Induction of metamorphosis with potassium ions requires development of competence and an anterior signalling centre in the ascidian Herdmania momus

 Increased K⁺ concentration in seawater induces metamorphosis in the ascidian Herdmania momus. Larvae cultivated at 24°C exhibit highest rates of metamorphosis when treated with 40 mM KCl-elevated seawater at 21°C. At 24°C, H. momus larvae develop competence to respond to KCl-seawater and initiate metamorphosis approximately 3 h after hatching. Larval trunks and tails separated from the anterior papillae region, but maintained in a common tunic at a distance of greater than 60 μm, do not undergo metamorphosis when treated with KCl-seawater; normal muscle degradation does not occur in separated tails while ampullae develop from papillae-containing anterior fragments. Normal programmed degradation of myofibrils occurs when posterior fragments are fused to papillae-containing anterior fragments. These data indicate that H. momus settlement and metamorphosis only occurs when larvae have attained competence, and suggest that an anterior signalling centre is stimulated to release a factor that induces metamorphosis. Received: 15 May 1996 / Accepted: 19 September 1996     

Photoadaptation and Protection against Active Forms of Oxygen in the Symbiotic Procaryote Prochloron sp. and Its Ascidian Host

Superoxide dismutase, ascorbate peroxidase, and catalase activities were studied in the symbiotic photosynthetic procaryote Prochloron sp. and its ascidian host Lissoclinum patella. The protein-specific activities of these antioxidant enzymes in the Prochloron sp. and L. patella collected at different depths from the Great Barrier Reef, Australia, were directly proportional to irradiance, whereas the pigment concentrations in the Prochloron sp. were inversely proportional to irradiance. The presence of a cyanide-sensitive superoxide dismutase, presumably a Cu-Zn metalloprotein, in the Prochloron sp. extends the possible phylogenetic distribution of this protein. The concentration of UV-absorbing mycosporine-like amino acids is inversely proportional to irradiance in both the host and symbiont, suggesting that these compounds may not provide sufficient protection against UV radiation in high-irradiance environments. The significant differences in the specific activities of these antioxidant enzymes, cellular photosynthetic pigment concentrations, and UV-absorbing compounds from high- and low-irradiance habitats constitute an adaptive response to different photic environments. These photoadaptive responses are essential to prevent inhibition of photosynthesis by high fluxes of visible and UV radiation.     

Differential Responses to Environmental Conditions by Fungal Cells Sensitized by Heat Shock or UV Irradiation

Cells of the ascomycele Ophiostoma multianulatum were sensitized to the supra-optimal temperature of 30°C either by heat shock or by UV irradiation. At this incubation temperature the death rate of the heat-shocked cells was higher than that of the irradiated cells. This difference was increased if hydrolysed casein was added to the incubation medium. The heat-shocked cells were also killed faster at 30°C, if nitrogen instead of air was bubbled through the cell suspension. Heat shock, in contrast to UV irradiation, strongly increased the sensitivity to a high concentration of sodium chloride.     

How incubation temperature influences the physiology and growth of embryonic lizards

Eggs of two small Australian lizards, Lampropholis guichenoti and Bassiana duperreyi, were incubated to hatching at 25 °C and 30 °C. Incubation periods were significantly longer at 25 °C in both species, and temperature had a greater effect on the incubation period of B. duperreyi (41.0 days at 25 °C; 23.1 days at 30 °C) than L. guichenoti (40.1 days at 25 °C; 27.7 days at 30 °C). Patterns of oxygen consumption were similar in both species at both temperatures, being sigmoidal in shape with a fall in the rate of oxygen consumption just prior to hatching. The higher incubation temperature resulted in higher peak and higher pre-hatch rates of oxygen consumption in both species. Total amount of oxygen consumed during incubation was independent of temperature in B. duperreyi, in which approximately 50 ml oxygen was consumed at both temperatures, but eggs of L. guichenoti incubated at 30 °C consumed significantly more (32.6 ml) than eggs incubated at 25 °C (28.5 ml). Hatchling mass was unaffected by either incubation temperature or the amount of water absorbed by eggs during incubation in both species. The energetic production cost of hatchling B. duperreyi (3.52 kJ · g⁻¹) was independent of incubation temperature, whereas in L. guichenoti the production cost was greater at 30 °C (4.00 kJ · g⁻¹) than at 25 °C (3.47 kJ · g⁻¹). Snout-vent lengths and mass of hatchlings were unaffected by incubation temperature in both species, but hatchling B. duperreyi incubated at 30 °C had longer tails (29.3 mm) than those from eggs incubated at 25 °C (26.2 mm). These results indicate that incubation temperature can affect the quality of hatchling lizards in terms of embryonic energy consumption and hatchling morphology. Accepted: 27 January 2000     

