Role of trehalose in the spores of Streptomyces

Abstract Dormant spores of Streptomyces antibioticus contain large amounts of trehalose (11–12% of dry weight) and can be subjected to a dehydration treatment without a significant loss of viability. Loss of dehydration resistance coincided with a decrease in the trehalose level of the spores, under different conditions of incubation. The viability of dehydration-sensitive cells was enhanced by the presence of exogenous trehalose during dehydration. The morphology and functional activity of isolated membranes of S. antibioticus can be retained when dehydrated in the presence of trehalose. It is suggested that, in dormant spores of S. antibioticus , trehalose may serve to protect cellular components during dehydration by acting as a substitute for water.     

Pathways of glucose catabolism during germination of Streptomyces spores

Abstract The participation of the different glucose-catabolic pathways during germination of Streptomyces antibioticus spores was studied. In dormant spores, glucose is catabolized through the pentose phosphate (PP) and the Embden-Meyerhof-Parnas (EMP) pathways, with an active tricarboxylic acid cycle. The relative participation of each catabolic pathway is regulated by germinative or non-germinative conditions. During spore germination, the pentose phosphate pathway continuously increased in its participation in the glucose catabolism and it was the major glucose-catabolic pathway in the exponential phase of growth. In addition, it showed the existence of an active tricarboxylic acid cycle in dormant spores, which was being drained for biosynthetic purposes.     

Properties of the germination inhibitor of Streptomyces viridochromogenes spores

Germinating spores of Streptomyces viridochromogenes excreted a substance into the surrounding medium which inhibited germination of another sample of the spores. The germination inhibitor (GI) was produced during submerged culture after exponential growth had ceased. The GI was purified 51-fold following extraction from growth liquor with chloroform. It was soluble in alcohol and water and had a molecular weight of less than 1000. The GI blocked growth and respiration of some Gram-positive bacteria and was an inhibitor of the membrane bound, but not solubilized, calcium-dependent ATPase of germinated spores and mycelia of the producing organism. Several sodium-potassium activated ATPases were also inhibited. All four activities (respiration, growth, germination inhibition, ATPase) co-purified during column and thin-layer chromatography. The GI activities released during germination and produced during growth were identical. A role for the GI antibiotic in regulation of dormancy of spores of the producing organism is discussed.     

Nutritionally defined conditions for germination of Streptomyces viridochromogenes spores.

Spores of Streptomyces viridochromogenes were removed from the surface of solid media with glass beads and suspended in a buffer-detergent solution. Addition of yeast extract and glucose resulted in rapid loss of refractility of the spores. Appearance of germ tubes followed. Germination was accompanied by a decrease in the optical density (OD) of the suspension. The OD decrease was used as an assay for germination. A defined germination medium (DGM) comprised of L-alanine, L-glutamic acid, adenosine, para-aminobenzoic acid, and calcium and magnesium ions provided a germination rate nearly equal to that of complex media. The germination rate was essentially the same if D-alanine and D-glutamate replaced the L-isomers. The optimum pH and temperature for germination were 7.0 and 35 C. Germination was absolutely dependent on the presence of CO2. Spores harvested after growth for longer periods than the usual time (10 days) became less germinable in DGM. The same was observed for spores grown at 37 C as compared with 30 C. Spores incubated in DGM for various time periods before being transferred to a buffer solution did not continue to germinate. Spores harvested after growth of eight species of Streptomyces did not show a decrease in OD when incubated in yeast extract medium. Another strain of S. viridochromogenes did exhibit an OD decrease in the medium. Comparative properties of spores of streptomycetes, fungi, and bacilli are discussed.     

Control of the circadian rhythm of carbon dioxide assimilation in Bryophyllum leaves by exposure to darkness and high carbon dioxide concentrations

