Developmental induction of Myxococcus xanthus myxospores.

Myxospore differentiation during the developmental cycle of Myxococcus xanthus is characterized by several distinguishable morphological stages. Two experimentally useful criteria of myxospore induction are the conversion of vegetative rods to optically refractile short rods or ovoids and the development of resistance to sonic lysis. The use of optical refractility as the first morphological criterion of myxospore induction has facilitated an analysis of induction on developmental plates. The time-dependent changes in the cell population from vegetative rods to the final products of development, autolysed cells and myxospores, were determined in liquid suspension by interrupting cells from developmental plates before the first appearance of myxospores. The treatment of cells involved a two-step induction system. The cells were first aerated in buffer at 32 degrees C (preinduction) and then aerated in 1% tryptone (Difco) at 32 degrees C (induction). At early plate times (0 to 18 h) there was little or no response to these treatments. After 18 h, many of the cells undergoing development on plates responded to preinduction in buffer by subsequent induction to myxospores in tryptone medium (intermediate cells). After 32 h, cells induced to myxospores in tryptone medium and did not require preinduction (competent cells). After 36 h, cells begin to undergo differentiation to myxospores on plates. These results indicate that there was a sequence of physiological changes in developing cells that are defined by the differential response of cells to treatment in liquid suspension. The liquid induction system described here provides a means to analyze the regulation of developmental myxospore induction.     

Fine Structure of Fruiting Bodies of Stigmatella aurantiaca (Myxobacterales)

The fruiting body of Stigmatella aurantiaca consists of a thick stalk supporting a number of individual cysts. The stalk is made of discontinuous tubules, of dimensions known for vegetative cells, which are oriented parallel to the longitudinal axis of the stalk. The red-brown cysts contain numerous, randomly oriented myxospores which are surrounded by thick, fibrous capsules. Their cell walls are wavy or ruffled and exhibit fewer budlike infoldings than reported for myxospores induced in liquid. We suggest that the extended time period available for metabolic and regulatory adjustments by the cell during morphogenesis within cysts accounts for the presence of considerably fewer deep cell wall infoldings than in glycerol-induced myxospores.     

Intracellular and Extracellular Nucleotides and Related Compounds During the Development of Myxococcus xanthus

Changes in nucleotide pools and extracellular nucleotides during the developmental cycle of the myxobacterium Myxococcus xanthus were determined using a high-pressure liquid chromatography nucleotide analyzer. A general increase in all nucleotide pools occurred during the morphological phase of glycerol conversion of vegetative cells to myxospores. The levels of the nucleoside triphosphate pools remained high as the myxospore matured and throughout subsequent germination. Oxidized nicotinamide adenine dinucleotide levels were elevated in the dormant myxospore and then declined during germination. The adenylate energy charge value was 0.85 +/- 0.02 for vegetative cells, germinating myxospores, and 6-h-old myxospores. It was interesting that the value for the so-called dormant myxospore was the same as that characteristic of physiologically active cells. The germinating myxospores excreted large quantities of uracil along with lesser quantities of purine nucleoside monophosphates. Although the source of the extracellular uracil cannot be determined from these experiments, it may have been derived from a shift in base ratios accompanying an assumed ribonucleic acid turnover during germination.     

Myxospore Formation in Myxococcus xanthus: Chemical Changes in the Cell Wall During Cellular Morphogenesis

Vegetative cells of Myxococcus xanthus (strain FB) were induced to form myxospores by the glycerol induction technique. Several structural changes took place in the peptidoglycan during myxospore formation. The percent of the peptidoglycan comprised of monomer (disaccharide peptide) decreased from about 20% to approximately 7%. The proportion of the total diaminopimelic acid possessing a free amino group decreased about 11%. A carbohydrate containing only glucose was found to be bound, possibly covalently, to the vegetative cell and myxospore peptidoglycan. The amount of carbohydrate relative to peptidoglycan decreased by two-thirds during myxospore formation. None of the above changes in the peptidoglycan were observed in a mutant (strain GNI) of M. xanthus which was unable to convert to myxospores when incubated in the glycerol induction medium, or in the parental wild type (FB) when it was incubated in induction medium lacking the myxospore inducer, glycerol.     

The role of extracellular material in chick neurulation. II. Surface morphology of neuroepithelial cells during neural fold fusion

Changes in cell surface morphology of the neuroepithelium during fusion of neural folds in the chick were studied. As the folds were about to meet, a thick extracellular coat material (ECM) appeared between the two leading edges. Cell membranes forming the fusion area were relatively smooth and heavily coated with ECM. By contrast, the apical surface of most cells lining the wall of the neural tube was folded with much less ECM. During the contact of neural folds, ECM was displaced from the space between the two leading edges, leaving a thin, closely adherent "typical" cell surface coat. Trypsin and concanavalin A inhibited proper alignment and fusion of apposing neural folds by modifying the surface of developing neuroepithelium. Results of this study support a hypothesis that ECM may serve temporarily as an adhesive to bind together the leading edges of neural folds until establishment of more intimate contacts (junctional complexes).     

