Effect of Incubation Temperature on Isolation of Campylobacter jejuni Genotypes from Foodstuffs Enriched in Preston Broth

Preston broth and agar incubated at either 37 or 42°C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42°C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37°C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37°C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42°C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42°C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.     

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : III. Responses of Protoplasts and Intact Chloroplasts

Protoplasts and intact chloroplasts isolated from Agropyron smithii Rybd. were utilized in an effort to determine the limiting factor(s) for photosynthesis at supraoptimal temperatures. Saturated CO₂-dependent O₂ evolution had a temperature optimum of 35°C for both protoplasts and intact chloroplasts. A sharp decline in activity was observed as assay temperature was increased above 35°C, and at 45°C only 20% of the maximal rate remained. The temperature optimum for 3-phosphoglycerate reduction by intact chloroplasts was 35°C. Above this temperature, 3-phosphoglycerate reduction was more stable than CO₂-dependent O₂ evolution. Reduction of nitrite in coupled intact chloroplasts had a temperature optimum of 40°C with only slight variation in activity between 35°C and 45°C. Reduction of nitrite in uncoupled chloroplasts had a temperature optimum of 40°C, but increasing the assay temperature to 45°C resulted in a complete loss of activity. Reduction of p-benzoquinone by protoplasts and intact chloroplasts had a temperature optimum of 32°C when measured in the presence of dibromothymoquinone. This photosystem II activity exhibited a strong inhibition of O₂ evolution as assay temperature increased above the optimum. It is concluded that, below the temperature optimum, ATP and reductant were not limiting photosynthesis in these systems or intact leaves. Above the temperature optimum, photosynthesis in these systems is limited in part by the phosphorylation potential of the stromal compartment and not by the available reductant.     

Modification of Rubisco and Altered Proteolytic Activity in O3-Stressed Hybrid Poplar (Populus maximowizii x trichocarpa)

Exposing hybrid poplar (Populus maximowizii x trichocarpa) plants to ozone (O3) resulted in an acceleration of the visual symptoms of senescence and a decrease in the activity and quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Whole plants, crude leaf extracts, and isolated intact chloroplasts of hybrid poplar clone 245 were used to test the hypothesis that O3-induced structural modifications of Rubisco affect the activity of this key photosynthetic enzyme. Proteolytic activity, per se, could not account for losses in Rubisco; acidic and alkaline protease activities declined or were unaffected in foliage of O3-treated poplar saplings. In vitro treatment of leaf extracts with O3 decreased total Rubisco activity and binding of the enzyme's transition-state analog, 2-carboxyarabinitol bisphosphate. Additionally, O3 increased the loss of Rubisco large subunit (LSU) when extracts were incubated at 37[deg]C. Treatment of isolated intact chloroplasts with O3 accelerated both the loss of the 55-kD Rubisco LSU and the accumulation of Rubisco LSU aggregates, as visualized by immunoblotting. The time-dependent modification in Rubisco structure was the primary response of the isolated organelles to O3 treatment, with little proteolytic degradation of the LSU detected.     

Effect of Incubation Temperature on the Detection of Thermophilic Campylobacter Species from Freshwater Beaches,Nearby Wastewater Effluents,and Bird Fecal Droppings

This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni, C. coli, and C. lari, in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni, 10% for C. coli, and 3% for C. lari) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.     

The Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase is Fragmented into 37-kDa and 16-kDa Polypeptides by Active Oxygen in the Lysates of Chloroplasts from Primary Leaves of Wheat

