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Peptidoglycan of Myxococcus xanthus: structure and relation to morphogenesis.

The chemical nature and distribution of the peptidoglycan in Myxococcus xanthus at various stages of the cellular life cycle were investigated. Vegetative cells and microcysts contained approximately 0.6% by weight of peptidoglycan. The overall composition of the peptidoglycan was similar in both cell types and was approximately 1 glutamic acid, 1 diaminopimelic acid, 1.7 alanine, 0.75 N-acetylglucosamine, and 0.75 N-acetylmuramic acid. (We have assumed that all the hexosamines are N-acetylated.) The sizes of the subunits (estimated by gel filtration) solubilized by muramidases were considerably larger (tetramer and oligomer) in the microcysts than in the vegetative cells (mostly dimer). There was a transient decrease in cross-linking (measured as an increase in the amount of free amino group of diaminopimelic acid) during the stage of microcyst formation when the cells converted from ovoids to spheres. At the same time, there occurred a large and rapid increase in a galactosamine derivative which may have reflected the synthesis of capsular material. Immediately prior to this period of morphogenesis, the cells became resistant to penicillin but remained sensitive to d-cycloserine. The walls of vegetative cells were completely disaggregated by trypsin and sodium lauryl sulfate, suggesting a discontinuous peptidoglycan layer. This was no longer apparent after the ovoid-sphere stage of microcyst formation. The relationship to morphogenesis of the chemical changes in the cell wall is discussed.

Amino Acids↗

Microcyst germination in Myxococcus xanthus.

Germination of glycerol-prepared microcysts of Myxococcus xanthus was studied. The sequence of morphological events during germination resembled that of germinating fruiting body-microcysts. The turbidity drop of a culture of germinating microcysts could be described by McCormick's formula derived for germinating Bacillus spores. The rate of uptake of labeled glycine and acetate did not change during germination. Temperature, aeration, and pH optima for germination were the same as for vegetative cell growth. Germination was induced by protein hydrolysates and the individual amino acids glycine, alanine, valine, aspartic acid, and glutamic acid. A number of organic compounds, including sugars, alcohols, aldehydes, ketones, organic acids, and chelating agents, did not induce germination. The inorganic ions HPO(4) (2-), Mg(++), Ca(++), and NH(4) (+) induced germination, although ionic strength was not a factor. Microcysts incubated in distilled water at concentrations greater than about 10(9) cells/ml germinated; supernatant fluid from such suspensions (germination factor) induced germination of less concentrated suspensions. The activity of germination factor was resistant to boiling, but was lost on charring and dialysis. Germination of microcysts and growth of vegetative cells was equally sensitive to a variety of metabolic inhibitors, including penicillin and chloramphenicol. Germination was more resistant than vegetative growth to inhibition by antibiotics of the streptomycin family and by actinomycin D.

Acetates↗

Cytochemistry of phosphatases in Myxococcus xanthus.

An Mg(2+)-dependent and a K(+)-stimulated adenosine triphosphatase were localized by cytochemistry at or near both surfaces of the cytoplasmic membrane of Myxococcus xanthus. An alkaline and an acid phosphatase resided at the external surface of the membrane or in the periplasm. All enzymes could be extracted from partially fixed cells with Mg(2+)-deficient buffers. Suboptimal external phosphate elicited dissociation of adenosine triphosphatase from the membrane but not that of the unspecific phosphatases. The dissociated enzymes migrated into the cytoplasm where they were associated mainly with cytoplasmic aggregates.

Acid Phosphatase↗

Deoxyribonucleic acid synthesis during microcyst germination in Myxococcus xanthus.

Deoxyribonucleic acid (DNA) synthesis was measured during microcyst germination in Myxococcus xanthus by radioactive thymidine incorporation, autoradiography, and chemical analysis. Microcysts contained an average of 6.6 conserved units of DNA, corresponding to 3 to 4 chromosomes per cell. Correlation of the DNA content and chromosome number of microcysts indicated that the molecular weight of the nonreplicating M. xanthus chromosome is 4.9 x 10(9) daltons. DNA synthesis was initiated 3.5 to 4 hr after induction of germination. From 4 to 6 hr, the rate of synthesis was constant and the accumulation was linear. After a lag period (6 to 6.5 hr), the rate of DNA synthesis increased, reaching a second plateau at 9 hr. From 9 to 11 hr, the rate was again constant and the accumulation was linear. Cellular division during germination showed an unusual kind of synchrony. A model is presented that accounts for chromosomal replication and cell division during microcyst germination.

Autoradiography↗

Comparative intermediary metabolism of vegetative cells and microcysts of Myxococcus xanthus.

Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO(2). In addition, microcysts and vegetative cells showed a different distribution of (14)C-label from incorporated acetate.