The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development

Upon nutrient limitation cells of the swarming soil bacterium Myxococcus xanthus form a multicellular fruiting body in which a fraction of the cells develop into myxospores. Spore development includes the transition from a rod-shaped vegetative cell to a spherical myxospore and so is expected to be accompanied by changes in the bacterial cell envelope. Peptidoglycan is the shape-determining structure in the cell envelope of most bacteria, including myxobacteria. We analyzed the composition of peptidoglycan isolated from M. xanthus. While the basic structural elements of peptidoglycan in myxobacteria were identical to those in other gram-negative bacteria, the peptidoglycan of M. xanthus had unique structural features. meso- or LL-diaminopimelic acid was present in the stem peptides, and a new modification of N-acetylmuramic acid was detected in a fraction of the muropeptides. Peptidoglycan formed a continuous, bag-shaped sacculus in vegetative cells. The sacculus was degraded during the transition from vegetative cells to glycerol-induced myxospores. The spherical, bag-shaped coats isolated from glycerol-induced spores contained no detectable muropeptides, but they contained small amounts of N-acetylmuramic acid and meso-diaminopimelic acid.     

Iodination of Myxococcus xanthus during development

Intact cells of Myxococcus xanthus were iodinated with [125I]lactoperoxidase to permit examination of the surface components accessible to labeling during cell development. Vegetative cells, starved on a defined solid medium, aggregated, formed fruiting bodies, and produced myxospores. Cells collected at different stages were iodinated, and their proteins were analyzed by one- and two-dimensional electrophoresis and autoradiography. One-dimensional electrophoresis revealed six iodinated bands in vegetative cell extracts. During development, 10 radioactive bands were detected, 4 of which migrated to the same positions as those of vegetative cells. Only six bands were detected in purified, labeled myxospores. Of these, one band possessed mobility similar to that of labeled vegetative cell proteins, whereas the other bands possessed mobility similar to that detected in developing cells. Analysis of two-dimensional gels indicated that at least 14 proteins were iodinated in vegetative cells, one of which was intensely labeled (protein b). Another of the proteins (protein a) was labeled throughout development. During development, about 30 proteins were iodinated and the prominently labeled ones were designated c, d, e, f, and g. The latter two (proteins f and g) were not detected in purified, iodinated myxospores. The data indicated a pronounced change in surface structure during development; some of the change may be involved in cellular interaction during aggregation.     

Intracellular proteolytic activity in developing myxospores of Myxococcus xanthus

1. Cell-free extracts from vegetative cells and developing myxospores of Myxococcus xanthus were found to contain similar amounts of proteolytic activity, approximately 80% of which was due to one or more neutral metal proteases.
2. Sixty per cent of the proteolytic activity was particulate.
3. The specific activity of the proteases was high throughout all stages of myxospore formation and displayed small increases in activity at two stages of development: (1) during cell shortening and (2) immediately following the conversion to spheres. The first peak in activity was apparent in assays conducted at pH 8 or 10 whereas the second peak was obvious only at pH 6.
4. A mutant which develops into myxospores only after a lag of approximately 7–8 h possessed levels of proteases similar to the wild type and displayed a peak in proteolytic activity after a delay of 7–8 h.
5. Low levels of serine protease activity were occasionally detected in both vegetative cells and myxospores; no sulfhydryl proteases were detectable in either cell type.
6. Extracellular proteases accumulated in the medium throughout myxospore development but differed from the intracellular proteases in pH optima and sensitivity to inhibitors.

     

Changes in Activity of Glyoxylate Cycle Enzymes During Myxospore Development in Myxococcus xanthus

Activities of the glyoxylate cycle enzymes isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) were assayed in extracts prepared at different stages of myxospore formation in liquid cultures of Myxococcus xanthus. Activities of both enzymes attained peak values during conversion of rods to spheres. Isocitrate lyase activity decreased after reaching its peak value. Malate synthase activity also declined but at a much slower rate. The loss of isocitrate lyase activity could be prevented by the addition of chloramphenicol to cultures early in myxospore formation (during the initial rise in enzyme activity), but not by such addition at later stages of myxospore formation. The increase in glyoxylate cycle enzymes was not observed in a mutant unable to form myxospores in liquid culture under conditions suitable for morphological conversion of the wild type, or in wild-type cells incubated in the absence of an inducer for myxospore formation. It is concluded that the changes in the glyoxylate cycle enzymes represent regulatory phenomena associated with the development of the myxospore.     

