Trehalose accumulation in vegetative cells and spores of Myxococcus xanthus.

The disaccharide trehalose is found in the spores and cysts of a variety of organisms. We analyzed developing cells of Myxococcus xanthus for trehalose accumulation. Vegetative cells grown in media with low osmotic strengths contained less than 5 micrograms of trehalose per mg of protein. Spores formed in fruiting bodies accumulated up to 1,100 micrograms of trehalose per mg of protein. Spores formed in liquid culture following the addition of glycerol contained up to 300 micrograms of trehalose per mg of protein. The trehalose contents of both spore types decreased rapidly during the early stages of germination. Trehalase activity was not detected in extracts of dormant or germinating spores. Trehalose accumulation in M. xanthus was also associated with elevated osmotic strength. Vegetative cells accumulated up to 214 micrograms of trehalose per mg of protein when grown in media containing elevated levels of solutes.     

Nutrition of Myxococcus xanthus, a fruiting myxobacterium.

The minimal requirements for vegetative growth of Myxococcus xanthus have been sought. Isoleucine, leucine, and valine were required, and vitamin B12 was needed for the synthesis of methionine. Pyruvate was an excellent energy source and an efficient source of cellular carbon. Acetate, aspartate, glutamate, and most tricarboxylic acid cycle intermediates could also be utilized, but were less efficient sources of carbon and energy than was pyruvate. Many mono- and disaccharides were tested, but, in agreement with earlier results, none served as carbon-energy sources. A minimal medium (A1) has been devised that includes the essential amino acids and vitamin B12, with pyruvate and aspartate as carbon-energy sources. In this medium, M. xanthus could propagate indefinitely, and on it vegetative cells formed colonies with greater than 75% efficiency; hence, it is likely that no organic cofactors other than those present in A1 are required in more than trace amounts.     

Germination of myxospores from the fruiting bodies of Myxococcus xanthus.

Germination of myxospores from fruiting bodies of Myxococcus xanthus was examined under a light microscope as well as by analyzing the incorporation of [3H]uracil into the RNA fraction. Efficient germination was observed in 0.2% Casitone containing 8 mM MgSO4 and 1 mM CaCl2 at 30 degrees C. Under this condition, spherical myxospores were converted into rod-shaped vegetative cells within 5 to 6 h. The germination was severely inhibited in the presence of 1 mM phenylmethylsulfonyl fluoride, a protease inhibitor, indicating that a serine protease(s) is required for the myxospore germination. EGTA (1 mM) also completely blocked germination, indicating that Ca2+ plays an important role in myxospore germination. In 1% Casitone without added Mg2+ and Ca2+ or 0.2% Casamino Acids with 8 mM MgSO4 and 1 mM CaCl2, myxospores lost their refractility under a phase microscope, while no RNA synthesis took place within 6 h, as judged by the incorporation of [3H]uracil. A group of proteins were found to be specifically synthesized during an early stage of germination. In addition, a new major spore-associated protein with a size of 41.5 kDa became detectable in the spore shell fraction 3 h after germination. The present results demonstrate that myxospore germination occurs in at least two steps: the loss of myxospore refractility, followed by an outburst of metabolic activities. The first step can occur even in the absence of energy metabolism, while the second step was blocked by rifampin, EGTA, and protease inhibitors.     

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.     

De novo purine synthesis in vegetative cells and myxospores of Myxococcus xanthus

This study was designed to determine whether vegetative cells and myxospores of Myxococcus xanthus were capable of classical de novo purine biosynthesis. To answer this question, vegetative and myxospore extracts of M. xanthus FBa were tested for their ability to synthesize the second de novo intermediate, 5'-phosphoribosylglycinamide, from beginning precursors either by way of phosphoribosyl-pyrophosphate amido transferase (EC 2.4.2.14) or ribose-5-phosphate amino transferase. Both the amido and amino transferase routes occurred in both types of extracts, and both enzymes appear to be present at about the same level (per milligram of protein) in vegetative cells, myxospores, and in a bacterial prototype, Salmonella typhimurium. The dose response of the vegetative and myxospore forms of both enzymes towards adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) suggests that the allosteric structure of both enzymes is changed little by sporulation. Both enzymes were inhibited to varying degrees by a variety of purine nucleotides besides AMP, GMP, and 3':5' cyclic AMP.     