The circadian rhythm of CO₂ assimilation in detached leaves of Bryophyllum fedtschenkoi at 15° C in normal air and continuous illumination is inhibited both by exposure to darkness, and to an atmosphere enriched with 5% CO₂. During such exposures substantial fixation of CO₂ takes place, and the malate concentration in the cell sap increases from about 20 mM to a constant value of 40–50 mM after 16 h. On transferring the darkened leaves to light, and those exposed to 5% CO₂ to normal air, a circadian rhythm of CO₂ assimilation begins again. The phase of this rhythm is determined by the time the transfer is made since the first peak occurs about 24 h afterwards. This finding indicates that the circadian oscillator is driven to, and held at, an identical, fixed phase point in its cycle after 16 h exposure to darkness or to 5% CO₂, and it is from this phase point that oscillation begins after the inhibiting condition is removed. This fixed phase point is characterised by the leaves having acquired a high malate content. The rhythm therefore begins with a period of malate decarboxylation which lasts for about 8 h, during which time the malate content of the leaf cells must be reduced to a value that allows phosphoenolpyruvate carboxylase to become active. Inhibition of the rhythm in darkness, and on exposure to 5% CO₂ in continuous illumination, appears to be due to the presence of a high concentration of CO₂ within the leaf inhibiting malic enzyme which leads to the accumulation of high concentrations of malate in the leaf cells. The malate then allosterically inhibits phosphoenolpyruvate carboxylase upon which the rhythm depends. The results give support to the view that malate synthesis and breakdown form an integral part of the circadian oscillator in this tissue.Abbreviations B. Bryophyllum - PEPCase phosphoenolpyruvate carboxylase     

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH₄)₂SO₄ (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH₂PO₄. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.     

Demulsification of oil-in-water emulsions by aerial spores of a Streptomyces sp.

Of a number of actinomycetes isolated from Antarctica and coastal regions of Korea, one isolate, identified as a Streptomycessp., demulsified an emulsion of kerosene/0.2% Triton X-100 (2:1, v/v) within 1 min. The aerial spores broke emulsions of low viscosity hydrocarbons such as kerosene and gasoline within 1 min, but after 3 min of contact they could demulsify less than 50% of emulsions of high viscosity hydrocarbons diluted with kerosene.     

Short communication: Effect of water activity on cell growth and phenol oxidase production by Streptomyces cyaneus var. viridochromogenes in surface culture

In surface cultures of Streptomyces cyaneus var. viridochromogenes, NaCl depressed water activity (a _w) without supporting growth. Reducing a _w from 0.987 to 0.951 led to 3- and 4-fold increases in intracellular and extracellular phenol oxidase activities, respectively.     

Sagebrush and grasshopper responses to atmospheric carbon dioxide concentration

Summary Seed- and clonally-propagated plants of Big Sagebrush (Artemisia tridentata var.tridentata) were grown under atmospheric carbon dioxide regimes of 270, 350 and 650 μl l⁻¹ and fed toMelanoplus differentialis andM. sanguinipes grasshoppers. Total shrub biomass significantly increased as carbon dioxide levels increased, as did the weight and area of individual leaves. Plants grown from seed collected in a single population exhibited a 3–5 fold variation in the concentration of leaf volatile mono- and sesquiterpenes, guaianolide sesquiterpene lactones, coumarins and flavones within each CO₂ treatment. The concentration of leaf allelochemicals did not differ significantly among CO₂ treatments for these seed-propagated plants. Further, when genotypic variation was controlled by vegetative propagation, allelochemical concentrations also did not differ among carbon dioxide treatments. On the other hand, overall leaf nitrogen concentration declined significantly with elevated CO₂. Carbon accumulation was seen to dilute leaf nitrogen as the balance of leaf carbon versus nitrogen progressively increased as CO₂ growth concentration increased. Grasshopper feeding was highest on sagebrush leaves grown under 270 and 650 μl l⁻¹ CO₂, but varied widely within treatments. Leaf nitrogen concentration was an important positive factor in grasshopper relative growth but had no overall effect on consumption. Potential compensatory consumption by these generalist grasshoppers was apparently limited by the sagebrush allelochemicals. Insects with a greater ability to feed on chemically defended host plants under carbon dioxide enrichment may ultimately consume leaves with a lower nitrogen concentration but the same concentration of allelochemicals. Compensatory feeding may potentially increase the amount of dietary allelochemicals ingested for each unit of nitrogen consumed.     