Stimulation of cell wall biosynthesis and structural changes in response to cytokinin- and elicitor-treatments of suspension-cultured Phaseolus vulgaris cells

Qualitative sugar flux into cell wall polysaccharides has been determined for two model systems. The first, treatment of suspension-cultured French bean (Phaseolus vulgaris L.) cells with an increase in the cytokinin/auxin ratio and in the concentration of sucrose, models some aspects of differentiation. Wall changes are characterised by up to a five-fold increase in thickness due to the laying down of extra wall material. Sugar flux following labelling of cells with [¹⁴C]-sucrose was examined during the period of maximum extractable catalytic activities of the enzymes of sugar nucleotide conversion determined previously. Increased secretion was observed in all major groups of polysaccharides, particularly the cellulosic fraction. Analysis of the sugars in the hemicellulosic fraction indicated that the newly synthesised polysaccharide was most probably xylan. It was confirmed by immunolocalisation of xylan in these walls. This treatment thus increases incorporation into the wall of components characteristic of secondary wall. In the second system, which models the defence response, suspension cultures were treated with an elicitor from the walls of Colletotrichum lindemuthianum. Again, sugar flux was determined by labelling cells with [¹⁴C]-sucrose and examined during the period determined previously of maximum extractable catalytic activities of the enzymes of sugar nucleotide conversion. Increased secretion into unextractable polymers was the major change and was consistent with the occurrence of oxidative processes leading to immobilisation of some wall components. Callose, a polysaccharide characteristic of the defence response was immunolocalised in these walls but not in those of control cells.     

Fruiting-body formation and myxospore differentiation and germination in Mxyococcus xanthus viewed by scanning electron microscopy.

Streaming cells, fruiting bodies, and single cells undergoing myxospore differentiation and germination were examined in the FB strain of Myxococcus xanthus by scanning electron microscopy. Myxospores differentiated in fruiting bodies differed in size, in kinetics of germination, in the fate of the myxospore capsule, and in the external structure of the walls of newly emerged cells when compared with myxospores differentiated in liquid medium after glycerol induction. Vegetative cells outgrowing from glycerol-induced myxospores were regularly pleomorphic, a condition that persisted through the first cell division.     

Immunomodulation by myxospores of Myxococcus xanthus

Glycerol-induced myxospores of Myxococcus xanthus caused non-specific modulation of humoral and cellular immune responses in laboratory animals. The number of cells which formed specific haemolysins in spleens of mice immunized with sheep erythrocytes was increased when 0.5 X 10(8) myxospores were inoculated 2 d after the erythrocytes, and decreased when myxospores were injected 2 d before or at the same time as the erythrocytes. Both the IgG primary response and the secondary response to erythrocytes were decreased in rabbits after pretreatment with 2 X 10(8) myxospores per rabbit. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed in mice after intraperitoneal (i.p.) injection of 0.3 X 10(8) myxospores. One day after i.p. injection of myxospores, neither an inflammatory response nor bone marrow cell depletion was observed in mice. These results support the idea that M. xanthus myxospores possess diverse immunomodulation properties apparently due to factors different from the classical LPS of Gram-negative bacteria.     

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB^A116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB^A116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB^A116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.     

Development of Stigmatella aurantiaca: effects of light and gene expression.

Stigmatella aurantiaca, a gliding, gram-negative bacterium, exhibits complex developmental changes upon starvation. In the light the cells aggregate and develop multicellular fruiting bodies with stalks and sporangia within 20 h. Between 23 and 27 h, sonication-resistant myxospores are synchronously formed inside the sporangia. On the other hand, in the dark, the cells aggregate and differentiate into myxospores between 13 and 27 h without forming stalks and sporangia. The pattern of protein synthesis during development in the light as well as in the dark was investigated. Three periods of synthesis, characterized by sharp increases and decreases in the rate of isotope incorporation into certain proteins, were distinguished. In the light these periods corresponded approximately to an early stage before the formation of aggregates, a middle period during which aggregates appeared and developed into fruiting bodies, and a late stage that corresponded to the appearance of myxospores. The pattern of protein synthesis in the dark could also be divided into three stages, but the middle stage was considerably shorter than in the light and showed diminished synthesis of certain proteins that were actively synthesized in the light. In particular, the synthesis of one protein was detected only in samples that developed in the light.     

Developmental changes of sugar contents in the gall on the leaf of elm (Zelkowa serrata Makino) formed byParacolopha morrisoni Baker (homopetra)

To understand cell wall polysaccharide synthesis and the role of gall in interaction with aphids, the changes of sugar contents in the galls during their growth and development were determined from May 2 to June 8, 1996. The sugar content in the symplastic (MeOH and hot water) fractions decreased as the developmental stages progressed. In the cell wall fraction, the amount of pectic substances (2-3 mg per gram fresh weight) did not change. The hemicellulosic substance increased by 40% from May 14 to May 31. Among the neutral sugar components of hemicellulosic polysaccharides, xylose and arabinose contents increased during development of the gall, suggesting that xylans with arabinose residues were massively synthesized. On the other hand, glucose content decreased during development of the gall. The cellulose substance consistently increased 5 folds from May 2 to 31. The relationship between the aphid and the changes in sugar contents of cell walls during the development of aphid and the gall formation was discussed.     

