Growth and division of Spiroplasma citri: elongation of elementary helices.

The smallest viable cell of Spiroplasma citri is a two-turn helix (elementary helix). This elementary helix grows into longer parental cells, which then divide by constriction. The helical morphology is conserved during this process. The growth pattern of S. citri membranes has been investigated by different methods of membrane labeling. When labeling is done with specific antibodies, a diffuse growth of the membrane is observed. On the contrary, pulse-labeling of the membrane with tritiated amino acids reveals a polar growth of the organism. Finally, labeling of oxydo reduction sites with potassium tellurite also indicates a polarity in the organism. These results are discussed, and a scheme for spiroplasma growth is proposed.     

外循环气升式反应器培养新疆紫草细胞

采用两步培养法进行新疆紫草细胞悬浮培养及5L外循环气升式反应器扩大培养,探讨了培养过程中细胞生长、紫草色素合成与培养液的电导率、可溶性糖含量变化之间的关系。第一步培养时细胞生长迅速,但也有一部分色素合成,电导率及可溶性糖含量迅速下降;第二步培养初期电导率也开始下降,但当色素合成达到高峰并有一部分外泌到培养基后,电导率又开始回升。可溶性糖捎耗很快,到后期巳测不出其存在。因此通过监测培养液中电导率及可溶性糖的变化情况,可以为新疆紫草细胞大规模培养与色素合成提供有用的参数指标。     

Exo-(1----3)-beta-glucanase, autolysin and trehalase activities during yeast growth and germ-tube formation in Candida albicans

Exo-(1----3)-beta-glucanase, beta-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and beta-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 degrees C. However, in situ trehalase, (1----3)-beta-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. beta-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (1----3)-beta-glucanase and autolysin activities were optimal at pH 5 X 6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1----3)-beta-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1----3)-beta-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1----3)-beta-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1----3)-beta-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1----3)-beta-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.     

The helix clock: a potential biomechanical cell cycle timer

A model based upon helical geometry that provides cylindrically shaped cells with a means to measure their length during growth and to time cell cycle events is presented. The helix clock arises from the change in pitch angle that accompanies the parallel packing of strands on a cylinder surface. A single strand inserted into the cylinder surface nearly perpendicular to the long axis of the cylinder starts the clock running. As additional strands are inserted parallel to those in place, the pitch angle of all strands must reorient. A limit is reached when all strands lie parallel to the long axis of the cylinder. By sensing either the pitch angle or a physical ramification thereof, cells can measure their length during growth and time events of the cell cycle. The helix clock model is discussed in relationship to the bacterial cell cycle. The idea that bacterial cells use one helix hand for cylinder elongation, the other for septation is presented. The negative twist so generated apparently drives folding in the helical bacterial macrofiber system of Bacillus subtilis.     

Release from quorum-sensing molecules triggers hyphal formation during Candida albicans resumption of growth

Candida albicans is a pathogenic fungus able to change morphology in response to variations in its growth environment. Simple inoculation of stationary cells into fresh medium at 37 degrees C, without any other manipulations, appears to be a powerful but transient inducer of hyphal formation; this process also plays a significant role in classical serum induction of hyphal formation. The mechanism appears to involve the release of hyphal repression caused by quorum-sensing molecules in the growth medium of stationary-phase cells, and farnesol has a strong but incomplete role in this process. We used DNA microarray technology to study both the resumption of growth of Candida albicans cells and molecular regulation involving farnesol. Maintaining farnesol in the culture medium during the resumption of growth both delays and reduces the induction of hypha-related genes yet triggers expression of genes encoding drug efflux components. The persistence of farnesol also prevents the repression of histone genes during hyphal growth and affects the expression of putative or demonstrated morphogenesis-regulating cyclin genes, such as HGC1, CLN3, and PCL2. The results suggest a model explaining the triggering of hyphae in the host based on quorum-sensing molecules.     

