Morphology of a spectrum of parasporal endotoxin crystals from cultures of Bacillus thuringiensis ssp. kurstaki isolate A3-4

Morphology of the parasporal -endotoxin crystals of Bacillus thuringiensis subsp. kurstaki isolate A3-4, a native (Taiwan) strain was studied by scanning (SEM) and transmission (TEM) electron microscopy. The typical bipyramidal crystals and an unusual parasporal inclusion with an embedded body were observed. The parasporal -endotoxin crystal with an embedded body, which was in different size and shape was well demonstrated in thin sections by transmission electron microscopy. The sporulated cells had multiple individually separated inclusions up to four crystals in one cell, which was unique to this isolate, and has not been reported before. The -endotoxin crystals included in the cell or released from the cell after batch fermentation or fed-batch fermentation did not show any altered morphological characteristics. However, judging from thin-sections of TEM, cells and the included parasporal crystals from fed-batch fermentation appeared larger than those from batch fermentation. It was observed that release of spore and parasporal crystals from the bacterial cell produced from batch cultures was earlier than that of fed-batch cultures. Preliminary bioassay results showed that the isolate cultures from both types of culture were equally effective against Plutella xylostella larvae. Based on the morphological observations, this strain may have a multiple insecticidal activities toward different insect species.     

Halophilic Archaea cultured from ancient halite,Death Valley,California

Halophilic Archaea cultured from ancient fluid inclusions in a 90‐m‐long (0‐ to 100 000‐year‐old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34 000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface‐sterilized halite crystals exclusively in one section of the core (13.0–17.8 m; 22 000–34 000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1–86.7 m (10 000–100 000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra‐laboratory reproducibility. Inter‐laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.     

Naturally occurring apatite as a source of orthophosphate for growth of bacteria and algae

Several naturally occurring calcium-phosphate apatites which varied in crystalline structure and ionic composition were added as crystals of different particle size to P-free (<1g/liter total P) nutrient media. Sufficient ortho-PO ₄ ^3– was released by the partial dissolution of apatite crystals at limnetic pH levels (pH 7.8) to support growth of several unialgal-mixed bacterial cultures. The biomass produced by mixed populations increased as the amount of available apatite was increased and as the pH of the media and the particle size of the apatite crystals were decreased. These findings suggest that although apatite characteristically displays reduced solubility under alkaline conditions, the tons of apatite which are continuously entering aquatic environments as erosion material may be contributing to the P loading of those ecosystems.     

Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium

Several aromatic compounds increased initial lignin degradation rates in cultures of Phanerochaete chrysosporium. This activation was connected to increased H₂O₂ production and glucose oxidation rates. Veratryl alcohol, a natural secondary metabolite of P. chrysosporium, also activated the lignin-degrading system. In the presence of added veratryl alcohol the ligninolytic system appeared 6–8 h earlier than in reference cultures. This effect was only seen when lignin was added after the primary growth was completed because lignin itself also caused earlier appearance of the degradative system. In cultures which received no added lignin or veratryl alcohol the ligninolytic activity only appeared once the alcohol started to accumulate. The degradation patterns of veratryl alcohol and lignin were similar. The activity levels of lignin degradation and glucose oxidation could be regulated by veratryl alcohol concentration. It is suggested that either veratryl alcohol itself or a metabolite derived from it is actually responsible for the low levels of ligninolytic activity in glucose grown cultures.     

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

Summary Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CM^R filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.     

Carbonic anhydrase,carbondioxide levels and growth of Nitrosomonas

The ammonia oxidizing bacterium Nitrosomonas europaea was grown either (a) with added bicarbonate in the absence of added CO₂ (bubbled through the culture), (b) with added bicarbonate plus low added CO₂ (0.03% v/v), or (c) without added bicarbonate with high added CO₂ (1% v/v). Cell doubling times of 12 h were observed in 1% cultures; doubling times of 2 to 3-fold longer wre found with 0.03% CO₂ and/or bicarbonate grown cultures. The specific activity of carbonic anhydrase was 40–80% lower in cultures grown on 1% CO₂. These results are compared with those in heterotrophic and photosynthetic microorganisms.Scientific Contribution Number 1241 from the New Hampshire Agricultural Experiment Station     