Ultrastructural changes of sparkling wine lees during long-term aging in real enological conditions

Ultrastructural changes of lees of three series of sparkling wines produced using the traditional method during long-term aging (4?years) were assessed by high-pressure freezing in combination with transmission electron microscopy. The stratified structure of the cell wall disappeared throughout aging. After 18?months, the microfibrous material of the cell wall appeared more diffuse and the amorphous midzone of the inner wall layer was progressively degraded. From 30?months onward, the cell wall consisted of a tangled structure of fibers. In spite of these changes, the cell wall of yeasts remained unbroken at 48?months of wine aging. Cell membrane breakage was observed for the first time in lees of Saccharomyces cerevisiae. An increase in the thickness of the periplasmic space owing to plasmolysis and of the number of cells with less cytoplasmic content was observed during aging. Morphological evidence of microautophagy was detected for the first time in S.?cerevisiae in enological conditions.     

The initiation of cell wall synthesis in parasynchronous cultures of Schizosaccharomyces pombe

Summary Schizosaccharomyces pombe has been grown in parasynchronous culture to study the synthesis of cell wall material. After a lag period of 2.5h following inoculation the cells began to grow, as measured by optical density, dry weight and cell size. The cell number remained constant until 4.5h after inoculation when approximately 70% of the population divided synchronously. Immunofluorescence studies of the growing cells have shown that new wall material is inserted at the cell apices from 2.5 h after inoculation; this result is supported by radio-isotope labelling data which indicated that synthesis of new cell wall material also commenced 2.5 h after inoculation. The incorporation experiments also demonstrated an interruption in cell wall synthesis during the cell separation stage. The composition of the cell wall material varied during the growth cycle, with maximum nitrogen levels at inoculation and following cell division. No serological differences could be detected in the cell walls during the growth cycle.     

Scanning microscopy of the developing vagina in postnatal gerbils

Scanning electron microscopy of postnatally developing gerbil vagina (birth to maturity) shows that longitudinal folds form prior to transverse folds; the process of fold formation is initiated on the dorsal wall and proceeds ventrally. From days 1 to 7 postnatally, the vaginal epithelium is composed of either flat or bulging cells, depending on the vaginal region. The luminal cell surface is covered with uniform stubby microvilli and solitary cilia. Between days 9 and 20, the flat cells with distinct cell boundaries spread toward more proximal areas, leading to the formation of mixed patches of cells with flat or rounded apices. Individual elongated microvilli or tufts of forked microvilli may sprout from their surfaces. Solitary cilia gradually disappear. The transition from immature to mature vaginal epithelium starts around day 20, when individual cells recess below the level of neighboring cells. This process spreads throughout the vagina during the following days, reflecting local changes in the subsurface layers of the epithelium preparatory to exfoliation. Around day 40 the actual exfoliation of the luminal cell layer starts. By this time the surface characteristics of many of the desquamating cells have changed. In addition to microvilli, microridges are being formed. The process of exfoliation is finished by about day 60. The newly appearing cell layers now transform into typical cornified cells of the cycling vaginal epithelium.     

Ultrastructural features of spermatocytes and spermatozoids in the fern Phyllitis scolopendrium (L.) Newm. subsp. scolopendrium

Phyllitis scolopendrium Newm. subsp. scolo-pendrium spermatozoids are cells 10 μm long in the form of spirals with about four turns. Their chromatin is partly honeycomb-shaped and partly highly condensed. The nuclear envelope over the latter has a regular, thin intermembrane space crossed by fibers that are probably involved in connecting the chromatin with elements of the microtubular ribbon. The cytoplasm is traversed by long cistern-shaped folds of the plasma membrane, believed to be involved in a late process of cell simplification through segregation and detachment of parts of the cytoplasm. The spermatozoids are embedded in 1–1.5 μm thick amorphous electron-transparent material containing cellulose fibrils. These fibrils are considered a network connected to the original spermatocyte wall and elements of elastic support for the amorphous material. The different polysaccharide composition of the inner and outer parts of the walls causes changes in the size and shape of the ring cells, so that the spermatozoids are pushed against and past the cap cell. The gametes are released through limited laceration of the cap cell. The laceration is due to the generally weak substructure of the cell wall. A light microscope sequence of spermatozoid release and scanning electron microscope features of newly released spermatozoids are shown. Received: 24 November 1999 / Revision accepted: 29 December 1999     

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.