Pressure and temperature effects on growth and viability of the hyperthermophilic archaeon Thermococcus peptonophilus

We studied the effects of high temperatures and elevated hydrostatic pressures on the physiological behavior and viability of the extremely thermophilic deep-sea archaeon Thermococcus peptonophilus. Maximal growth rates were observed at 30 and 45 MPa although no significant increases in cell yields were detected. Growth at 60 MPa was slower. The optimal growth temperature shifted from 85° C at 30 MPa to 90–95° C at 45 MPa. Cell viability during the stationary phase was also enhanced under high pressure. A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated at increasing growth temperatures. The viability of cells during starvation, at high temperature (90, 95° C), and at low temperature (10° C) was enhanced at 30 and 45 MPa as compared to atmospheric pressure. These results show that the extremely thermophilic archaeon T. peptonophilus is a barophile. Received: 21 October 1996 / Accepted: 5 February 1997     

Adaptive Response to Cold Temperatures in Vibrio vulnificus

The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation. When a V. vulnificus culture was shifted from 35°C to 6°C conditions, it underwent transition to a non-culturable state. Cells adapted to 15°C prior to change to 6°C condition, however, remain viable and culturable. In addition, cultures adapted to 15°C were able to survive better upon freezing at −78°C compared with cultures frozen directly from 35°C. Inhibition of protein synthesis by addition of chloramphenicol in a V. vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance. These results suggest that cold-adaptive “protective” proteins may enhance survival and tolerance at cold temperatures. In addition, removal of iron from the growth medium by adding 2,2′-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels. This suggests that iron plays an important role in adaptation at cold temperatures. Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with ³⁵S-methionine during exposure at 15°C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein. These results suggest that, for V. vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA. In this study, for the first time we have shown that exposure to an intermediate cold temperature (15°C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6°C). This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V. vulnificus infection from consuming raw oysters. Received: 30 July 1998 / Accepted: 1 October 1998     

Alteration of low-temperature susceptibility of the cyanobacterium Synechococcus sp. PCC 7002 by genetic manipulation of membrane lipid unsaturation

Cyanobacteria acclimate to low temperature by desaturating their membrane lipids. Mutant strains of Synechococcus sp. PCC 7002 containing insertionally inactivated desA (Δ12 acyl-lipid desaturase) and desB (ω3 acyl-lipid desaturase) genes were produced, and their low-temperature susceptibility was characterized. The desA mutant synthesized no linoleic acid or α-linolenic acid, and the desB mutant did not produce α-linolenic acid. The desA mutant grew more slowly than the wild-type at 22° C and could not grow at 15° C. The desB mutant could not continuously grow at 15° C, although no observable phenotype appeared at higher temperatures. It has been shown that expression of the desA gene occurs at 38° C and is up-regulated at 22° C, and that the desB gene is only expressed at 22° C. These results indicate that the expression of the desA and desB genes occurs at higher temperatures than those at which a significant decline in physiological activities is caused by the absence of their products. The temperature dependency of photosynthesis was not affected by these mutations. Since chlorosis and inability to grow at 15° C with nitrate was suppressed by the substitution of urea as a nitrogen source, it is very likely that the chilling susceptibility of the desaturase mutants is attributable to nutrient limitation. Received: 24 April 1997 / Accepted: 5 August 1997     

Leaf movement of bush bean: a biometeorological perspective

 Leaf movements of bush bean plants were studied at the relatively low photon flux density of 0.2 mmol/m² per s, and air temperatures of 25° and 35° C in a growth chamber. A beta-ray gauge system was used to monitor continuously pulvinus water status and bending. Leaf angles were below the horizontal and were linearly related to the soil water content (R≥−0.91 at 25° C and R≥−0.93 at 35° C). The beta-ray transmission maxima coincided with the stem temperature minima in darkness and vice versa when brightness prevailed as the growth chamber temperature varied with the photoperiod. Leaf angle increased linearly with increased beta-ray transmission. The Q₁₀ temperature coefficient, a measure of the metabolic energy requirement for leaf movement between 25° and 35° C was estimated at 1.8, and the corresponding mean Arrhenius constant at 423 kJ/mol for bush bean. Received: 19 July 1996 / Accepted: 9 September 1996     

Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis

 Artificial cold adaptation of a mesophilic protease, subtilisin BPN′, was attempted by means of random mutagenesis of its entire gene coupled with screening of cleared-zone-forming colonies on skim-milk plates at a low temperature. Out of sixty clones screened at 10 °C, one mutant enzyme (termed M-15) was found to acquire higher proteolytic activities, specifically dependent on low temperatures ranging from 10 °C to 1 °C, in comparison with those of the wild-type. DNA sequencing analysis revealed that, by this mutation, the 84th amino acid residue, valine, was substituted by isoleucine, which is located 1.5 nm from the center of the catalytic triad in the tertiary structure of subtilisin. By kinetic analysis of the purified enzyme samples, the higher proteolytic activities of M-15 at low temperatures were found to be due to the decrease in the K _m value. There was no difference in thermostability between the wild-type and mutant enzymes, when tested by heat treatment. Circular dichroism spectra also showed no difference between them at 10 °C, indicating that the mutation of V84I had no effect on the secondary structure of subtilisin. Received: 22 April 1996 / Received last revision: 29 July 1996 / Accepted: 24 August 1996     

Effect of temperature on the survival and infectivity of <Emphasis Type="Italic">Pseudotheraptus devastans</Emphasis> vector

The aim of this study was to investigate under a controlled environment, the effect of temperature on the survival and infectivity of Pseudotheraptus devastans Distant, a cassava anthracnose disease vector. The insect P. devastans was collected from young cassava (Manihot esculenta Crantz) field plots, at the International Institute of Tropical Agriculture, (IITA), Ibadan, Nigeria. A mixture of the different developmental stages of eggs, first to fifth instar nymphs, and adults, were incubated in controlled environment chambers, under various constant temperatures of: 15, 17, 22, 25, 27, 30, and 35°C. Relative humidity at different temperature conditions were recorded and maintained at 90%, 85%, 80%, 75%, 70%, 65%, and 60%, respectively. A significant increase in insect survival was observed between 22 and 27°C temperature conditions while a significant decrease in survival was observed at 15°C and above 30°C. Lesion number, lesion diameter and infectivity among the insect stages varied as a function of temperature and relative humidity. Infectivity was highest at 22–25°C maintained at 75–80% RH and lowest at 15°C and above 30°C maintained respectively, at 65% RH and 90% RH. There was considerable low vector infectivity due to low survival of the insects at extreme temperatures.     

Temperature-sensitive mutants of Corynebacterium ammoniagenes ATCC 6872 with a defective large subunit of the manganese-containing ribonucleotide reductase

Chemical mutagenesis of the nucleotide-producing strain Corynebacterium ammoniagenes ATCC 6872 with N-methyl-N-nitro-N-nitrosoguanidine followed by an enrichment protocol yielded 46 temperature-sensitive (ts) clones. A rapid assay for the allosterically regulated Mn-ribonucleotide reductase (RRase) was developed with nucleotide-permeable cells of C. ammoniagenes in order to screen for possible defects in DNA precursor biosynthesis at elevated temperature. Three mutants (CH 31, CH 32, and CH 33) grew well at 30° C but did not proliferate at 40° C because they did not reduce ribonucleotides to 2′-deoxyribonucleotides. They were designated nrd ^ts (nucleotide reduction defective). When the cultures were shifted from 30 to 40° C, the nrd ^ts mutants immediately ceased to incorporate radiolabeled nucleic acid precursors into the DNA fraction, while DNA chain elongation was barely affected. Thus, exhaustion of the deoxyribonucleotide pool ultimately inhibited cell division, leading to a filamentous growth morphology. In contrast to the wild-type, all three nrd ^ts mutants displayed a distinctly enhanced sensitivity of ribonucleotide reduction towards hydroxyurea (in permeabilized cells and in vitro) at 30° C. The results from assays for biochemical complementation of heat-inactivated (2 min, 37° C) mutant enzyme with either the small or the large subunit of wild-type Mn-RRase located the mutational defect on the large subunit. Received: 28 December 1995 / Revision received: 22 January 1997 / Accepted: 29 January 1997     