Control and possible applications of a novel carrot-spoilage basidiomycete, Fibulorhizoctonia psychrophila

A novel cold-tolerant fungus, Fibulorhizoctonia psychrophila, was isolated from a refrigerated carrot storage facility and identified as an anamorph of Athelia, often classified in Rhizoctonia s.l. Growth of this fungus was observed between 0 and 20°C with an optimum at 9–12°C, while incubation of mycelium grown at 15–32°C resulted in absence of growth even after the fungus was transferred back to 15°C. Growth was inhibited in the presence of the antifungals sorbic acid or natamycin, in particular when the fungus was incubated at 18°C. F. psychrophila produces polysaccharide degrading enzymes that, when compared to enzymes from the ascomycete fungus Aspergillus niger, retain a larger proportion of their activity at lower temperatures. This indicates that F. psychrophila could be used as a source for novel industrial enzymes that are active at 4–15°C.     

Embryonic and larval development of a cold adapted Antarctic ascidian

Ova of the Antarctic ascidian Cnemidocarpa verrucosa were mature at 240–245 μm. At 0 to −1.5°C, embryos hatched as swimming tadpoles at 8 days from fertilization, which is close to the ages at which some Antarctic echinoderm and nemertean embryos hatch as blastulae. Comparisons of Antarctic and temperate ascidian larvae suggest that the ascidian’s development rate is affected by low environmental temperatures to about the same extent as embryos and larvae of an echinoid, nemertean, and calanoid copepods. The ascidian’s tadpoles were bright orange and large, >2 mm in length including tunic and >1.5 mm in length without tunic. The large and brightly colored tadpoles were conspicuous when swimming, which supports the hypothesis that larvae of C. verrucosa are chemically defended against predators. Metamorphosed juveniles were found in cultures within 16 days from fertilization, when some unsettled tadpoles still moved but were less active. The potential pelagic period may therefore be 16 or more days with 8 days as an unhatched embryo and up to 8 or more days as a tadpole. The resting metabolic rate of tadpole larvae was 15 pmol O₂ h⁻¹ individual⁻¹ which is equivalent to larval respiration rates in Antarctic echinoderms. A low resting metabolic rate suggests a potential mechanism for the extended larval lifespan in C. verrucosa.     

The identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae)

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.     

Temperature-dependent phosphate solubilization by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil

Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C, it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions (higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters in most cases at different temperatures.     

Synergistic Action of Recombinant α-Amylase and Glucoamylase on the Hydrolysis of Starch Granules

Barley α-amylase 1 mutant (AMY) and Lentinula edodes glucoamylase (GLA) were cloned and expressed in Saccharomyces cerevisiae. The purified recombinant AMY hydrolyzed corn and wheat starch granules, respectively, at rates 1.7 and 2.5 times that of GLA under the same reaction conditions. AMY and GLA synergistically enhanced the rate of hydrolysis by ∼3× for corn and wheat starch granules, compared to the sum of the individual activities. The exo-endo synergism did not change by varying the ratio of the two enzymes when the total concentration was kept constant. A yield of 4% conversion was obtained after 25 min 37°C incubation (1 unit total enzyme, 15 mg raw starch granules, pH 5.3). The temperature stability of the enzyme mixtures was ≤50°C, but the initial rate of hydrolysis continued to increase with higher temperatures. Ca⁺⁺ enhanced the stability of the free enzymes at 50°C incubation. Inhibition was observed with the addition of 10 mM Fe⁺⁺ or Cu⁺⁺, while Mg⁺⁺ and EDTA had lesser effect. Reference to a company and/or products is only for purposes of information and does not imply approval of recommendation of the product to the exclusion of others that may also be suitable. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

A new xylanase from thermoacidophilic Alicyclobacillus sp. A4 with broad-range pH activity and pH stability

We have identified a highly pH-adaptable and stable xylanase (XynA4) from the thermoacidophilic Alicyclobacillus sp. A4, a strain that was isolated from a hot spring in Yunnan Province, China. The gene (xynA4) that encodes this xylanase was cloned, sequenced, and expressed in Escherichia coli. It encodes a 338-residue polypeptide with a calculated molecular mass of 42.5 kDa. The deduced amino acid sequence is most similar to (53% identity) an endo-1,4-β-xylanase from Geobacillus stearothermophilus that belongs to family 10 of the glycoside hydrolases. Purified recombinant XynA4 exhibited maximum activity at 55°C and pH 7.0, had broad pH adaptability (>40% activity at pH 3.8–9.4) and stability (retaining >80% activity after incubation at pH 2.6–12.0 for 1 h at 37°C), and was highly thermostable (retaining >90% activity after incubation at 60°C for 1 h at pH 7.0). These properties make XynA4 promising for application in the paper industry. This is the first report that describes cloning and expression of a xylanase gene from the genus Alicyclobacillus.     