Lysates of chloroplasts isolated from wheat (Triticum aestivumL. cv. Aoba) leaves were incubated on ice (pH 5.7) for 0 to60 min in light (15 µmol quanta m^–2 s^–1),and degradation of the large subunit (LSU) of ribulose-l,5-bis-phosphatecarboxylase/oxygenase (Rubisco: EC 4.1.1.39 [EC] ) was analyzed byapplying immunoblotting with site-specific antibodies againstthe N-terminal, internal, and C-terminal amino acid sequencesof the LSU of wheat Rubisco. The most dominant product of thebreakdown of the LSU and that which was first to appear wasan apparent molecular mass of 37-kDa fragment containing theN-terminal region of the LSU. A 16-kDa fragment containing theC-terminal region of the LSU was concomitantly seen. This fragmentationof the LSU was inhibited in the presence of EDTA or 1,10-phenanthroline.The addition of active oxygen scavengers, catalase (for H₂O₂)and n-propyl gallate (for hydroxyl radical) to the lysates alsoinhibited the fragmentation. When the purified Rubisco fromwheat leaves was exposed to a hydroxyl radical-generating systemcomprising H₂O₂, FeSO₄ and ascorbic acid, the LSU was degradedin the same manner as observed in the chloroplast lysates. Theresults suggest that the large subunit of Rubisco was directlydegraded to the 37-kDa fragment containing the N-terminal regionand the 16-kDa fragment containing the C-terminal region ofthe LSU by active oxygen, probably the hydroxyl radical, generatedin the lysates of chloroplasts. (Received October 28, 1996; Accepted February 7, 1997)     

Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: Immunocytochemical and ultrastructural observations

Summary The possible involvement of vacuolar cysteine proteinases in degradation of ribulose-bisphosphate carboxylase (Rubisco) in senescing French bean leaves was studied by ultrastructural and immunocytochemical analyses with antibodies raised against the large subunit (LSU) of Rubisco and SH-EP, a cysteine proteinase fromVigna mungo that is immunologically identical to one of the major proteinases of French bean plants. Primary leaves of 10-day-old plants were detached and placed at 25 °C in darkness for 0, 4, and 8 days to allow their senescence to proceed. The leaves at each senescence stage were subjected to the conventional electron microscopic and immunocytochemical studies. The results indicated that the chloroplasts of senescing French bean leaves were separated from the cytoplasm of the cell periphery and taken into the central vacuole and that the Rubisco LSU in the chloroplasts was degraded by vacuolar enzymes such as an SH-EP-related cysteine proteinase that developed in senescing leaves. The present results together with the results of previous biochemical studies using vacuolar lysates support the view that Rubisco is degraded through the association of chloroplasts with the central vacuole during the senescence of leaves that were detached and placed in darkness.     

In vitro Protein Synthesis by Plastids of Phaseolus vulgaris. II. The Probable Relation Between Ribonuclease Insensitive Amino Acid Incorporation and the Presence of Intact Chloroplasts

Amino acid incorporation into protein by chloroplasts from primary leaves of Phaseolus vulgaris L., var. Black Valentine is only partially inhibited by 400 μg/ml ribonuclease. The rate of incorporation, in the presence of ribonuclease, is progressively inhibited with time, and ceases after about half an hour. Preincubation of chloroplasts at 25°, in the absence of ribonuclease, increases the inhibitory effect of ribonuclease on the initial rate of incorporation of amino acid into protein. Examination of electron micrographs of freshly prepared chloroplast suspensions shows that chloroplasts are largely intact. However, after incubation at 25° for 1 hour the chloroplasts are disrupted, as indicated by loss of their stroma contents. It is concluded that the intact chloroplast membrane is relatively impermeable to ribonuclease. Amino acid incorporating activity probably becomes inhibited as the inside of the chloroplast is made accessible to ribonuclease by breakage of membranes during incubation at 25°.     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Cleavage of a viral polyprotein by a cellular proteolytic activity.

The 200,000-dalton polyprotein encoded by the bottom component RNA of cowpea mosaic virus was synthesized in rabbit reticulocyte lysates, and this in vitro-synthesized protein was isolated from the lysate reaction mixture by sucrose density gradient centrifugation. Incubation of the isolated polyprotein with buffer caused no change in the protein, but incubation with reticulocyte lysates or with fractionated lysate proteins resulted in cleavage of the protein into the expected cleavage products (32,000- and 170,000-dalton proteins). This finding indicated that reticulocytes contain a proteolytic activity that is needed for the primary cleavage reaction. A cleavage assay in which we used partially purified preparations showed that cleavage was an ATP-dependent reaction.     