Experimental Colonization and Alteration of Orthopyroxene by the Pleomorphic Bacteria Ramlibacter tataouinensis

The colonization of orthopyroxene crystals by a pleomorphic bacterium and the mineralogical products resulting from a prolonged interaction have been studied. We used Ramlibacter tataouinensis (strain TTB310), which is an aerobic β-Proteobacterium moving over surfaces by gliding motility and whose life cycle includes rods and spherical cysts. Analysis of cultures grown on solid media with micrometer-sized pyroxene and quartz crystals scattered over the surface revealed a taxis of the bacteria toward the crystals. Given the mineralogical non-specificity of the interaction, a mechanism of elasticotaxis is inferred. After the rods had adhered to the pyroxene surface, they differentiated into cysts leading to the formation of microcolonies that were centered on a crystal grain. This suggests an original coupling between the life cycle of R. tataouinensis and the interaction with the crystals. The alteration of orthopyroxene was studied by high-resolution transmission electron microscopy at the interface between cysts and pyroxene crystals. The pyroxene surface showed an amorphous layer that was more developed than that of abiotic control samples processed under the same conditions. Moreover, chemical analyses showed that the dissolution of pyroxene was reduced in the presence of R. tataouinensis. The origin and the significance of the amorphous layers is discussed.     

Purine Composition of the Crystalline Cytoplasmic Inclusions of Paramecium tetraurelia

Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.     

Autoclaved fungal mycelia increase diosgenin production in cell suspension cultures of Dioscorea deltoidea

The addition of autoclaved mycelia of non-host specific fungi to cell suspension cultures of Dioscorea deltoidea improved diosgenin production by as much as 72% compared to control cultures. Phytoalexin elicitors laminarin, arachidonic acid and chitin added to D. deltoidea cultures had no stimulating effect on the diosgenin level.     

The nature of the microbial stimulus affecting sporophore formation in Agaricus bisporus (Lange) Sing

An apparatus is described in which pure cultures of Agaricus bisporus were maintained on composted media in filtered atmospheres free from (a) noxious concentrations of carbon dioxide, and (b) contaminating microorganisms. When grown on compost alone, cultures of A. bisporus did not produce sporophores. Their formation was however stimulated by a covering layer of an unsterilized mixture of peat and chalk (=‘casing’ soil). Autoclaving or fumigating ‘casing’ with propylene oxide decreased populations of contaminating bacteria and prevented sporophore formation. Populations of micro-organisms isolated from unsterile ‘casing’ contained bacteria which when added to pure cultures of A. bisporus stimulated fruit-body formation. Numbers of these stimulators increased when cultured on a carbon-free liquid medium exposed to atmospheres with ethanol, ethyl acetate and acetone or containing the volatile metabolites of A. bisporus. The ability to utilize these volatile chemicals was exploited in a selective technique for isolating sporophore stimulators where aqueous suspensions of mixed bacterial populations were exposed to atmospheres of these materials for 5 days, before aliquots were added to agar media subsequently gelled. The stimulatory bacteria were identified as, or closely related to, Pseudomonas putida.     

Effects of static magnetic field on magnetosome formation and expression of mamA,mms13, mms6 and magA in Magnetospirillum magneticum AMB‐1

Magnetotactic bacteria produce nanometer‐size intracellular magnetic crystals. The superior crystalline and magnetic properties of magnetosomes have been attracting much interest in medical applications. To investigate effects of intense static magnetic field on magnetosome formation in Magnetospirillum magneticum AMB‐1, cultures inoculated with either magnetic or non‐magnetic pre‐cultures were incubated under 0.2 T static magnetic field or geomagnetic field. The results showed that static magnetic field could impair the cellular growth and raise C_mag values of the cultures, which means that the percentage of magnetosome‐containing bacteria was increased. Static magnetic field exposure also caused an increased number of magnetic particles per cell, which could contribute to the increased cellular magnetism. The iron depletion in medium was slightly increased after static magnetic field exposure. The linearity of magnetosome chain was also affected by static magnetic field. Moreover, the applied intense magnetic field up‐regulated mamA, mms13, magA expression when cultures were inoculated with magnetic cells, and mms13 expression in cultures inoculated with non‐magnetic cells. The results implied that the interaction of the magnetic field created by magnetosomes in AMB‐1 was affected by the imposed magnetic field. The applied static magnetic field could affect the formation of magnetic crystals and the arrangement of the neighboring magnetosome. Bioelectromagnetics 30:313–321, 2009. © 2009 Wiley‐Liss, Inc.     