Acetates↗

Action spectrum for carotenogenesis in Myxococcus xanthus.

An action spectrum was measured for photoinduction of colored carotenoids in dark-grown, early stationary-phase cells of Myxococcus xanthus. Maximum activity was observed at 405 to 410 nm with subsidiary maxima at 512, 533, 548, 585, and 635 nm. These maxima correspond closely in position and magnitude with absorption maxima of protoporphyrin IX, which had previously been isolated from M. xanthus cells and had been shown to increase during the stationary phase of the culture. Late stationary-phase, dark-grown cells undergo photolysis which had been shown to have an action spectrum resembling the absorption spectrum of protoporphyrin IX. The similarity of the action spectra of photolysis and photoinduced carotenogenesis in M. xanthus and of other photoinduced biological phenomena is discussed.

Bacteria↗

Resistance of vegetative cells and microcysts of Myxococcus xanthus.

The resistance of vegetative cells and of microcysts of Myxococcus xanthus to several destructive agents was compared. Fruiting-body microcysts were 300 times more resistant to 60 C, 5.4 times more resistant to ultraviolet light, and 19.3 times more resistant to sonic vibration than were vegetative cells. Whereas resistance to sonic vibration developed during the conversion of rods to refractile spheres, resistance to heat did not appear until after the conversion was complete. Both vegetative cells and microcysts of the yellow variant of this strain were more resistant to ultraviolet irradiation than was the tan variant.

Bacteria↗

Pigmentation phenotype instability in Myxococcus xanthus.

Cells of Myxococcus xanthus FB2 produce tan or yellow colonies. Subcultures of tan colonies yielded tan and yellow colonies and subcultures of most yellow colonies yielded only yellow colonies. Strain FB2 variants in which the color type is more stable were obtained. Yellow cells were distinguishable from tan by the presence of pigment(s) with an absorption maximum at 379 nm. Fluctuation Test experiments and the presence of this pigment(s) in liquid cultures of FB2 indicated that tan phenotype cells spontaneously became or segregated yellow cells in liquid culture. The frequency of appearance of yellow cells was increased in low density cultures (less than 10(6)/ml). The increase cannot be explained by differences in growth rates of the two phenotypes. No evidence that cell-cell contact or culture medium constituents affect the appearance of the yellow phenotype was found. Ultraviolet irradiation of FB2 resulted in an increased proportion of cells producing yellow colonies among the survivors. Greater UV resistance of yellow cells and UV-induced conversion of tan to yellow accounts for this increase. Low level photoreactivation of viability and of the tan phenotype occurred. Incubation of FB2 in medium containing mitomycin C, nalidixic acid, phenethyl alcohol, or at 36.5 degrees C also resulted in conversion of tan to yellow cells.

Myxococcales↗

Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in Myxococcus xanthus.

Exogenous cyclic nucleotide phosphodiesterase (PD) accelerated fruiting body (FB) formation and increased territory size of aggregates in Myxococcus xanthus. Both guanosine 3'5'-monophosphate (cGMP) and guanosine 5'-monophosphate (GMP) were antagonistic to the PD effect. Adenosine 3'5'-monophosphate (cAMP) increases FB numbers twofold in the absence but not in the presence of PD. PD induction is not affected by methionine or isoleucine, which inhibit, or by threonine, which stimulates, FB formation. There is an increase and subsequent decrease in cAMP levels during early glycerol-induced microcyst development but 10 mM theophylline or caffeine not only inhibited microcyst development but induced germination in the presence of glycerol. On the basis of these results and the reports of other investigators a tentative model is proposed based on a dual role for cyclic nucleotides in the development in M. xanthus.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

The function of fimbriae in Myxococcus xanthus. II. The role of fimbriae in cell-cell interactions.

Anti-fimbriae antiserum specifically inhibited swarming but no gliding motility per se in Myxococcus xanthus. However, formation of motile aggregates on agar and clumps in liquid media correlated with the presence of fimbriae. Ethylenediaminetetraacetic acid which inhibited swarming also inhibited fimbriae formation. Direct electron-microscopic observations revealed that fimbriae establish contact with apposing cell surfaces. Intact but not depolymerized fimbriae exhibited hemagglutination activity against guinea pig erythrocytes. This activity was inhibited by mannose, N-acetyl-D-galactosamine, and to a lesser degree by fructose, raffinose, melibiose, and alpha-methyl-D-mannoside. It is concluded that fimbriae are organelles which function to establish and maintain intercellular contacts, perhaps by a lectin-like function, during the coordinated movement of cell aggregates' (swarming) in myxobacteria. This hypothesis is supported by the observations of other workers that genes determining movement of cells in groups also control fimbriation in M. xanthus.

Acetylglucosamine↗

Cyclic adenosine 3',5'-monophosphate binding protein in developing myxospores of Myxococcus xanthus.