Morphological characterization and germination of aerial and submerged spores of the entomopathogenic fungus Verticillium lecanii

Under solid-state and liquid-state cultivations, the entomopathogenic fungus Verticillium lecanii F091 produced different types of spores. The aerial spores (AS) on cooked rice formed clusters on the tips of conidiophores, while the submerged spores (SS) were dispersed in the medium. The aerial spore appeared relatively uniform in size, which was 6.1 ± 0.9 m long, and 2.2 ± 0.3 m wide. The submerged spore varied in shape and size, with a mean length of 5.0 ± 1.0 m and width of 1.9 ± 0.5 m. Under scanning electron microscopy, the AS had a tendency to have rough, brittle surface characteristics; however, the SS appeared smooth on the surface. These spores were compared in two different germination media. On SMAY (Sabouraud maltose, agar, yeast extract, and neopeptone) coated coverslips, the AS did not show germ tubes until 8 h of incubation; while the SS showed many germ tubes. However, over 90% spore germination ratio was reached for both types of spores at 18-h of incubation. In the liquid medium, the SS germinated rapidly and many spores even produced spores on the spores; while the AS germinated, grew, and branched in the submerged culture gradually, and some sporulated on the tips of the short branches, or on the mycelia until 18 h of incubation. Evidently, the germination, growth patterns of aerial or submerged spores differed greatly under the different culture conditions. The virulence of the pathogen in relation to the type of spore of V. lecanii is discussed.     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.     

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO₂ assimilation in Chlamydomonas has been developed in order to evaluate whether a CO₂-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO₂, low photorespiration, little O₂ inhibition of photosynthesis, and low CO₂ compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO₂-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO₂, and active transport of HCO ₃ ^- . Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO₂ diffusion or reversal of HCO ₃ ^- transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO₂-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO₂-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO₂-concentrating system. These calculations indicate that the system may be no more energy-costly than C₄ photosynthesis.Abbreviations Chl chlorophyll - RuBPC/O ribulose 1,5-bisphosphate carboxylase-oxygenase - CA carbonic anhydrase     

Effect of carbon dioxide on pigment and membrane content in Synechococcus lividus

The effect of carbon dioxide on pigment and membrane content in Synechococcus lividus was studied by depriving cells of CO₂ and examining cell populations biochemically and by electron microscopy. After 120 h of CO₂ deprivation, S. lividus lost all detectable chlorophyll a and C-phycocyanin. Such bleached cultures were mustard yellow, the result of approximately 1.8 times more carotenoid per cell than green control cultures.Although cells from beached cultures appeared morphologically identical to control green cells when examined by light microscopy, electron microscopic examination revealed them to be devoid of detectable thylakoid membrane. Thylakoid membrane could not be recovered by physical isolation or revealed by freeze etching of bleached S. lividus. In addition, inclusion bodies characteristically found in S. lividus were also absent.Reintroduction of CO₂ into bleached cultures resulted in a rapid resynthesis of both chlorophyll a and C-phycocyanin. Electron microscopic examination of these regreening cultures revealed that thylakoid membrane was also rapidly resynthesized. Growth of regreened cultures did not occur until there was the synthesis of a full complement of chlorophyll a, C-phycocyanin, and thylakoid membrane.A time course study of the cytological events occurring during bleaching and regreening is presented.     

Uranous ion oxidation and carbon dioxide fixation byThiobacillus ferrooxidans

The stoichiometric oxidation of uranous-to uranyl-uranium byThiobacllus ferrooxidans is demonstrated. Fixation of¹⁴CO₂ and the effect of inhibitors demonstrate that energy is conserved during the oxidation and used for energy-dependent reverse electron flow and carbon dioxide fixation.Abbreviations HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - 8-HQ 8-hydroxyquinoline - TTFA thenoyltrifluoroacetone     

Antibiotics-free stable polyhydroxyalkanoate (PHA) production from carbon dioxide by recombinant cyanobacteria

A practical antibiotics-free plasmid expression system in cyanobacteria was developed by using the complementation of cyanobacterial recA null mutation with the EscherichiacolirecA gene on the plasmid. This system was applied to the production of polyhydroxyalkanoate (PHA), a biodegradable plastic, and the transgenic cyanobacteria stably maintained the pha genes for PHA production in the antibiotics-free medium, and accumulated up to 52% cell dry weight of PHA.     