Straight-chain fatty acids are dispensable in the myxobacterium Myxococcus xanthus for vegetative growth and fruiting body formation

Inactivation of the MXAN_0853 gene blocked the production in Myxococcus xanthus of straight-chain fatty acids which otherwise represent 30% of total fatty acids. Despite this drastic change in the fatty acid profile, no change in phenotype could be observed, which contrasts with previous interpretations of the role of straight-chain fatty acids in the organism's development.     

Spatial organization of Myxococcus xanthus during fruiting body formation

Microcinematography was used to examine fruiting body development of Myxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. A pilA (MXAN_5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. A csgA (MXAN_1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.     

Heat-shock-induced proteins from Myxococcus xanthus

Optimal conditions for two-dimensional gel electrophoresis of total cellular proteins from Myxococcus xanthus were established. Using these conditions, we analyzed protein patterns of heat-shocked M. xanthus cells. Eighteen major spots and 15 minor spots were found to be induced by heat shock. From N-terminal sequences of 15 major spots, DnaK, GroEL, GroES, alkyl hydroperoxide reductase, aldehyde dehydrogenase, succinyl coenzyme A (CoA) synthetase, 30S ribosomal protein S6, and ATP synthase alpha subunit were identified. Three of the 18 major spots had an identical N-terminal sequence, indicating that they may be different forms of the same protein. Although a DnaK homologue, SglK, has been identified in M. xanthus (R. M. Weimer, C. Creghton, A. Stassinopoulos, P. Youderian, and P. L. Hartzell, J. Bacteriol. 180:5357-5368, 1998; Z. Yang, Y. Geng, and W. Shi, J. Bacteriol. 180:218-224, 1998), SglK was not induced by heat shock. In addition, there were seven substitutions within the N-terminal 30-residue sequence of the newly identified DnaK. This is the first report to demonstrate that succinyl CoA synthetase, 30S ribosomal protein S6, and ATP synthase alpha subunit are heat shock inducible.     

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).     

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.     

Developmentally induced autolysis during fruiting body formation by Myxococcus xanthus.

The developmental events during fruiting body construction by the myxobacterium M. xanthus is an orderly process characterized by several sequential stages: growth leads to aggregation leads to formation of raised, darkened mounds of cells leads to autolysis leads to myxospore induction. The temporal sequence of autolysis followed by myxospore induction is consistent with the interpretation that developmental autolysis provides essential requirements for the surviving cells to induce to myxospores. At intermediate developmental times on agar plates a fraction of the cell population is irreversibly committed to lyse; i.e., lysis continues in liquid growth medium or in magnesium-phosphate buffer. Lysis is cell concentration independent and is therefore likely to be by an autolytic mechanism. The lysis sequence can be preliminarily characterized as having an early stage during which deoxyribonucleic acid synthesis continues and a later irreversible stage during which deoxyribonucleic acid synthesis does not occur. Irreversible lysis in liquid growth medium or in magnesium-phosphate buffer is initiated on agar plates during nutrient deprivation and such lysis results in the induction of a fraction of the population to myxospores. This induction is dependent upon the concentration of lysis products, thus providing evidence that developmentally induced autolysis is required for myxospore induction.     

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.     

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.     

Pattern formation: fruiting body morphogenesis in Myxococcus xanthus

When Myxococcus xanthus cells are exposed to starvation, they respond with dramatic behavioral changes. The expansive swarming behavior stops and the cells begin to aggregate into multicellular fruiting bodies. The cell-surface-associated C-signal has been identified as the signal that induces aggregation. Recently, several of the components in the C-signal transduction pathway have been identified and behavioral analyses are beginning to reveal how the C-signal modulates cell behavior. Together, these findings provide a framework for understanding how a cell-surface-associated morphogen induces pattern formation.     