The interaction of cyclic adenosine 3',5'-monophosphate (cAMP) with specific protein molecules was examined in the high-speed supernatant fraction of extracts made at stages throughout glycerol-induced myxospore development in Myxococcus xanthus. Experiments using 8-azido[32P]cAMP, a photoaffinity analogue of cAMP, and SDS - polyacrylamide gel electrophoresis showed that the nucleotide interacts with only a single protein band of 12 500 molecular weight. Both the identiy and amount of this protein remained constant throughout development. The binding protein was specific for cAMP; other nucleotides did not compete with cAMP for binding sites. A Scatchard analysis showed evidence of only a single class of binding sites with a high affinity for cAMP.

Binding Sites↗

Protein and lipid methylation by methionine and S-adenosylmethionine in Myxococcus xanthus.

Methylation of lipids and proteins has been examined in Myxococcus xanthus using radioactive methionine and S-adenosylmethionine as methyl donors. S-adenosylmethionine is shown to be taken up by these cells and utilized directly. This permits detection of methylation in the presence of protein synthesis. Patterns of methylation obtained using methionine and S-adenosylmethionine during vegetative growth are compared by polyacrylamide gel electrophoresis, and inhibitors of protein synthesis and S-adenosylmethionine synthesis are examined for their effects on methylation. The ability to investigate methylation using exogenous S-adenosylmethionine will be advantageous in studying the role of methylation under conditions of growth and development where ongoing protein synthesis is required.

Bacterial Proteins↗

Purification and partial characterization of an antibiotic produced by Myxococcus coralloides.

A strain of Myxococcus coralloides producing an antibiotic capable of inhibiting growth of Gram-positive bacteria was isolated. Antibiotic production occurred during vegetative growth but not during myxospore formation. The antibiotic was extracted from the growth medium with chloroform and purified by adsorption on silicic acid and by preparative silica gel thin-layer chromatography. The purified antibiotic showed a resistance to heat, acid, alkali and proteolytic enzymes. Chromatographic and electrophoretic behavior as well as infrared, ultraviolet and mass spectra are presented.

Anti-Bacterial Agents↗

The myxovirescins, a family of antibiotics from Myxococcus virescens (Myxobacterales).

The myxobacterium, Myxococcus virescens strain Mx v48 produced a family of at least 12 closely related antibiotics, the myxovirescins. At a concentration of 1 to 5 micrograms/ml, the main component, myxovirescin A, was bactericidal for many Gram-negative bacteria, in particular enterobacteria, and at 20 to 50 micrograms/ml it also inhibited some pseudomonads and Gram-positive bacteria. The antibiotics seem to interfere with cell wall synthesis. The molecular formula of myxovirescin A was C35H61NO8. It is a new antibiotic.

Anti-Bacterial Agents↗

Chemical properties of Myxococcus xanthus antibiotic TA.

Antibiotic TA was purified and crystallized from culture fluids of Myxococcus xanthus TA. The antibiotic (C34H57O9N, M.W. 623.8) contained the following functional groups: ketone, lactone, secondary amide, methoxy-substituted diene (lambda max 239 nm), primary alcohol and three secondary alcohols, two of which were cis-vicinal. Mild alkaline hydrolysis opened the lactone with concomitant loss of antibiotic activity. Periodate oxidation also destroyed biological activity.

Anti-Bacterial Agents↗

The myxalamids, new antibiotics from Myxococcus xanthus (Myxobacterales). I. Production, physico-chemical and biological properties, and mechanism of action.

From the cell mass and culture supernatant of Myxococcus xanthus strain Mx X12 an antibiotic activity against yeasts, molds and some Gram-positive bacteria could be extracted. It consisted of 4 biologically active compounds which were named myxalamid A, B, C and D. The main component, myxalamid B, was shown to block in beef heart submitochondrial particles the respiratory chain at the site of complex I, i.e. NADH: ubiquinone oxidoreductase. The myxalamids are new antibiotics.

Animals↗

Amino acid precursors of Myxococcus xanthus antibiotic TA.

The production of Myxococcus xanthus antibiotic TA was stimulated by addition of alanine, serine and glycine to Casitone medium. These three amino acids served as the major biosynthetic precursors of the antibiotic. Alanine and serine were incorporated via acetate. In Casitone medium supplemented with alanine and serine, 29 to 30 of the 34 carbon atoms of antibiotic TA were derived from these two amino acids. Both carbon atoms of glycine were incorporated into antibiotic TA by a mechanism not involving acetate as an intermediate. Antibiotic TA was split into two fragments by alkaline hydrolysis followed by periodate oxidation. Radioactive alanine was incorporated into both fragments, whereas glycine was incorporated only into the smaller, polar fragment.

Acetates↗