Ornithine decarboxylase in Paracoccidioides brasiliensis

Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37° C in the yeast phase and at 30° C in the mycelial phase. In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P. brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone. The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition. Received: 18 December 1995 / Accepted: 8 March 1996     

Clostridium vincentii sp. nov., a new obligately anaerobic, saccharolytic, psychrophilic bacterium isolated from low-salinity pond sediment of the McMurdo Ice Shelf, Antarctica

A gram-positive, motile, rod-shaped, strictly anaerobic bacterium was isolated from an enrichment initiated with sediment taken from below the cyanobacterial mat of a low-salinity pond on the McMurdo Ice Shelf, Antarctica. The organism grew optimally at 12° C, at pH 6.5, and at an NaCl concentration of < 0.5% (w/v). It survived freeze-thawing at low salt concentrations, but not exposure to temperatures over 25° C for more than 20 h or short-term exposure to temperatures > 50° C. Out of a variety of polysaccharides tested as growth substrates, only xylan supported growth. The organism also grew on a variety of mono- and disaccharides including the cyanobacterial cell wall constituent, N-acetyl glucosamine. Fermentation products on a mol product per 100 mol of hexose monomer fermented basis were: acetate, 72; formate, 72; butyrate, 55; hydrogen, 114; and CO₂, 100. Not detectable in the culture medium (< 2 mol per 100 mol of monomer) were lactate, propionate, ethanol, n-propanol, n-butanol, and succinate. The G+C content of the DNA from the bacterium was 33 mol%, and a phylogenetic analysis indicated that it grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. It differed from other species of this genus with regard to growth temperature optimum, substrate range, and fermentation pattern, and is therefore designated as a new species of Clostridium for which the name Clostridium vincentii is proposed. The type strain is lac-1 (DSM 10228). Received: 6 August 1996 / Accepted: 30 October 1996     

Desulforhopalus vacuolatus gen. nov., sp. nov., a new moderately psychrophilic sulfate-reducing bacterium with gas vacuoles isolated from a temperate estuary

A new type of gas-vacuolated, sulfate-reducing bacterium was isolated at 10° C from reduced mud (E₀ < 0) obtained from a temperate estuary with thiosulfate and lactate as substrates. The strain was moderately psychrophilic with optimum growth at 18–19° C and a maximum growth temperature of 24° C. Propionate, lactate, and alcohols served as electron donors and carbon sources. The organism grew heterotrophically only with hydrogen as electron donor. Propionate and lactate were incompletely oxidized to acetate; traces of lactate were fermented to propionate, CO₂, and possibly acetate in the presence of sulfate. Pyruvate was utilized both with and without an electron acceptor present. The strain did not contain desulfoviridin. The G+C content was 48.4 mol%. The differences in the 16S rRNA sequence of the isolate compared with that of its closest phylogenetic neighbors, bacteria of the genus Desulfobulbus, support the assignment of the isolate to a new genus. The isolate is described as the type strain of the new species and genus, Desulforhopalus vacuolatus. Received: 4 March 1996 / Accepted: 17 June 1996     

Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH optimum was 7.6–7.8. The apparent K _m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t _50%) at 70° C was approximately 8 h. Received: 9 September 1996 / Accepted: 27 December 1996     

Oral temperatures of the elderly in nursing homes in summer and winter in relation to activities of daily living

 This study was conducted to clarify the seasonal difference in body temperature in summer and winter, and to document the thermal environment of the elderly living in nursing homes. The subjects were 57 healthy elderly people aged ≥63 years living in two nursing homes in Japan. One of the homes was characterized by subjects with low levels of activities of daily living (ADL). Oral temperatures were measured in the morning and afternoon, with simultaneous recording of ambient temperature and relative humidity. Oral temperatures in summer were higher than in winter, with statistically significant differences (P<0.05) of 0.25 (SD 0.61) °C in the morning and 0.24 (SD 0.50) °C in the afternoon. Differences between oral temperatures in summer and winter tended to be greater in subjects with low ADL scores, even when their room temperature was well-controlled. In conclusion, the oral temperatures of the elderly are lower in winter than summer, particularly in physically inactive people. It appears that those with low levels of ADL are more vulnerable to large changes in ambient temperature. Received: 28 March 1996 / Accepted: 12 November 1996     