Adaptation and acclimation of growth and photosynthesis of five Antarctic red algae to low temperatures

Temperature requirements for growth, photosynthesis and dark respiration were determined for five Antarctic red algal species. After acclimation, the stenothermal species Gigartina skottsbergii and Ballia callitricha grew at 0 or up to 5 °C, respectively; the eurythermal species Kallymenia antarctica, Gymnogongrus antarcticus and Phyllophora ahnfeltioides grew up to 10 °C. The temperature optima of photosynthesis were between 10 and 15 °C in the stenothermal species and between 15 and 25 °C in the eurythermal species, irrespective of the growth temperature. This shows that the temperature optima for photosynthesis are located well below the optima from species of other biogeographical regions, even from the Arctic. Respiratory rates rose with increasing temperatures. In contrast to photosynthesis, no temperature optimum was evident between 0 and 25 °C. Partial acclimation of photosynthetic capacity to growth temperature was found in two species. B. callitricha and Gymnogongrus antarcticus acclimate to 0 °C, and 5 and 0 °C, respectively. But acclimation did in no case lead to an overall shift in the temperature optimum of photosynthesis. B. callitricha and Gymnogongrus antarcticus showed acclimation of respiration to 5 °C, and P. ahnfeltioides to 5 and 10 °C, resulting in a temperature independence of respiration when measured at growth temperature. With respect to the acclimation potential of the species, no distinction can be made between the stenothermal versus the eurythermal group. (Net)photosynthetic capacity:respiration (P:R) ratios showed in all species highest values at 0 °C and decreased continuously to values lower than 1.0 at 25 °C. In turn, the low P:R ratios at higher temperatures are assumed to determine the upper temperature growth limit of the studied species. Estimated daily carbon balance reached values between 4.1 and 30.7 mg C g⁻¹ FW day⁻¹ at 0 °C, 16:8 h light/dark cycle, 12–40 μmol m⁻² s⁻¹. Received: 4 November 1999 / Accepted: 7 March 2000     

Genotypic relationships between Prochloron samples from different localities and hosts as determined by DNA-DNA reassociations

Genotypic relationships between seven Prochloron samples isolated from different didemnid ascidian hosts collected at the Palau archipelago and Munda (Solomon Islands) and one cyanobacterial (Synechocystis) strain were determined by DNA-DNA reassociations. Thermal stability values of DNA-DNA hybrids indicate that all Prochloron samples involved are mutually very closely related and only slightly related with the Synechocystis strain. It is concluded that the Prochloron samples tested are representatives of one and the same species.     

Thermoactive β-N-acetylhexosaminidase production by a soil isolate of Penicillium monoverticillium CFR 2 under solid state fermentation: parameter optimization and application for N-acetyl chitooligosaccharides preparation from chitin

Two fungal strains were evaluated for β-N-acetylhexosaminidase production by solid state fermentation using different agro-industrial residues such as commercial wheat bran (CWB) and shrimp shell chitin waste (SSCW), of which Penicillium monoverticillium CFR 2 a local soil isolate showed significantly (P ≤ 0.001) higher β-N-acetylhexosaminidase activity on CWB medium as compared with the activity of Fusarium oxysporum CFR 8. Fermentation parameters such as incubation temperature, incubation time, initial moisture content and inoculum concentration were optimized by statistically designed experiments, using 3**(4–1) fractional factorial design of Response Surface Methodology. The high R² (0.9512) observed during validation experiment showed the usefulness of the model. Highest level of enzyme activity (311.84 U/g IDS) was predicted at 75% (w/w) initial moisture content, 26 °C incubation temperature, 168 h incubation time and initial inoculum, at the highest concentration tested (2.95 ml spore suspension/5 g substrate). Statistical optimization yielded a 4.5 fold increase in β-N-acetylhexosaminidase activity. The crude β-N-acetylhexosaminidase showed optimum temperature of 57 ± 1 °C and pH of 3.6 and retained 50% activity after 1 h of incubation at 57 ± 1 °C. SDS–PAGE zymogram revealed crude enzyme was a monomer with an apparent molecular weight ~110 kDa. The crude enzyme formed 6.81 ± 0.03 mM/l of N-acetyl chitooligosaccharides from colloidal chitin in 24 h of incubation. HPLC analysis revealed hydrolysate contained 37.57% N-acetyl chitotriose and 62.43% N-acetyl chitohexose, indicating its potential for specific N-acetyl chitooligosaccharides production.     

A new thermolabile alkaline phospholipase D from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS628

A phospholipase D (PLD₆₂₈), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7 ∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD₆₂₈ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD₆₂₈ lacks the transphosphatidylation activity. PLD₆₂₈ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.     

