Biomedical subjects
Publications and source records attributed to M Dworkin.
Suspension cultures of Stigmatella aurantiaca can be induced to form myxospores by addition of the monovalent cations Li(+), Na(+), NH(4) (+), K(+), or Rb(+).
We have examined germination, protein synthesis and ribonucleic acid (RNA) synthesis by microcysts of the fruiting myxobacterium Myxococcus xanthus. The morphological aspects of microcyst formation were completed at about 2 hr after induction had begun. In such microcysts, germination, RNA synthesis, and protein synthesis were inhibited by actinomycin D (Act D). At 6 hr after induction, germination and protein synthesis had become relatively resistant to Act D, whereas RNA synthesis was inhibited by about 95%. Experiments with (3)H-Act D indicated that the deoxyribonucleic acids of both young and old microcysts bind Act D equally. Resistance of germination to Act D was acquired 4 to 5 hr after induction of microcyst formation, and was due to an Act D-sensitive synthesis at that time. Vegetative cells and microcysts were pulsed with uridine-5-(3)H and chased for 60 min; the RNA was extracted and analyzed by means of sucrose density gradient centrifugation and gel electrophoresis. Both microcysts and vegetative cells were found to contain grossly the same types of RNA in the same proportions. RNA pulse-labeled in microcysts was more stable than that in vegetative cells. No particular portions of the microcyst pulse-labeled RNA were selectively stabilized. These data indicate that a stable messenger RNA required for synthesis of germination proteins was synthesized during microcyst formation. This may be the same as the RNA synthesized 4 to 5 hr after initiation of microcyst formation. We suggest that the existence of such stable messenger RNA in microcysts is consistent with the limited biosynthetic activities of such cells.
Suspension cultures of Stigmatella aurantiaca (Chondromyces aurantiacus) were induced to form myxospores by addition of glycerol to the growing culture. The cells were fixed at various stages during conversion, thin sections were prepared, and changes in fine structure were studied. Vegetative cells are quite similar in their ultrastructure to Myxococcus xanthus. During transformation into myxospores, three important cytological changes were observed. Granules of storage material, probably polysaccharide and polyphosphate, accumulated; a 200 to 300-mum thick capsule was laid down, and the outer triple layer of the cell wall became locally folded. These cell wall folds were often densely packed and lay in pockets formed by the cytoplasmic membrane. We have suggested the possibility that the cell may store in these folds wall material which has become superfluous by the decrease in surface area during conversion.
Growth of 20 of 22 strains of gliding bacteria (including fruiting myxobacteria) was inhibited by relatively low concentrations of actinomycin D.
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.
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.
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.
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.
Burchard, Robert P. (University of Minnesota, Minneapolis), and Martin Dworkin. Light-induced lysis and carotenogenesis in Myxococcus xanthus. J. Bacteriol. 91:535-545. 1966.-Myxococcus xanthus, grown vegetatively in the light, developed an orange carotenoid after the cells entered stationary phase of growth; pigment content increased with age. Cells grown in the dark did not develop carotenoid and could be photolysed by relatively low-intensity light only during stationary phase; rate of photolysis increased with age. Photolysis adhered to the reciprocity law, was temperature-independent and oxygen-dependent, and required the presence of nonspecific, monovalent cations; it was inhibited by one of several divalent cations. Logarithmic-phase cells were photosensitized by 100,000 x g pellet preparations of sonic-treated stationary-phase cells grown in the light and dark. A porphyrin with a Soret band at 408 mmu was isolated from photosensitive cells; logarithmic-phase cells contained about 1/16 the amount of porphyrin of stationary-phase cells. The purified material had spectral and chemical properties of protoporphyrin IX and photosensitized logarithmic-phase cells. Its spectrum was similar to the action spectrum for photolysis. We concluded that protoporphyrin IX is the natural endogenous photosensitizer. Carotenogenesis was stimulated by light in the blue-violet region of the visible spectrum and was inhibited by diphenylamine, resulting in photosensitivity of the cells. Photoprotection by carotenoid was lost in the cold. A mutant which synthesized carotenoid in the light and dark was photosensitive only after growth in diphenylamine. The ecological significance of these phenomena is discussed.
