Temperature effects on solute accumulation by Candida utilis

Summary A study was made of the effect of temperature on accumulation of glucosamine and 2-aminoisobutyrate by Candida utilis NCYC 321 grown at 30° C or 10° C. Exponential-phase cells contained greater proportions of C_16:1 and C_18:3 acids, and smaller proportions of C_13:1 and C_18:2 acids, when grown in a defined medium at 10° C compared with 30° C. Cells grown at 30° C or 10° C were able to accumulate extracellular (10 mM) glucosamine and 2-aminoisobutyrate against concentration gradients. 2-Aminoisobutyrate was not metabolised by the cells; glucosamine was accumulated probably as a mixture of glucosamine 1- and 6-phosphates. Rates of accumulation of glucosamine and 2-aminoisobutyrate by cells grown at 30° C or 10° C decreased markedly when the test temperature was decreased from 30° C to 15° C. The rate of accumulation of glucosamine by cells grown at 10° C was considerably lower at each of the test temperatures compared with the corresponding rates for cells grown at 30° C; the rate of accumulation of 2-aminoisobutyrate was much less affected by the temperature at which the cells were grown and then only when measured at temperatures below about 20° C. Apparent K _m values for accumulation of glucosamine by cells grown at 30° C or 10° C decreased considerably when the test temperature was lowered from 20° C to 15° C. The extent of the decrease in K _m value was approximately the same for cells grown at 30° C or 10° C. Apparent K _m values for accumulation of 2-aminoisobutyrate were hardly affected by test temperature. Apparent V _max values for accumulation of glucosamine or 2-aminoisobutyrate were much lower when measured at 15° C than at 30° C. When measured at 30° C, apparent V _max values for accumulation of either solute were slightly lower with cells grown at 10° C compared with cells grown at 30° C; when measured at 15° C, the values were slightly greater with cells grown at 10° C. Net accumulation of glucosamine, at 30° C or 20° C, by cells grown at 30° C or 10° C ceased after 4–6 h. Cells grown at either temperature continued to accumulate 2-aminoisobutyrate at 30° C or 20° C for at least 12 h. The rate of efflux of glucosamine by cells grown at 30° C was slower when measured at 20° C compared with 30° C. With cells grown at 10° C, the rate of efflux at 30° C was slower than with cells grown at 30° C; when measured at 20° C, the rates were about equal. The temperature at which the cells were grown did not affect the ability of d-glucose, d-mannose or d-ribose to compete with d-glucosamine, or with the ability of l-alanine to compete with 2-aminoisobutyrate, when tested at 30° C or 20° C. Cells grown 30° C or 10° C had very similar ATP contents. The results are discussed in relation to the effect of temperature on the rate of solute accumulation by micro-organisms.Abbreviation AIB 2-Aminoisobutyrate     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

Relationship Between Expression of Cell Surface Hydrophobicity Protein 1 (CSH1p) and Surface Hydrophobicity Properties of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>

Candida dubliniensis and Candida albicans cause most of the oral candidiasis infections in AIDS patients. Unlike C. albicans, which variably expresses cell surface hydrophobicity (CSH) depending on environmental conditions, C. dubliniensis is hydrophobic under all environmental conditions. C. dubliniensis produces CdCSH1p, a protein related to CaCSH1p that contributes to CSH expression of C. albicans. We investigated whether environmental conditions affect CdCSH1p expression, CSH avidity, and adhesion to fibronectin (Fn). C. dubliniensis CD36 was grown at 23°C and 37°C in four different media. CdCSH1p expression was affected by growth temperature, with cells grown at 37°C expressing the protein, but cells grown at 23°C did not. Hydrophobic avidity for two media was higher in cells grown at 37°C than at 23°C. Cells grown at 23°C were generally less adherent than 37°C-grown cells to Fn. The results suggest CdCSH1p but not hydrophobic avidity may have a role in adhesion of C. dubliniensis to Fn.     

Upper temperature limits for growth and diazotrophy in the thermophilic cyanobacterium HTF Chlorogloeopsis

The effect of temperature and oxygen on diazotrophic growth of the thermophilic cyanobacterium HTF (High Temperature Form) Chlorogloeopsis was investigated using cells grown in light-limited continuous culture at a dilution rate of 0.02 h^-1. Diazotrophy was more sensitive to elevated temperatures than growth with combined nitrogen. The maximum temperature for growth of cultures gassed with CO₂-enriched air was more than 55 °C but less than 60 °C with N₂ as the sole nitrogen source, but between 60°C and 65°C when nitrate was present in the medium. The effect of temperature on nitrogenase activity, photosynthesis and respiration in the dark was determined using cells grown at 55°C. Maximal rates of all three processes were observed at 55°C and rates at 60°C during shortterm incubations were not less than 75% of the maximum. However, nitrogenase activity at 60°C was unstable and decayed at a rate of 2.2 h^-1 under air and at 0.3 h^-1 under argon. Photosynthesis and respiration were more stable at 60°C than anoxic nitrogen fixation. The upper temperature limits for diazotrophic growth thus seem to be set by the stability of nitrogenase.Abbreviations chl chlorophyll a - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - Taps N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid     

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.     