Production by Bacillis subtilis of brown sporulation-associated pigments

The mode of production of the brown pigments of Bacillus subtilis 168 L-4, pigments frequently used as phenotypic markers for sporulation in this organism, has been studied. A defined liquid medium which promoted maximal pigment formation was developed. Five brown components, which could be resolved by thin-layer chromatography, were produced in the culture broth. Removal of cells from the medium at the end of logarithmic growth did not alter the type or amount of the pigments formed, indicating that the cells excreted pigment precursors into the medium during growth. Pigment formation from the precursors was found to occur by an oxygen-requiring, base-dependent, Mn2+-requiring, nonenzymatic pathway. Pigment production was also stimulated by the presence of tyrosine and histidine in the medium. The increases in extracellular pH often associated with spore formation in B. subtilis might be the cause of the concomitant appearance of brown pigments.     

Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.     

Morphological and biochemical responses of Bacillus subtilis to selenite stress

When introduced into a chemically defined minimal medium supplemented with 1 mM sodium selenite (79 ppm Se(o)), Bacillus subtilis was found to undergo a series of morphological and biochemical adaptations. The morphological changes included the formation of "round bodies" associated with the detoxification of selenite to elemental selenium. Round bodies observed transiently were not apparent during balanced growth of cells adapted previously to selenite-containing medium. Under balanced growth conditions, cell structures similar to "round bodies", could be produced by treating cells with lysozyme. The selenite-induced structural alterations in cells were accompanied by an increase in the content of thioredoxin and the associated enzyme, NADP-thioredoxin reductase. The results suggest that the biovalence transformation of high levels of selenite may involve a dithiol system.     

Lysis of Saccharomyces cerevisiae with 2-deoxy-2-fluoro-D-glucose, an inhibitor of the cell wall glucan synthesis

The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.     

YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA

Control of DNA replication initiation is essential for cell growth. A unifying characteristic of DNA replication initiator proteins is their distinctive AAA+ nucleotide‐binding domains. The bacterial initiator DnaA assembles into a right‐handed helical oligomer built upon interactions between neighbouring AAA+ domains to form an active initiation complex. Recently we developed a unique cross‐linking assay that specifically detects ATP‐dependent DnaA helix assembly. Here we have utilized this assay to show that two DnaA regulatory proteins in Bacillus subtilis, YabA and DnaD, inhibit DnaA helix formation. These results, in combination with our previous finding that the regulatory factor Soj/ParA also targets DnaA filament formation, highlight the critical importance of regulating DnaA helix formation during the initiation reaction. Moreover, these observations lead us to suggest that DnaA oligomerization may be the main regulatory step of the initiator assembly pathway in B. subtilis, in contrast to the prevailing model of bacterial DNA replication based on Escherichia coli DnaA where ATP binding appears to be the targeted activity.     

Kinetic studies of polyhydroxybutyrate granule formation in Wautersia eutropha H16 by transmission electron microscopy

Wautersia eutropha, formerly known as Ralstonia eutropha, a gram-negative bacterium, accumulates polyhydroxybutyrate (PHB) as insoluble granules inside the cell when nutrients other than carbon are limited. In this paper, we report findings from kinetic studies of granule formation and degradation in W. eutropha H16 obtained using transmission electron microscopy (TEM). In nitrogen-limited growth medium, the phenotype of the cells at the early stages of granule formation was revealed for the first time. At the center of the cells, dark-stained "mediation elements" with small granules attached were observed. These mediation elements are proposed to serve as nucleation sites for granule initiation. TEM images also revealed that when W. eutropha cells were introduced into nitrogen-limited medium from nutrient-rich medium, the cell size increased two- to threefold, and the cells underwent additional volume changes during growth. Unbiased stereology was used to analyze the two-dimensional TEM images, from which the average volume of a W. eutropha H16 cell and the total surface area of granules per cell in nutrient-rich and PHB production media were obtained. These parameters were essential in the calculation of the concentration of proteins involved in PHB formation and utilization and their changes with time. The extent of protein coverage of the granule surface area is presented in the accompanying paper.     