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Burchard, Robert P. (University of Minnesota, Minneapolis), and M. Dworkin. A bacteriophage for Myxococcus xanthus: isolation, characterization and relation of infectivity to host morphogenesis. J. Bacteriol. 91:1305-1313. 1966.-A bacteriophage (MX-1) infecting Myxococcus xanthus FB(t) has been isolated from cow dung. The bacteriophage particle is approximately 175 mmu long. A tail about 100 mmu in length is encased in a contractile sheath and terminates in a tail plate. The head is polyhedral with a width of about 75 mmu. The nucleic acid of the bacteriophage is deoxyribonucleic acid and has a guanine plus cytosine content of 55.5%. The bacteriophage requires 10(-3)m Ca(++) and 10(-2)m monovalent cation for optimal adsorption. Grown on vegetative cells of M. xanthus FB(t) at 30 C in 2% Casitone medium, the bacteriophage has a latent period of 120 min and a burst size of approximately 100. Host range studies indicate that three strains of M. xanthus including a morphogenetic mutant are sensitive to the bacteriophage, whereas M. fulvus, Cytophaga, Sporocytophaga myxococcoides, and a fourth strain of M. xanthus are not. Of the two cellular forms characteristic of the Myxococcus life cycle, the bacteriophage infect only the vegetative cells; they do not adsorb to microcysts. Ability to adsorb bacteriophage is lost between 65 and 75 min after initiation of the relatively synchronous conversion of vegetative cells to microcysts. The bacteriophage does not adsorb to spheroplasts. After the appearance of visible morphogenesis and before the loss of bacteriophage receptor sites, addition of bacteriophage results in the formation of microcysts which give rise to infective centers only upon germination. The possibility that the infected microcysts are harboring intact bacteriophages has been eliminated.
Dworkin, Martin (University of Minnesota, Minneapolis), and William Sadler. Induction of cellular morphogenesis in Myxococcus xanthus. I. General description. J. Bacteriol. 91:1516-1519. 1966.-The details of a process for converting vegetative rods of Myxococcus xanthus to microcysts rapidly (120 min), quantitatively, and synchronously are presented. The conversion is induced by 0.5 m glycerol. On the basis of a number of morphological and physiological parameters, the microcysts thus produced are identical with those produced within fruiting bodies. Microcyst formation requires Mg(++) or Ca(++) and aeration, and is considerably stimulated by the presence of the growth medium. The process is reversible, by the removal of the inducer, and will take place only with exponentially growing cells. Mutants unable to form microcysts have been isolated. These are also deficient in their ability to form fruiting bodies.
Sadler, William (University of Minnesota, Minneapolis), and Martin Dworkin. Induction of cellular morphogenesis in Myxococcus xanthus. II. Macromolecular synthesis and mechanism of inducer action. J. Bacteriol. 91:1520-1525. 1966.-Net changes in ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein syntheses in cells of Myxococcus xanthus during induced, synchronous conversion to microcysts are described. The net synthesis of all three macromolecules was temporarily halted for a brief period during the initiation of shape change. Synthesis then resumed and leveled off when refractile microcysts began to appear. The conversion was completely sensitive, throughout the process, to low concentrations of chloramphenicol and actinomycin D. The uptake of amino acids and uracil was linear throughout the conversion, suggesting that the plateaus in rates of net synthesis of protein and RNA represented a period of rapid turnover. The most effective inducers of microcyst formation were fully saturated aliphatic compounds containing 2 to 4 carbon atoms and at least one primary or secondary alcohol group. Studies with labeled inducer indicated that the inducer need not be taken up by the cells to be effective, and probably interacts with some peripheral structure of the cell. The possibility that induction involves an alteration of a membrane-DNA complex is discussed.
Monoclonal antibodies (MCA) have been developed against cell surface antigens (CSA) of Myxococcus xanthus undergoing fruiting body formation. Three of these antibodies are directed against CSA which increase during development, six against CSA which decrease and three against CSA which show no change during development. Western-type immunoblots have been done to determine the molecular weights of the CSA to which the various MCA bind. Various applications of these MCA to the study of myxobacterial cell interactions are discussed.