Production of a thermostable ATPase by the facultative thermophile,Bacillus coagulans

The properties of the ATPase in the facultative thermophile, Bacillus coagulans, grown at thermophilic or mesophilic temperatures were similar. Arrhenius plots did not show discontinuities indicative of thermoadaptation. Magnesium stimulation of the enzyme was dependant on the assay temperature but independant of the growth temperature. The ATPase in cells grown at 35°C or 55°C was equally thermostable at 65°C. In contrast, the ATPase from the mesophile, Bacillus megaterium (T _max=42°C) was completely inactivated at 55°C in 5 min.     

Degradation of long chain alkanes by bacilli

Summary Five different species of bacilli tested at 30°C degrade hydrocarbons by cooxidation. Subterminal degradation pathway of these bacilli was proved by estimating isomeric esters, developed from Baeyer-Villiger oxidation of ketones, and also by finding diols, ketols, and diketones. Comparing the composition of fatty acids in lipids of bacilli grown with and without alkane as well as growing them on labeled n-tridecane suggested a subterminal, partly a di-subterminal pathway.It was also shown that Bacillus stearothermophilus cannot grow on alkane as sole carbon source even in a thermophilic temperature range and that oxidation products are formed in amounts twenty times less when grown on cooxidation medium at 50°C, compared to product formation on the same medium at 30°C.     

A new isolate of Hydrogenobacter,an obligately chemolithoautotrophic,thermophilic, halophilic and aerobic hydrogen-oxidizing bacterium from seaside saline hot spring

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from a seaside saline hot spring in Izu Peninsula, Japan. The isolate was a Gram-negative, non-motile, non-spore-forming rod cell measuring 0.3 to 0.5 by 1.0 to 2.5 m. The optimal temperature for growth was around 70°C, and no growth was observed at 40°C or 80°C. Elemental sulfur or thiosulfate could be an alternative to molecular hydrogen as the sole energy source. The DNA base composition of the isolate was 46.0 mol% G+C. 2-Methylthio-3-VI,VII-tetrahydromultiprenyl⁷-1,4-naphthoquinone (methionaquinone) was the major component of the quinone system. C_18:0, C_18:1 and C_20:1 were the major components of the cellular fatty acids. These properties clearly indicate that the isolate belongs to genus Hydrogenobacter, but differed from H. thermophilus in some respects. Specifically, the isolate was a halophile which grew optimally at around 0.3–0.5 M NaCl, while H. thermophilus could not grow at such NaCl concentration levels. A new species name H. halophilus is proposed for this new halophilic isolate.     

Activation,synthesis and turnover of nitrate reductase controlled by nitrate and ammonium in Chlorella vulgaris

Cells of Anacystis nidulans grown at 25 or 30°C were examined both by thin-section and freeze-fracture electron microscopy. Cells grown at either temperature appeared similar when fixed at the growth temperature prior to observation. When cells were chilled to near 0°C for 30 min prior to fixation, those previously grown at 25° appeared unchanged as judged by thin sectioning while those grown at 39° showed considerable morphological alteration. Freeze fracture showed particle aggregation (more pronounced in 39°-grown cells) indicating lipid-phase separation in cells chilled prior to fixation. The phase separation was totally reversed by rewarming the chilled, 25°-grown cells to their growth temperature but was only partially reversed by rewarming chilled, 39°-grown cells. These results correlate with other effects of chilling seen in Anacystis cells grown at different temperatures.     

Mannan structural complexity is decreased when Candida albicans is cultivated in blood or serum at physiological temperature

The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30 °C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37 °C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30 °C and 37 °C, then cultivated overnight at 30 °C in YPD. The mannan was extracted and characterized using 1D and 2D ¹H NMR techniques. At 30 °C cells grown in blood and serum contain less acid-stable terminal β-(1→2)-linked d-mannose and α-(1→2)-linked d-mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer β-Man-(1→2)-α-Man-(1→ side chains. The decrement in acid-stable mannan side chains is greater at 37 °C than at 30 °C. Cells grown on blood at 37 °C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37 °C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.     