Beta-D-Allose inhibits fruiting body formation and sporulation in Myxococcus xanthus

Myxococcus xanthus, a gram-negative soil bacterium, responds to amino acid starvation by entering a process of multicellular development which culminates in the assembly of spore-filled fruiting bodies. Previous studies utilizing developmental inhibitors (such as methionine, lysine, or threonine) have revealed important clues about the mechanisms involved in fruiting body formation. We used Biolog phenotype microarrays to screen 384 chemicals for complete inhibition of fruiting body development in M. xanthus. Here, we report the identification of a novel inhibitor of fruiting body formation and sporulation, beta-d-allose. beta-d-Allose, a rare sugar, is a member of the aldohexose family and a C3 epimer of glucose. Our studies show that beta-d-allose does not affect cell growth, viability, agglutination, or motility. However, beta-galactosidase reporters demonstrate that genes activated between 4 and 14 h of development show significantly lower expression levels in the presence of beta-d-allose. Furthermore, inhibition of fruiting body formation occurs only when beta-d-allose is added to submerged cultures before 12 h of development. In competition studies, high concentrations of galactose and xylose antagonize the nonfruiting response to beta-d-allose, while glucose is capable of partial antagonism. Finally, a magellan-4 transposon mutagenesis screen identified glcK, a putative glucokinase gene, required for beta-d-allose-mediated inhibition of fruiting body formation. Subsequent glucokinase activity assays of the glcK mutant further supported the role of this protein in glucose phosphorylation.     

Separation and properties of the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus

We have developed methods for separating the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus. The total membrane fraction from ethylenediaminetetraacetic acid-lysozyme-treated cells was resolved into three major fractions by isopycnic density centrifugation. Between 85 and 90% of the succinate dehydrogenase and cyanide-sensitive reduced nicotinamide adenine dinucleotide oxidase activity was found in the first (I) fraction (rho = 1.221 g/ml) and 80% of the membrane-associated 2-keto-3-deoxyoctonate was found in the third (III) fraction (rho = 1.166 g/ml). The middle (II) fraction (rho = 1.185 g/ml) appeared to be a hybrid membrane fraction and contained roughly 10 to 20% of the activity of the enzyme markers and 2-keto-3-deoxyoctonate. No significant amounts of deoxyribonucleic acid or ribonucleic acid were present in the three isolated fractions, although 26% of the total cellular deoxyribonucleic acid and 3% of the total ribonucleic acid were recovered with the total membrane fraction. Phosphatidylethanolamine made up the bulk (60 to 70%) of the phospholipids in the membrane fractions. However, virtually all of the phosphatidylserine and cardiolipin were found in fraction I. Fraction III appeared to contain elevated amounts of lysophospholipids and contained almost three times the amount of total phospholipid as compared with fraction I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved approximately 40 polypeptides in the total membrane fraction. Two-thirds of these polypeptides were enriched in fraction I, and the remainder was enriched in fraction III. Fraction II contained a banding pattern similar to the total membrane fraction. Electron microscopy revealed that vegetative cells of M. xanthus possessed an envelope similar to that of other gram-negative bacteria; however, the vesicular appearance of the isolated membranes was somewhat different from those reported for Escherichia coli and Salmonella typhimurium. The atypically low bouyant density of the outer membrane of M. xanthus is discussed with regard to the high phospholipid content of the outer membrane.     

FibA and PilA act cooperatively during fruiting body formation of Myxococcus xanthus

The extracellular matrix (ECM) of Myxococcus xanthus is essential for social (S-) motility and fruiting body formation. An ECM-bound protein, FibA, is homologous to M4 zinc metalloproteases and is important for stimulation by a phosphatidylethanolamine (PE) chemoattractant and for formation of discrete aggregation foci. In this work, we demonstrate that a correlation exists between a reduced ability to respond to PE and the observed defects in fruiting body morphogenesis. Furthermore, the fibA aggregation defect is accentuated by the absence of either PilA, the structural subunit of type IV pili, or DifD, a chemosensory response regulator. The inability to form fruiting bodies is not due to a loss of S-motility, but rather the loss of PilA and pili as pilT fibA mutants form fruiting bodies. The FibA active site residue E342 is important for fruiting body morphogenesis in the absence of PilA. Mutants exhibiting defects in fruiting body morphogenesis also produce fewer viable spores. It is proposed that FibA and PilA act as extracellular sensors for developmental signals.