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.     

Aberrant Forms of Streptococcus cremoris HP

The cheese starter strain, Streptococcus cremoris HP, produced variant colonies when streaked on the surface of solid media and incubated at 30 or 37°C or in the presence of penicillin. Serial plating and incubation at 37°C or in the presence of penicillin resulted in the production of variants. Subculture followed by incubation at 25°C or in the absence of penicillin resulted in the reversion or partial reversion to the parent form. Colony morphology and cell morphology exhibited the characteristics of the L-phase. Evidence suggested that the aberrant forms of S. cremoris at 30°C were transitional phase variants but at 37°C and in the presence of penicillin they were L-phase variants. Electron micrographs showed that the cell walls of the variant cells were defective and that there were differences in the density and the organization of the cytoplasmic constituents compared with the parent cell.     

Influence of growth temperature on respiratory characteristics of mitochondria from callus-forming potato tuber discs

The uninhibited respiration of mitochondria, isolated from potato tuber discs (Solanum tuberosum L. cv. Bintje) incubated on a callus-inducing medium at 28°C, is higher than that of mitochondria from tissue incubated at 8°C. This respiration is composed of a CN-sensitive and a CN-resistant part. The capacity of the CN-resistant alternative oxidase pathway is larger in mitochondria from 28°C tissue than in mitochondria from 8°C tissue (35% and 8% of uninhibited respiration, respectively). The alternative pathway is operative both in mitochondria from 28°C tissue and 8°C tissue.

The observed difference in uninhibited respiration, is not only caused by lower values of respiration via the alternative pathway in mitochondria from 8°C tissue, but also by lower values of respiration via the cytochrome pathway.

A positive correlation has been demonstrated between the incubation temperature (ranging from 4-37°C) and the relative capacity of respiration via alternative pathway in the mitochondria. Induction of alternative pathway is not directly correlated with growth (in terms of increase in fresh weight) of the potato tuber discs.

     

Leaf Cytosolic Fructose-1,6-bisphosphatase : A Potential Target Site in Low Temperature Stress

Leaf cytosolic fructose-1,6-bisphosphatase (FBPase), partially purified from both spinach (Spinacia oleracea, var Hipack) and peas (Pisum sativum, var Progress No. 9), is reversibly inactivated by exposure to low temperature. Thus, even though assays were conducted at 22°C, samples incubated at 0 to 12°C had greatly reduced activity relative to controls maintained at 22°C. Following incubation at 22°C prior to assay, the inactivated samples regained their initial activity. Chloroplast FBPase, by contrast, was unaffected by low temperature treatment. This feature as well as lack of a response of cytosolic FBPase to thioredoxins f or c_f and to chloroplast FBPase antibody indicate that the FBPase isozymes of leaves are different proteins.     

Temperature-Driven Adaptation of the Bacterial Community in Peat Measured by Using Thymidine and Leucine Incorporation

The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25°C to 35, 45, or 55°C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures. Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature. Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community. Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures. The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55°C (thermophilic activity) and 25°C (mesophilic activity). Shifting the peat incubation temperature from 55 to 25°C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity. The availability of substrate for bacterial growth varied over time and among different peat samples. To avoid confounding effects of substrate availability, a temperature adaptation index was calculated. This index consisted of the log₁₀ ratio of TdR incorporation at 55 and 25°C. The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift. There were no differences between the slopes of the temperature adaptation indices over time for peat samples incubated at 55°C 3 or 11 days before incubation at 25°C. Thus, different levels of bacterial activity did not affect the temperature-driven adaptation of the bacterial community.     

