starvation, but it is copper up-regulated during growth. Phenotypic analyses of deletion mutants reveal that CuoB is involved in the primary copper-adaptive response; CuoA and CuoC are necessary for the maintenance of copper tolerance; and CuoC is required for normal development. These roles seem to be carried out through cuprous oxidase activity.     

Cell-cell interactions that direct fruiting body development in Myxococcus xanthus.

The soil bacterium, Myxococcus xanthus initiates a developmental program when nutrients are limited. This results in the formation of a multicellular fruiting body structure filled with differentiated, environmentally resistant spores. At least four cell-cell signals, cell motility, and aggregation functions are required for the completion of fruiting body formation.     

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.     

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.     

Role of phase variation in the resistance of Myxococcus xanthus fruiting bodies to Caenorhabditis elegans predation

The phenomenon of phase variation between yellow and tan forms of Myxococcus xanthus has been recognized for several decades, but it is not known what role this variation may play in the ecology of myxobacteria. We confirm an earlier report that tan variants are disproportionately more numerous in the resulting spore population of a M. xanthus fruiting body than the tan vegetative cells that contributed to fruiting body formation. However, we found that tan cells may not require yellow cells for fruiting body formation or starvation-induced sporulation of tan cells. Here we report three differences between the yellow and tan variants that may play important roles in the soil ecology of M. xanthus. Specifically, the yellow variant is more capable of forming biofilms, is more sensitive to lysozyme, and is more resistant to ingestion by bacteriophagous nematodes. We also show that the myxobacterial fruiting body is more resistant to predation by worms than are dispersed M. xanthus cells.     

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.     

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 .