Mild temperature “stress” and callose synthesis

Seedlings of Zea mays L., Sorghum vulgare, Pisum sativum L., Phaseolus aureus, Glycine max L. and Lycopersicum esculentum were grown at 20°C and at 26°C. The seedlings were fixed in glutaraldehyde and sections were examined for aniline-blue-induced fluorescence, which is supposedly indicative of -1,3-glucans or callose. There was much more aniline-blue fluorescence in Zea, Glycine and Phaseolus seedlings grown at 20°C compared with 26°C whereas Pisum and Lycopersicum seedlings grown at 26°C showed more fluorescence than those grown at 20°C. In Zea, large deposits of fluorescent material were particularly noticeable in the walls of elongating cells around the shoot apex and in root-cap cells, and appeared to be closely associated with a few of the pitfields. The remaining pitfields showed the normal, low level of aniline-blue fluorescence.     

The effect of vesicular-arbuscular mycorrhizae and chilling on two hybrids of Zea mays L.

In order to investigate the effect of vesicular-arbuscular mycorrhizae on the chilling resistance of Zea mays, seeds of two hybrids (Pioneer 3902 and Pride 5) were grown in soil inoculated with Glomus mosseae. Germination tests at 10° C and 25° C showed that Pride 5 was more resistant to chilling than Pioneer 3902. Plants grown at 25° C for 6 weeks were given a 1-week chilling treatment at 10° C and the responses of mycorrhizal and nonmycorrhizal plants of the two hybrids were compared. At 10° C, the mycorrhizal plants had greater biomass, carbohydrate, and protein content than the nonmycorrhizal plants.     

Thermophilic Biodesulfurization of Various Heterocyclic Sulfur Compounds and Crude Straight-Run Light Gas Oil Fraction by a Newly Isolated Strain Mycobacterium phlei WU-0103

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50°C. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45°C. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography–mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45°C with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.     

Growth and hyoscyamine production of ‘hairy root’ cultures of Datura stramonium in a modified stirred tank reactor

Summary The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg·g^–1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g^–1 wet weight). Growth at 30°C was initially faster than at either 25°C or 35°C and by day 12, the drained weight of roots in the fermentor at 30°C was about fourfold greater than at 25°C and twice that at 35°C. The ultimate hyoscyamine levels attained (approximately 0.5 mg·g^–1 wet weight) were similar at both 25°C and 30°C but some 40% lower at 35°C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25°C and the highest production rate of 8.2 mg hyoscyamine l^–1 per day was obtained for roots grown at 30°C. In continuous fermentation at 25°C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30°C or 35°C. Offprint requests to: M. G. Hilton     

Production of sterigmatocystin by Aspergillus versicolor QM 432 used in fungus resistance tests for United States military specifications

A study was made to determine whether Aspergillus flavus (QM 380; NRRL 3537; ATCC 9643) and Aspergillus versicolor (QM 432; NRRL 573; ATCC 16,020), mold strains routinely combined in fungus resistance tests for United States military specification (MIL-STD-810B; MIL-STD-331A), were capable of producing aflatoxins or sterigmatocystin when grown separately or in mixed culture fermentations of cracked corn (11 days at 25 °C or 28 °C). Substantial quantities of sterigmatocystin (average of 3 replicates= 1,895 ppb) were detected when A. versicolor was the sole colonist. No sterigmatocystin was detected when A. versicolor was simultaneously inoculated with other molds. Aflatoxins were not detected in any of the treatment combinations.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

A correlation between photosynthetic temperature adaptation and seasonal phenology patterns in the shortgrass prairie

Summary The temperatures at which chlorophyll fluorescence yield is substantially increased and the temperatures at which the quantum yield for CO₂ uptake is irreversibly inhibited were measured for three shortgrass prairie species. The experimental taxa include, a cool season species (Agropyron smithii), a warm season species (Bouteloua gracilis), and a species which grows throughout the cool and warm seasons (Carex stenophylla). Agropyron smithii exhibited lower high temperature damage thresholds (43°C in cool grown plants, 46°C in warm grown plants), relative to the other two species. Bouteloua gracilis exhibited the highest tolerance to high temperature, with threshold values being 44–49°C for cool grown plants and 53–55°C for warm grown plants. Carex stenophylla exhibited threshold values which were intermediate to the other two species (43–47°C for cool grown plants, and 51–53°C for warm grown plants). Seasonal patterns in the fluorescence rise temperatures of field grown plants indicated acclimation to increased temperatures in all three species. The results demonstrate a correlation between the high temperature thresholds for damage to the photosynthetic apparatus, and in situ seasonal phenology patterns for the three species.     