Degradation of Ribulose-l,5-bisphosphate Carboxylase/Oxygenase in the Lysates of the Chloroplasts Isolated Mechanically from Wheat (Triticum aestivum L.) Leaves

Intact chloroplasts were isolated mechanically from the primaryleaves of 8- to 12-day old seedlings of wheat (Triticum aestivumL.) and purified by Percoll gradient centrifugation. The chloroplastswere lyzed by osmotic shock and the reaction mixtures containingthe lysates were incubated in the pH range of 5.3 to 9.4 at37°C. The degradation of ribulose-l,5-bisphosphate carboxylase/oxygenase(RuBisCO, EC 4.1.1.39 [EC] ) and its degradation products in the mixtureswere examined by using SDS-polyacrylamide gel electrophoresis.RuBisCO-hydrolase activity in the lysates was very weak, andit was difficult to assess the activity by measuring the lossof the amount of the large subunit of RuBisCO on the gels afterstaining with Coomassie Brilliant Blue. By using immunoblottingmethod, however, degradation products of RuBisCO could be detectedin the reaction mixtures. The hydrolase activity was pronouncedin the presence of 0.1 % (w/v) of SDS in the reaction mixtures.Among the products, the 35 kDa fragment was conspicuous andfound in the wide range of pHs. This degradation of RuBisCOwas inhibited in the presence of leupeptin and N-ethylmaleimide. (Received October 3, 1988; Accepted November 25, 1988)     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Dithiothreitol triggers photooxidative stress and fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact pea chloroplasts

In intact chloroplasts isolated from mature pea leaves (Pisum sativum L.), the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was rapidly fragmented into several products upon illumination in the presence of 1 mM dithiothreitol (DTT). Very similar effects on LSU stability could be observed when illuminated chloroplasts were poisoned with cyanide which, like DTT, inhibits important plastid antioxidant enzymes, or when a light-dependent hydroxyl radical-producing system was added to the incubation medium. Moreover, DTT-stimulated light degradation of LSU was markedly delayed in the presence of scavengers of active oxygen species (AOS). It is therefore suggested that light degradation of LSU in the presence of DTT is mainly due to inhibition of the chloroplast antioxidant defense system and the subsequent accumulation of AOS in intact organelles. When chloroplasts were isolated from nonsenescent or senescent leaves, LSU remained very stable upon incubation without DTT, indicating that the antioxidant system was still functional in the isolated chloroplasts during leaf ageing. Our data support the notion that AOS might be important for the degradation of Rubisco in vivo under oxidative stress.     

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.     

Nature of photooxidative events in leaves treated with chlorosis-inducing herbicides

Leaves of rye seedlings (Secale cereale L.) grown in the presence of four chlorosis-inducing herbicides under a low light intensity of 10 lux formed chlorophyll. When segments of such dim-light-grown leaves were exposed to 30,000 lux at either 0°C or 30°C, treatments with aminotriazole or haloxidine (group 1) showed no or only minor changes of their chlorophyll contents. In treatments with San 6706 or difunon (group 2), however, rapid photodestruction of chlorophyll occurred both at 0°C and at 30°C and was accompanied by an increase of malondialdehyde that was not seen in the presence of group 1 herbicides. Unlike the in vivo behavior, virtually equal rates of chlorophyll breakdown were observed for aminotriazole and San 6706 treatments in suspensions of isolated chloroplasts from 10 lux-grown leaves after exposure to strong light. The free radical scavengers p-benzoquinone and hydroquinone and the d-penicillamine copper complex exerting superoxide dismutating activity effectively prevented photooxidation of chlorophyll in 10 lux-grown herbicide-treated leaf segments or even restored an accumulation of chlorophyll at 30,000 lux. Ascorbate and several singlet oxygen or hydroxyl radical scavengers had no protective effects. Deuterium oxide and H₂O₂ did not enhance the degradation of chlorophyll. Superoxide dismutase activity was decreased in leaves bleached in the presence of group 2 herbicides.