On-line monitoring of growth of Escherichia coli in batch cultures by bioluminescence

Bioluminescence was used to monitor growth of Escherichia coli in batch cultures on-line. Light emission of a strain engineered for constitutive bioluminescence was monitored with a simple set-up consisting of a photodiode, a photodetector amplifier and a recorder. Bioluminescence and colony forming units (CFU) of the cultures increased and decreased proportionally and were correlated during every growth phase at temperatures between 28 °C and 40 °C. Up to the late log (deceleration) phase, both light emission and CFU increased rapidly. Beyond the stationary phase these characteristics decreased very slowly at lower temperatures, while at higher ones they declined more rapidly. Towards the end of the cultivation, light emission of the cultures dropped to undetectable levels, even though CFU were recovered. This was particularly marked at lower temperatures where non-luminescent cultures retained very high CFU. This indicates that the actual metabolism of cells in a culture can be at a very low level or completely shut down, yet cells retain their capability to be culturable. The on-line technology described here has a number of potential uses in the laboratory and industry. Received: 30 September 1999 / Received revision: 29 November 1999 / Accepted: 3 December 1999     

A microcarrier culture method for the production of large quantities of viable Chlamydia pneumoniae

 We studied the propagation of Chlamydia pneumoniae strain TW-183 in HEp2 cells grown on microcarrier beads. Infection of the cells in microcarrier culture was optimized by addition of 7.5% polyethylene glycol 4000 (PEG4000) during adsorption. The yield in microcarrier culture was similar to that of microtitre-plate culture using centrifugation-assisted infection (120×10⁶ and 225×10⁶ bacteria/10⁶ HEp2 cells respectively), as was the burst size (505 and 449 bacteria produced/infecting bacterium respectively). However, up to 64% savings in labour time and 27% savings in culture medium were achieved if the microcarrier culture method was used instead of the microtitre-plate culture method. The optimal yield of viable bacteria could only be achieved at a narrow range of multiplicities of infection (0.24–1.14 inclusion-forming units/cell), independent of the mode of infection (centrifugation-assisted infection or PEG4000-facilitated infection by adsorption) and independent of incubation temperature (35°C or 37°C). The yield of microcarrier cultures was the same at an incubation temperature of 35°C or 37°C in contrast to an increased production at 35°C in the microtitre-plate culture method using centrifugation-assisted infection. In conclusion, the microcarrier culture method is useful to produce large quantities of viable Chlamydia pneumoniae economically. Received: 27 December 1995/Received revision: 4 April 1996/Accepted: 15 April 1996     

Growth at low temperature causes nitrogen limitation in the cyanobacterium Synechococcus sp. PCC 7002

The coloration of cells of the cyanobacterium Synechococcus sp. PCC 7002 changed from normal blue-green to yellow-green when cells were grown at 15° C in a medium containing nitrate as the sole nitrogen source. This change of coloration was similar to a general response to nutrient deprivation (chlorosis). For the chlorotic cells at 15° C, the total amounts of phycobiliproteins and chlorophyll a decreased, high levels of glycogen accumulated, and growth was arithmetic rather than exponential. These changes in composition and growth occurred in cells grown at low (50 μE m^–2 s^–1) as well as high (250 μE m^–2 s^–1) light intensity. After a temperature shift-up to 38° C, chlorotic cells rapidly regained their normal blue-green coloration and normal exponential growth rate within 7 h. When cells were grown at 15° C in a medium containing urea as the reduced nitrogen source, cells grew exponentially and the symptoms of chlorosis were not observed. The decrease in photosynthetic oxygen evolution activity at low temperature was much smaller than the decrease in growth rate for cells grown on nitrate as the nitrogen source. These studies demonstrate that low-temperature-induced chlorosis of Synechococcus sp. PCC 7002 is caused by nitrogen limitation and is not the result of limited photosynthetic activity or photodamage to the photosynthetic apparatus, and that nitrogen assimilation is an important aspect of the low-temperature physiology of cyanobacteria. Received: 24 April 1997 / Accepted: 5 August 1997     