Mitochondrial behaviour during the yeast-hypha transition of Candida albicans

Yeast cells of Candida albicans were brought to germ tube formation and hyphal growth in liquid synthetic medium. The behaviour of mitochondria and mitochondrial nucleoids (mt-nucleoids) during morphological conversion was examined by fluorescence staining with 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) and 4',6-diamidino-2-phenylindole (DAPI). Parent yeast cells possessed one or very few branched giant mitochondria which were stained intensely with DASPMI. When a germ tube emerged from the parent cell, one end of a giant mitochondrion extended into the germ tube and developed into the elongated form. In mycelia, apical hyphal cells contained giant mitochondria, whereas older hyphal compartments near the parent cells were vacuolated and possessed small, peripherally located mitochondria. The vacuolated hyphal compartments resynthesized cytoplasm before producing branches and contained giant mitochondria. The cytological model for germ tube formation and hyphal growth proposed by Gow and Gooday (1984) is discussed.     

Effects of low X-ray doses inSaccharomyces cerevisiae

Summary Three strains ofSaccharomyces cerevisiae with different capacities for repair of radiation damage (RAD, rad18, and rad52) have been tested for their colony forming ability (CFA) and growth rates after application of small X-ray doses from 3.8 mGy to 40 Gy. There was no reproducible increase in CFA observable after application of doses between 3.8 mGy and 4.7 Gy. X-ray doses of 40 Gy causing an inactivation of CFA from 90% to 50%, depending on the repair capacity of the strains used, caused a reduced increase in optical density during 2 h buffer treatment in comparison to unirradiated cells. This reduction however, is reversible as soon as the cells are transferred into nutrient medium. One hour after transfer into growth medium the portions of cells with large buds (G2 and M phase) and cells with small buds (S phase) are drastically different in irradiated cells from those obtained in unirradiated cells. The time necessary for separation of mother and daughter cells is prolonged by X-ray irradiation and the formation of new buds is retarded.     

Buffer capacity of cotton cells and effects of extracellular pH on growth and somatic embryogenesis in cotton cell suspensions

Summary This research was designed to: a) characterize the normal pH changes that occur when cotton cell are grown in culture; b) determine if cotton cells can regulate the pH of their extracellular medium; and c) explore the effects of starting pH on cellular differentiation in culture, including formation of somatic embryos. When an aliquot of cotton cell suspension culture (Gossypium hirsutum L. cv Coker 312) was inoculated into fresh Murashige and Skoog (MS) medium at pH 4.5, the pH stabilized near 5.5 during the log phase of growth and then rose to pH 7.25. Cotton cells actively adjust medium with initial pH between 3 and 8 to near pH 5.5 in the early culture period. By acid/base titration, it has been shown that living cotton cells increase the buffer capacity of water and MS medium. Therefore, the metabolic activity of living cells accounts for the adjustment and stabilization of pH during the log phase of growth. The starting pH of the culture medium affects longterm viability, growth, and differentiation of the cells; pH 3 to 5 is best for cell viability, pH 3 to 4 enhances cell elongation; and pH 4, 7, or 8 stimulates somatic embryogenesis. Cultured cotton cells and the pH of their extracellular medium are in a complex, interactive relationship. This study was supported by the Texas Advanced Technology Program and the USDA-ARS. The journal no. for this paper is T-4-280, The College of Agriculture, Texas Tech University.     

Astaxanthin production by a green alga,Haematococcus pluvialis accompanied with morphological changes in acetate media

Two acetate containing media were developed for astaxanthin production by a green unicellular alga, Haematococcus pluvialis. The basal medium, a vegetative growth medium facilitated the algal cell growth, whereas the modified medium was likely to induce morphological changes with the formation of large cysts and bleached cells which seemed to consequently enhance the carotenoid biosynthesis. In the two-stage culture, the injection of ferrous ion with acetate into the basal medium on the fourth day, was greatly stimulative for both the algal cell growth and the astaxanthin formation at a high light intensity. In addition, carotenoid precursors, mevalonate and pyruvate were effective on the carotenoid formation in the modified medium. Pyruvate was an especially good carbon source both for the algal cell growth and the carotenoid synthesis.     