Fusarium andDidymella-neglected spores in the air

Summary This paper reports about the occurence ofFusarium- andDidymella spores in the air of Essen/FRG. During the spore season 1990, the spore concentration was measured on several days with a volumetric pollen trap by hourly analysis. The calculated amount of spores per hour is compared to the data of a pluviometer and the values of the relative humidity during the same period.The occurence of both spore types in the air and high relative humidity (>80%) are correlated in a highly significant way (P<0.001). The dispersion of spores starts when rain begins or directly after the precipitation.Didymella reaches higher concentrations thanFusarium in the air (Maximum values:Didymella 30000 spores/m³,Fusarium only 800 spores/m³). During the emission of the spores the temperature varied between 10°C und 20°C degrees. Didymella andFusarium must be an important allergenic source in the outdoor area, because of their allergen-loaded biological aerosols. The question of providing well defined extracts ofDidymella exitialis is given to the pharmaceutical industry.     

The negligible role of carbon dioxide and ethylene in ajmalicine production by Catharanthus roseus cell suspensions

Summary Removal of gaseous metabolites in an aerated fermenter affects ajmalicine production by Catharanthus roseus negatively. Therefore, the role of CO₂ and ethylene in ajmalicine production by C. roseus was investigated in 3 l fermenters (working volume 1.8 l) with recirculation of a large part of the exhaust air. Removal of CO₂, ethylene or both from the recirculation stream did not have an effect on ajmalicine production. Inhibition of ethylene biosynthesis in shake flasks with Co²⁺, Ni²⁺ or aminooxyacetic acid did not affect ajmalicine production. However, the removal of CO₂ did enhance the amount of extracellular ajmalicine.     

Importance of carbon dioxide for the cultivation of Thermoactinomyces vulgaris from spores

Thermoactinomyces vulgaris has been used for the production of the serine protease Thermitase [EC 3.4.21.14] by fermentation. Spore germination of this strain is influenced by the gas composition of the medium. Since the outgrowth of the germ tubes occurs only in the presence of a raised CO₂ concentration, the cultivation in aerated stirred fermenters is not reproducible. However, if cultivation takes place in a closed fermentation system, initially unaerated, in which the CO₂ formed by spore respiration accumulates, reproducible germination can be obtained. From an O₂-balance, indirect conclusions can be drawn with regard to the effect of CO₂, and the process of germination can be described. The cultivation method described permits almost complete germination of all inoculated spores and reproducible rates of biomass and enzyme production in the subsequent fermentation. Thus, stabilization of the procedure with increased enzyme yields is ensured.     

Selective isolation and characterisation of members of the Streptomyces violaceusniger clade associated with the roots of Paraserianthes falcataria

Large numbers of putatively novel streptomycetes were isolated from environmental samples collected from in and around the root system of the tropical angiosperm, Paraserianthes falcataria. Representative isolates were assigned to 37 multi-membered and 107 single membered colour groups based on their ability to form pigments on oatmeal and peptone yeast extract iron agars. The largest taxon, colour group 3, encompassed 94 isolates which had morphological properties typical of members of the Streptomyces violaceusniger clade. Twelve representatives of this taxon chosen on the basis of Curie-point pyrolysis mass spectrometric data were compared with representatives of the validly described species which constitute the Streptomyces violaceusniger clade. Six out of the twelve representative strains were readily distinguished from one another and from the marker strains using a combination of genotypic and phenotypic properties. These organisms were consequently considered to merit species status as Streptomyces asiaticus sp. nov., Streptomyces cangkringensis sp. nov., Streptomyces indonesiensis sp. nov., Streptomyces javensis sp. nov., Streptomyces rhizosphaerius sp. nov. and Streptomyces yogyakartensis sp. nov.     

Sporogenesis in Streptomyces melanochromogenes

The mode of spore differentiation in a strain of Streptomyces melanochromogenes was followed by analysis of ultrathin sections of sporulating aerial hyphae at various stages of sporogenesis. A special accent was laid on the formation of the sporulation septum and its alterations in the course of spore delimitation and separation. Distinct differences in formation and substructure have been observed between the cross walls of vegetative hyphae and the sporulation septa.Cross walls of vegetative hyphae are formed in a way typical for Gram-positive bacteria by a centripetal annular ingrowth of cytoplasmic membrane, on which wall material immediately is deposited. The development of the sporulation septa is characterized by the accumulation of amorphous material in addition to the newly synthesized wall layer inside the invaginating cytoplasmic membrane. This amorphous septal material will later be decomposed presumably by two lytic systems which cause the separation of the spores. The central region of the finished sporulation septum is perforated by microplasmodesmata. Spores are released by a break down of the surface sheath. The complete spores are enveloped by a twolayered cell wall and the spiny surface sheath.