Purification and Characterization of an Extracellular β-Glucosidase from the Wood-Grown Fungus Xylaria regalis

Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the K _m was 1.72 mM and V _max was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca²⁺, Mg²⁺, Mn²⁺, Cd²⁺ and β-mercaptoethanol, and inhibited by Ag⁺, Hg²⁺, SDS, and p-chloromercuribenzoate (PCMB). Received: 30 March 1996 / Accepted: 3 May 1996     

Progeny production and adult longevity of the mealybug parasitoidsAnagyrus pseudococci,Leptomastix dactylopii,andLeptomastidea abnormis [Hym.: Encyrtidae] in relation to temperature

Progeny production increased and adult longevity decreased with rising temperature within the range 18°C to 30°C for the 3 mealybug parasitoidsAnagyrus pseudococci (Girault),Leptomastix dactylopii Howard andLeptomastidea abnormis (Girault). The Weibull distribution gave a good fit to survival curves for the 3 parasitoids and statistical comparison of Weibullb andc parameters at different temperatures allowed changes in the scale and shape of the curves to be detected. In general, ♀♀ lived longer than ♂♂ for all 3 species, except at high temperature. FemaleL. abnormis attained their maximum progeny production at 24°C and maintained this level up to 34°C. They lived longer than the other 2 parasitoid species at 30°C and showed a type I survival curve throuhout the range of temperatures examined.A. pseudococci andL. dactylopii both required high temperatures (30°C) to attain their maximal progeny production, but werepseudococci tended towards type II, with a larger proportion of the population dying within the first few days.L. dactylopii lived longest at 26°C, with ♀♀ showing a type I survival curve at all temperatures and ♂ survival curves changing from type I to type II at 30°C. The implications of these findings for the population dynamics of the different parasitoids are briefly discussed.      

Evaluation of mean skin temperature formulas by infrared thermography

 To study the reliabiliity of formulas for calculating mean skin temperature (T _sk), values were computed by 18 different techniques and were compared with the mean of 10,841 skin temperatures measured by infrared thermography. One hundred whole-body infrared thermograms were scanned in ten resting males while changing the air temperature from 40° C to 4° C. Local, regional average and mean skin temperatures were obtained using an image processing system. The agreement frequency, defined as the percentage of the calculated T _sk values which agreed with the corresponding infrared thermographic T _sk within ±0.2° C, ranged for with the various formulas from 7% to 80%. In many sites, the local skin temperature did not coincide with the regional average skin temperature. When the local skin temperatures which showed the highest percentage similarity to the regional average skin temperature within ±0.4° C were applied to the formula, the agreement frequency was markedly improved for all formulas. However, the agreement frequency was not affected by changing the weighting factors from specific constants to individually measured values of regional surface area. By applying the physiologically reliable accuracy range of ±0.2° C in the moderate and ±0.4° C in the cool condition, agreement frequencies of at least 95% were observed in formulas involving seven or more skin temperature measurement sites, including the hand and foot. We conclude that calculation of a reliable mean skin temperature must involve more than seven skin temperature measurement sites regardless of ambient temperature. Optimal sites for skin temperature measurement are proposed for various formulas. Received: 2 December 1996 / Accepted: 25 June 1997     

Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic archaeum that oxidizes Fe2+ at neutral pH under anoxic conditions

A novel coccoid, anaerobic, Fe²⁺-oxidizing archaeum was isolated from a shallow submarine hydrothermal system at Vulcano, Italy. In addition to ferrous iron, H₂ and sulfide served as electron donors. NO₃ ^– was used as electron acceptor. In the presence of H₂, also S₂O₃ ^2– could serve as electron acceptor. The isolate was a neutrophilic hyperthermophile that grew between 65° C and 95° C. It represents a novel genus among the Archaeoglobales that we name Ferroglobus. The type species is Ferroglobus placidus (DSM 10642). Received: 7 March 1996 / Accepted: 4 September 1996