Occurrence of Sulfolobus acidocaldarius,an extremely thermophilic acidophilic bacterium,in New Zealand hot springs

Several hot springs in the Rotorua-Taupo regions, North Island, New Zealand, were tested for the presence of extremely thermophilic acidophilic bacteria. In the majority of the springs, ranging in temperature from 43–96°C and in pH from 2.1–6.9, direct microscopic observations revealed the presence of both rod-shaped and spherical bacteria. Isolations were attempted at 70°C and pH 2.0 and 7.0, with either yeast extract for heterotrophic growth, or elemental sulfur as the sole source of energy for autotrophic growth. Eight of the samples produced grwoth at pH 2.0 with either yeast extract or sulfur, but none of the samples grew at pH 7.0. All the isolates obtained, resembled Sulfolobus acidocaldarius, a thermophilic acidophilic bacterium which has previously been reported from various regions in the Northern Hemisphere. Immunofluorescence examination of six of these isolates revealed varying degrees of cross reactions with two already characterized Sulfolobus isolates from the Yellowstone National Park, U.S.A. This paper is the first published record of Sulfolobus from the Southern Hemisphere.     

Phenomenon of Temperature-Dependent Chlorophyll Deficiency in Festuca pratensis

The composition and amount of pigments were studied in temperature-dependent chlorophyll-deficient seedlings of wild type (control) and several mutant lines of Festuca pratensis Huds. at room (25°C) and high (35°C) temperatures. In seedlings of all mutant lines grown at 25°C, chlorophyll b content was lower and the concentration of carotenoids was higher than in control seedlings. At 35°C, the concentration of all pigments decreased in a row from dark-green to xantha phenotypes, and this trend was retained when the temperature was lowered to 25°C. The phenotype xantha completely lacked violaxanthin and neoxanthin. The observed effects are related to the protective dissipative function of the xanthophyll cycle.     

Rapid acclimation of root hydraulic conductivity to low temperature

Root hydraulic conductance of many species is substantially reduced by exposure to low temperatures. The objective of this research was to investigate the decrease and recovery of root hydraulic conductivity in spinach (Spinacia oleracea L.) root systems upon exposure to low temperature. Root hydraulic conductivity (Lp) was determined for detached whole root systems as the slope of the flux and an applied pressure gradient. Water flux (Jv), of root systems grown at 20C, decreased immediately upon exposure to 5C. After 2-5 h Jv recovered and reached a stable value after 12 h exposure to 5°C. In separate experiments, the root Lp of plants acclimated for 7 d at 5°C was 125% greater than that of isolated root systems acclimated for 12 h at 5°C. Lp of plants grown and measured at 5°C was about 50% of the Lp of plants grown and measured at 20°C. The rapid acclimation to low temperatures observed in detopped root systems was also indicated in intact plants at 20/5°C (shoot/root temperatures) using mass flow porometry. Acclimation of the root system after exposure to 5°C was apparent by recovery of stomatal opening. These results indicate that spinach root systems have the ability to acclimate rapidly to changes in temperature and to continue acclimating during prolonged exposure to low temperature.     

Effects of temperature and CO2 enrichment on kinetic properties of phospho-enol-pyruvate carboxylase in two ecotypes of Echinochloa crus-galli (L.) Beauv., a C4 weed grass species

Summary Two populations of Echinochloa crus-galli (Québec, Mississippi) were grown at the Duke University Phytotron under 2 thermoperiods (28°/22°C, 21°/15°C day/night) and 2 CO₂ regimes (350 and 675 l l^-1). Thermostability, energy of activation (E _a ),K _m (PEP), K _m (Mg⁺⁺), and specific activity of phospho-enol-pyruvate carboxylase (PEP_c) were analyzed in partially purified enzyme preparations of plants grown for 5 weeks. Thermostability of PEP_c from extracts (in vitro) and leaves (in situ) was significantly higher in Mississippi plants. In vitro denaturation was not appreciably modified by thermal acclimation but CO₂ enrichment elicited higher thermostability of PEP_c. In situ thermostability was significantly higher than that of in vitro assays and was higher in Mississippi plants acclimated at 28°/22°C and in plants of the two ecotypes grown at 675 l l^-1 CO₂. E _a (Q ₁₀ 30°/20°C) for PEP_c was significantly lower in Québec plants as compared to Mississippi and no acclimatory shifts were observed. Significantly higher K _m's (PEP) in 20°C assays were obtained for Mississippi as compared to Québec plants but values were similar at 30°C and 40°C assays. K _m (Mg⁺⁺) decreased at higher assay temperatures and were significantly lower for PEP_c of the Québec ecotype. No significant changes in K _m (Mg⁺⁺) values were associated with modifications in temperature on CO₂ regimes. PEP_c activity measured at 30°C was significantly higher for Québec plants when measured on a leaf fresh weight, leaf area or protein basis but not on a chlorophyll basis. Significantly higher PEP_c activity for both genotypes was observed for plants acclimated at 21°/15°C or grown at 675 l l^-1 CO₂. Net photosynthesis (Ps) and net assimilation rates (NAR) were higher in Québec plants and were enhanced by CO₂ enrichment. NAR was higher in plants acclimated at low temperature, while an opposite trend was observed for Ps. PEP_c activities were always in excess of the amounts required to support observed rates of CO₂ assimilation.