The relatedness of Prochloron sp. Isolated from different didemnid ascidian hosts

The relationship of Prochloron sp. isolated from four different didemnid ascidian hosts, namely Lissoclinum patella, Lissoclinum voeltzkowi, Diplosoma virensand Trididemnum cyclops was elucidated by comparative analysis of their 16S ribosomal ribonucleic acid (RNA). The oligonucleotide catalogues of the 16S rRNA obtained are almost identical, indicating a very close relationship among the prochlorophytes investigated. Phylogenetically Prochloron is a member of Cyanobacteriales.     

COMBINED EFFECTS OF ULTRAVIOLET RADIATION AND TEMPERATURE ON MORPHOLOGY,PHOTOSYNTHESIS, AND DNA OF ARTHROSPIRA (SPIRULINA) PLATENSIS (CYANOPHYTA)1

Natural levels of solar UVR were shown to break and alter the spiral structure of Arthrospira (Spirulina) platensis (Nordst.) Gomont during winter. However, this phenomenon was not observed during summer at temperatures of ～30°C. Since little has been documented on the interactive effects of solar UV radiation (UVR; 280–400 nm) and temperature on cyanobacteria, the morphology, photosynthesis, and DNA damage of A. platensis were examined using two radiation treatments (PAR [400–700 nm] and PAB [PAR + UV‐A + UV‐B: 280–700]), three temperatures (15, 22, and 30°C), and three biomass concentrations (100, 160, and 240 mg dwt [dry weight] · L^?1). UVR caused a breakage of the spiral structure at 15°C and 22°C, but not at 30°C. High PAR levels also induced a significant breakage at 15°C and 22°C, but only at low biomass densities, and to lesser extent when compared with the PAB treatment. A. platensis was able to alter its spiral structure by increasing helix tightness at the highest temperature tested. The photochemical efficiency was depressed to undetectable levels at 15°C but was relatively high at 30°C even under the treatment with UVR in 8 h. At 30°C, UVR led to 93%–97% less DNA damage when compared with 15°C after 8 h of exposure. UV‐absorbing compounds were determined as negligible at all light and temperature combinations. The possible mechanisms for the temperature‐dependent effects of UVR on this organism are discussed in this paper.     

Tyrosine 39 of GH13 <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Thermococcus hydrothermalis</Emphasis> contributes to its thermostability

The presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine (present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ± 0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from 85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could contribute to the thermostability of the α-amylase from T. hydrothermalis.     

Production of extracellular alkaline lipase by a new thermophilicBacillus sp

A thermophilic soil isolate—Bacillus sp. RS-12, grew optimally at 50°C and not below 40°C. Production of an extracellular lipase by this organism was substantially enhanced when the type and concentration of carbon and nitrogen sources and initial pH of the culture medium were consecutively optimized. The lipase production was found to be growth-associated with maximum secretion in the late exponential growth phase,i.e. 15h of incubation. The enzyme activity as high as 0.98 nkat/mL was obtained under optimum conditions. Tween 80 (0.5%) and yeast extract (0.5%) were found to be the best carbon and nitrogen sources inducing maximum enzyme yield with initial pH 8.0 at 50°C. The kinetic characteristics of the crude lipase indicated the highest activity at 50–55°C and pH 8.0. It had a half life of 60, 18 and 15 min at 65, 70 and 75°C, respectively.     

A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1

A complete gene, xyl10C, encoding a thermophilic endo-1,4-β-xylanase (XYL10C), was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. XYL10C shares highest nucleotide and amino acid sequence identities of 57.3 and 49.7%, respectively, with a putative xylanase from Aspergillus fumigatus Af293 of glycoside hydrolase family 10. A high expression level in P. pastoris (73,400 U ml⁻¹) was achieved in a 3.7–l fermenter. The purified recombinant XYL10C was thermophilic, exhibiting maximum activity at 85°C, which is higher than that reported from any fungal xylanase. The enzyme was also highly thermostable, exhibiting ~100% of the initial activity after incubation at 80°C for 60 min and >87% of activity at 90°C for 10 min. The half lives of XYL10C at 80 and 85°C were approximately 45 and 3 h, respectively. It had two activity peaks at pH 3.0 and 4.5–5.0 (maximum), respectively, and was very acid stable, retaining more than 80% activity after incubation at pH 1.5−6.0 for 1 h. The enzyme was resistant to Co²⁺, Mn²⁺, Cr³⁺ and Ag⁺. The specific activity of XYL10C for oat spelt xylan was 18,831 U mg⁻¹. It also had wide substrate specificity and produced simple products (65.1% xylose, 25.0% xylobiose and 9.9% xylan polymer) from oat spelt xylan.