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen-starved in the presence of sodium. The carbon-to-nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen- and sodium-deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.     

Ultrastructure of the Marine Cryptomonad Chroomonas salina Cultured under Conditions of Photoautotrophy and Glycerol-Heterotrophy*

SYNOPSIS. Chroomonas salina was cultured in seawater medium enriched with nitrate, phosphate, silicate, trace-metal ions, and vitamins, under 3 conditions: (A) light without other organic additions (photoautotrophic); (B) light and added glycerol (photoheterotrophic); (C) in darkness but with added glycerol (chemoheterotrophic). The heterotrophic cultures were initiated from a stock maintained on glycerol in continuous darkness for 41/4 years. The autotrophic culture was initiated from a corresponding stock maintained under continuous illumination without any organic growth substrate. The fine structure of organisms from simultaneously initiated cultures was compared after 1, 2, 3, and 4 weeks of growth. “Young'’cells from the autotrophic and heterotrophic cultures of comparable maturity had no recognizable ultrastructural difference. In organisms from both the photoautotrophic and photoheterotrophic cultures there was a progressive accumulation of starch and lipid with aging, but whereas in cells from the former the production of starch was arrested after early growth and lipid was concentrated thereafter, in those from the latter both metabolites continued to be produced with consequent rapid degeneration of the cytoplasm followed by autolysis. By contrast, flagellates grown in the chemoheterotrophic culture accumulated only starch, with vacuole formation replacing the lipid stores. In all cases, the lipid bodies appeared to differ from the membrane-bound droplets normally observed, which actually diminished with aging. Starch accumulation appeared to cause more rapid cytopathologic changes and autolysis. No evidence of chloroplastic phycobilisome-type aggregations was noted in organisms from any culture at any age.     

Improved paclitaxel production in a two-phase suspension culture ofTaxus cuspidata using silicone cubes

Two-phase cultures ofTaxus cuspidata were performed using silicone cubes as a second phase in shake flasks for paclitaxel production. Among various taxanes, paclitaxel was selectively adsorbed on the silicon cubes. When silicone cubes were added to suspension culture ofTaxus cuspidata, paclitaxel production increased about 45 folds. The maximum paclitaxel production was 3.95 mg/L when 10% of silicone cubes were added to the culture at the 7th day from inoculation.     

The Role of a Green Alga in the Precipitation of Calcite and the Coprecipitation of Phosphate in Freshwater

The precipitation of calcite from a calcium bicarbonate solution, similar in ionic strength to natural hardwaters, was observed in a series of experiments utilizing an automated culture apparatus. Seeded growth experiments, using calcite seed crystals, were performed at a range of phosphate concentrations to observe inhibitory effects. These experiments demonstrated a linear relationship of increasing inhibition with increasing initial phosphate concentration. A further series of experiments was performed in which an actively photosynthesizing culture of a unicellular green alga (Chlorococcum sp.) was added to the culture vessel in order to initiate precipitation. Experiments to observe spontaneous precipitation, occurring in the absence of both seed and alga additions, were carried out to compare with precipitation rates in the algal experiments. A control experiment was also performed to investigate whether precipitation occurrred in algal cultures maintained in darkness. The carbonate site mechanistic model, developed for calcite precipitation in abiotic conditions, was used to analyse the results of the algal experiments and found to be applicable.     

THE EFFECT OF IRON NUTRITION ON PHOTOSYNTHESIS AND NITROGEN FIXATION IN CULTURES OF TRICHODESMIUM (CYANOPHYCEAE)1

Cultures of Trichodesmium NIBB 1067 were grown in the synthetic medium AQUIL with a range of iron added from none to 5 × 10^?7 M Fe for 15 days. Chlorophyll-a, cell counts, and total cell volume were two or three times higher in medium with 10^?7 M Fe than with no added Fe. Oxygen production rate per chlorophyll-a was over 60% higher with higher iron. Increased iron stimulated photosynthesis at all irradiances from about 12–250 μE · m^?2· s^?1. Nitrogen fixation rate, estimated from acetylene reduction, for 10^?7 and 10^?8 M Fe cultures was approximately twice that of the cultures with no added Fe. The range of rates of O₂ production and N₂ fixation in cultures at the iron concentrations we used were similar to the rates from natural samples of Trichodesmium from both the Atlantic, and the Pacific oceans. This similarity may allow this clone to be used, with some caution, for future physiological ecology studies. This study demonstrates the importance of iron to photosynthesis and nitrogen fixation and suggests that Trichodesmium plays a central role in the biogeochemical cycles of iron, carbon and nitrogen.     

Effect of anti-microtubular drug amiprophos-methyl on somatic embryogenesis and DNA ploidy levels in alfalfa and carrot cell suspension cultures

A short treatment with the anti-microtubular drug amiprophos-methyl (APM) blocked somatic embryogenesis in alfalfa(Medicago sativa L.) and carrot (Daucus carota L.). The interruption was temporary and restoration of somatic embryogenesis was observed in long-term cultures. In addition to the effect on somatic embryogenesis, APM treatment induced polyploidization the extent of which was concentration dependent. In long-term alfalfa cultures, APM-induced loss of somatic embryogenesis led to ploidy instability and to a shift to DNA aneuploidy. Critical stages of somatic embryogenesis sensitive to disruption of microtubule-mediated processes were determined in carrot cell cultures. Complete embryogenic arrest occurred when APM was added within the first 5 d of embryogenesis from single cells. The role of the cytoskeleton in the first events of somatic embryogenesis and the relation between totipotency and ploidy stabilityin vitro is discussed.     

INTERACTIONS AMONG PHOSPHATE UPTAKE,PHOTOSYNTHESIS, AND CHLOROPHYLL FLUORESCENCE IN NUTRIENT‐LIMITED CULTURES OF THE CHLOROPHYTE MICROALGA DUNALIELLA TERTIOLECTA1

Phosphate‐limited and phosphate‐sufficient continuous cultures of the marine chlorophyte microalga Dunaliella tertiolecta Butcher were examined for their responses to the addition of phosphate. Phosphate‐limited cultures showed a marked quenching of chl fluorescence following a pulse of phosphate. This response was absent from cells growing under phosphate‐sufficient conditions. Both the extent of fluorescence quenching (where present) and the initial rate of change in quenching were dependent on the concentration of phosphate added to cell suspensions and on the degree of limitation (growth rate in continuous culture). The addition of phosphate also brought about a transient decrease in photosynthetic oxygen evolution and a stimulation in respiration, which were relaxed as the added phosphate was depleted from the external medium. The applicability of using nutrient‐induced fluorescence transients as a tool to identify the nutrient status of phytoplankton populations is discussed.     

INTERACTION (ALLELOPATHY) BETWEEN MARINE DIATOMS: THALASSIOSIRA PSEUDONANA AND PHAEODACTYLUM TRICORNUTUM1

Two marine diatoms were studied singly and in mixed culture. Thalassiosira pseudonana (Hust.) Hade & Heimdal was capable of a higher growth rate (μ_max) than Phaeodactylum tricornutum Bohlin. In two-species batch cultures P. tricornutum took over in the latter portion of the exponential phase, possibly due to allelopathy. The filtrate from this species caused an initial lag phase and a reduced terminal population density for T. pseudonana. Two-species continuous cultures showed verification of these interactions. At high dilution rate (i.e., high growth rate) P. tricornutum washed out when added at low density, whereas T. pseudonana maintained constant cell density. However, when sufficient density of P. tricornutum was added as a contaminant, both species washed out. At a lower dilution rate P. tricornutum increased in density when added and eventually reached a stable population; T. pseudonana then washed out.