Production of brown tyrosine pigments by the yeast Yarrowia lipolytica

AIMS: To study the mechanism of production of brown pigments from tyrosine in the yeast Yarrowia lipolytica. METHODS AND RESULTS: Pigment formation was followed during growth in tyrosine medium, and the presence of the pigment precursor in the medium was assessed by evaluating pigment formation after removing the cells at different times of incubation. It was observed that the pigment precursor accumulated outside the cells during the exponential phase of growth, but pigment formation only occurred during the stationary phase of growth and resulted from the oxidation of the precursor. Pigment formation was repressed by glucose and L-glutamine, and promoted by lactic acid, L-asparagine and glycine. Spectra of 1H and 13C-NMR revealed that the brown pigment was derived from tyrosine and was a polymer composed of a core of aromatic residues. CONCLUSION: The results indicate that pigments result from the extracellular accumulation and auto-oxidation of an intermediate of tyrosine catabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the mechanism of pigment production from tyrosine in a yeast species.     

Regulation of the formation of protease byBacillus megaterium

The formation of protease takes place in washed cells ofBacillus megaterium incubated in a nitrogen-free medium. The rate of enzyme synthesis is decreased much less than that of cell proteins as compared with growing cells. The synthesis of protease in a nitrogen-free medium requires the presence of glucose. The omission of glucose results in stopping of the enzyme formation and substantial decrease of the rate of protein synthesis. Protease is not synthesized when the washed cells are incubated in a phosphate, free medium. The incubation of the cells in a nitrogen-free medium results in a decrease of the concentration of amino acids in the pool. In a phosphate-free medium the content of free amino acids increases temporarily and decreases again later. When the culture grown in the medium containing threonine or threonine and isoleucine in addition to NH₄ ions is transferred into the medium without amino acids, no protease formation is found during derepression of enzymes synthesizing both amino acids. The cells grown in a medium containing casamino acids begin to form the enzyme after a short lag period when transferred into the medium containing NH₄ as a sole nitrogen source or into a nitrogen-free medium.     

TRANSFORMATION OF NONPOLAR FILAMENTS OF THE BLUE-GREEN ALGA GLOEOTRICHIA ECHINULATA U. WISC. 1052 INTO DOUBLE HELICES12

Normally straight filaments of Gloeotrichia echinulata U. Wisc. 1052 transform into double helices when a critical culture density has been attained. In inorganic Zehnder-Gorham's medium No. 11, the alga initially is morphologically uniform and forms single, lightly curved, polar filaments. When a stage of close proximity of the filaments is reached, excessively long filaments are observed that are nonpolar. Some of these nonpolar filaments form helices. Two independent single helices may entwine to form a double helix. Double helices also form when both ends of an excessively long filament meet and start entwining until a loop is left at about the original middle of the filament. In another manner of double-helix formation, a straight filament makes a hairpin bend at about its middle; then 2 helices form starting at the bend and they entwine into a double helix, leaving a loop at the location of the original bend. Under proper culture conditions, the structures of double helices show an amazing regularity. Once double-helix formation has started, some strong force brings the process to completion. In a mature culture, helices disintegrate into apparently healthy, short pieces of filaments or single cells, while the straight or slightly curved polar filaments still persist. Helical transformation of nonpolar filaments does not appear to be a sign of nutritional or other stress but rather appears to serve a specific purpose. One might speculate that a genetic exchange between opposite cells takes place.     

A short sequence in the N-terminal region is required for the trimerization of type XIII collagen and is conserved in other collagenous transmembrane proteins

The recombinant transmembrane protein type XIII collagen is shown to reside on the plasma membrane of insect cells in a 'type II' orientation. Expressions of deletion constructs showed that sequences important for the association of three alpha1(XIII) chains reside in their N- rather than C-terminal portion. In particular, a deletion of residues 63-83 immediately adjacent to the transmembrane domain abolished the formation of disulfide-bonded trimers. The results imply that nucleation of the type XIII collagen triple helix occurs at the N-terminal region and that triple helix formation proceeds from the N- to the C-terminus, in opposite orientation to that of the fibrillar collagens. Interestingly, a sequence homologous to the deleted residues was found at the same plasma membrane-adjacent location in other collagenous transmembrane proteins, suggesting that it may be a conserved association domain. The type XIII collagen was secreted into insect cell medium in low amounts, but this secretion was markedly enhanced when the cytosolic portion was lacking. The cleavage occurred in the non-collagenous NC1 domain after four arginines and was inhibited by a furin protease inhibitor.     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid