Upper Temperature Limit For Growth Research Articles

Requirement of de novo synthesis of pyruvate carboxylase in long-term succinic acid production in Corynebacterium glutamicum.

Protein turnover through de novo synthesis is critical for sustainable cellular functions. We previously found that glucose consumption rate in Corynebacterium glutamicum under anaerobic conditions increased at temperature higher than the upper limit of growth temperature. Here, we showed that production of lactic and succinic acids increased at higher temperature for long-term (48h) anaerobic reaction in metabolically engineered strains. At 42°C, beyond the upper limit of growth temperature range, biomass-specific lactic acid production rate was 8% higher than that at 30°C, the optimal growth temperature. In contrast, biomass-specific succinic acid production rate was highest at 36°C, 28% higher than that at 30°C, although the production at 42°C was still 23% higher than that at 30°C. As enzymes are usually unstable at high temperatures, we investigated whether protein turnover of metabolic enzymes is required for the production of lactic and succinic acids under these conditions. Interestingly, when de novo protein synthesis was inhibited by addition of chloramphenicol, after 6h, only succinic acid production was inhibited. Because glycolytic enzymes are involved in both lactic and succinic acids synthesis, enzymes in the anaplerotic pathway and the tricarboxylic acid (TCA) cycle leading to succinic acid synthesis were likely to be responsible for its decreased production. Among the five enzymes examined, the specific activity of only pyruvate carboxylase was drastically decreased after 48h at 42°C. Thus, the de novo synthesis of pyruvate carboxylase is required for long-term production of succinic acid. Graphical abstract KEY POINTS: • Long-term reaction for organic acids can be improved at temperature beyond ideal growth conditions. • De novo synthesis of pyruvate carboxylase is required for long-term succinic acid production.

Thermal and solvent stress cross-tolerance conferred to Corynebacterium glutamicum by adaptive laboratory evolution.

Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance.

Isolation and characterization of bacteria from ancient siberian permafrost sediment.

In this study, we isolated and characterized bacterial strains from ancient (Neogene) permafrost sediment that was permanently frozen for 3.5 million years. The sampling site was located at Mammoth Mountain in the Aldan river valley in Central Yakutia in Eastern Siberia. Analysis of phospolipid fatty acids (PLFA) demonstrated the dominance of bacteria over fungi; the analysis of fatty acids specific for Gram-positive and Gram-negative bacteria revealed an approximately twofold higher amount of Gram-negative bacteria compared to Gram-positive bacteria. Direct microbial counts after natural permafrost enrichment showed the presence of (4.7 ± 1.5) × 108 cells g−1 sediment dry mass. Viable heterotrophic bacteria were found at 0 °C, 10 °C and 25 °C, but not at 37 °C. Spore-forming bacteria were not detected. Numbers of viable fungi were low and were only detected at 0 °C and 10 °C. Selected culturable bacterial isolates were identified as representatives of Arthrobacter phenanthrenivorans, Subtercola frigoramans and Glaciimonas immobilis. Representatives of each of these species were characterized with regard to their growth temperature range, their ability to grow on different media, to produce enzymes, to grow in the presence of NaCl, antibiotics, and heavy metals, and to degrade hydrocarbons. All strains could grow at −5 °C; the upper temperature limit for growth in liquid culture was 25 °C or 30 °C. Sensitivity to rich media, antibiotics, heavy metals, and salt increased when temperature decreased (20 °C > 10 °C > 1 °C). In spite of the ligninolytic activity of some strains, no biodegradation activity was detected.

Algal communities at Gouqi Island in the Zhoushan archipelago, China

The kelp bed of Gouqi Island lies in the east of the East China Sea, in the Shengsi Archipelago. It is a key component of the island-reef ecosystem. This study evaluated kelp beds on intertidal and subtidal zones from 2004 to 2006. We evaluated seasonal variations in the community structure of macroalgae, and their relationship with water temperature, nitrogen and phytoplankton. The Gouqi Island kelp bed consists mainly of species of Sargassum. Sargassum horneri was the dominant species in subtidal zones, representing 90% of the total biomass. The dominant species in intertidal zones were S. fusiforme, S. horneri, S. thunbergii and Undaria pinnatifida. Fifty-four phytoplankton species were found in the Gouqi Island kelp bed, of which 51 species were found inside the kelp beds, 43 species outside the kelp beds, and 40 species were present both inside and outside the kelp beds. Diatoms were dominant both inside and outside the kelp beds. Except in winter, Skeletonema costatum was dominant, and its abundance in autumn was over 98% of the total abundance. Water temperature was found to be the primary factor influencing the growth of S. horneri in the Gouqi Island kelp bed. S. horneri grew slowly from November to March, and rapidly from March to June. The upper temperature limit for growth of S. horneri was about 18°C. Higher water temperatures can result in a decline in the kelp bed. Nitrogen seemed to be a limiting factor for macroalgal growth. This was particularly true for S. horneri and phytoplankton. Since nitrate is the primary nutrient for S. horneri, its absorption by S. horneri resulted in seasonal changes of nitrate in the ecosystem.

Growth, structural and optical properties of Cu 3N films

Thin films of Cu 3N were grown on (0 0 1) MgO and (0 0 1) Cu substrates by molecular beam epitaxy of copper in the presence of nitrogen obtained from a radio frequency atomic source. The different aspects of the growth and the properties of the copper nitride films were investigated in relation with growth parameters such as: substrate temperature, growth rate and flux of nitrogen. On MgO substrates, despite a mismatch as high as ∼10%, the Cu 3N films grow as epitaxial layers even at room temperature. The upper temperature limit for growth was found to be ∼250 °C. It is likely that the growth mechanism proceeds in a layer-by-layer fashion similar to the growth of γ ′-Fe 4N layers on MgO substrates using the same growth method. This results in the growth of smooth layers of Cu 3N. The films, grown as a pure phase, have a behavior typical for an insulator with an optical gap of 1.65 eV. If a low amount of Cu impurities is present in the Cu 3N insulating matrix, the films still have the overall behavior of an insulator, but with a reduced optical band gap. For higher amounts of metal impurities, seen as Cu crystallites in the epitaxial Cu 3N phase, the optical band gap reduces even further, and additionally, an extra absorption peak due to Cu appears. The films grown on (0 0 1) Cu substrates, despite the better lattice match, did not show a striking improvement of the crystal quality.

Phytophthora inundata sp. nov., a part heterothallic pathogen of trees and shrubs in wet or flooded soils

A Phytophthora pathogen of trees and shrubs previously designated Phytophthora sp. O-group is formally named as P. inundata sp. nov. P. inundata falls within the P. gonapodyides-P. megasperma major ITS Clade 6, its present nearest known relative being P. humicola. It has non-papillate sporangia, fairly large oogonia (average ca 40 microns) with thick walled oospores, amphigynous antheridia, a distinctive colony type, a high optimum temperature for growth of 28-30 degrees C, fast growth at the optimum, and a high upper temperature limit for growth of ca 35-37 degrees. A study of the breeding system of eight P. inundata isolates showed them to be classically heterothallic with A1 and A2 compatibility types. However some P. inundata A1 x A2 combinations failed to mate even though the same isolates mated successfully with P. drechsleri testers. Others were 'silent' A1s or A2s, unable to produce their own gametangia but able to induce gametangial formation in the opposite sexual compatibility type of another species. This indicates a partial breakdown of the sexual mechanism in the species. Two isolates (one A1 and one A2) were unpredictably and chimaerically self-fertile, suggesting A1 + A2 chromosomal heteroploidy. The association of P. inundata with ponds and rivers and with root and collar roots of trees and shrubs after flooding is discussed.

Isolation, properties and kinetics of growth of a thermophilic Bacillus

A strictly aerobic, thermophilic, spore-forming bacteria was isolated from a soil sample collected in Campos dos Goytacazes City, Rio de Janeiro, Brazil. The cells of this organism were Gram-positive, catalase positive, actively motile, 1.2 µm wide and 5.3 µm long. Growth occurred at pH values ranging from 6.5 to 9.0, and optimum growth occurred at about pH 7.0. The optimum temperature for growth was around 55oC, and the upper temperature limit for growth was around 70oC. Yeast extract stimulated growth on glucose and was obligately required at supraoptimal temperatures (65oC). The results of 16S rRNA sequence comparisons indicated that the isolate was closely related to Bacillus caldoxylolyticus and Bacillus sp strain AK1, and these three organisms exhibited levels of ribossomal DNA sequence homology of 94%.

Short Communication: Salt Extends the Upper Temperature Limit for Growth of Lactococcus lactis ssp. cremoris on Solid M17 Medium

We have determined conditions for plating of the Lactococcus lactis ssp. cremoris laboratory strain MG1363 on solid M17 broth at 38°C, which is required for the optimal use of the pGhost plasmids. The addition of 1% NaCl (or KCl, potassium acetate, or sucrose at 170mM) to M17 agar plates results in extension of the upper temperature limit for growth from 37 to 40°C; no decrease in plating efficiency was detected from 30 to 39°C.

Bacillus thermoamylovorans sp. nov., a Moderately Thermophilic and Amylolytic Bacterium

A moderately thermophilic, facultatively anaerobic, amylolytic bacterium was isolated from palm wine, a tropical alcoholic beverage that was sampled in Senegal. The cells were gram positive, catalase positive, non-spore forming, rod shaped, and slightly motile with peritrichous flagella. The strain which we examined did not possess cytochrome and produced L-(+)-lactate, acetate, ethanol, and formate but not hydrogen during carbohydrate fermentation. Growth occurred at pH values ranging from 5.4 to 8.5, and optimum growth occurred at around pH 7.0. The optimum temperature for growth was around 50 degrees C, and the upper temperature limit for growth was 58 degrees C. The guanine-plus-cytosine content of the DNA was 38.8 +/- 0.2 mol%. A sequence analysis of the 16S rRNA gene revealed that the new organism is closely related phylogenetically to members of genus Bacillus. Despite the lack of spores, we propose that on the basis of phylogenetic characteristics, the new isolate should be classified as a new Bacillus species, Bacillus thermoamylovorans. The type strain is strain DKP (= Collection of Institut Pasteur CNCM I-1378).

Bacterial Sulfate Reduction Above 100°C in Deep-Sea Hydrothermal Vent Sediments

The currently known upper temperature limit for growth of organisms, shared by a number of archaebacteria, is 110 degrees C. However, among the sulfate-reducing bacteria, growth temperatures of greater than 100 degrees C have not been found. A search for high-temperature activity of sulfate-reducing bacteria was done in hot deep-sea sediments at the hydrothermal vents of the Guaymas Basin tectonic spreading center in the Gulf of California. Radiotracer studies revealed that sulfate reduction can occur at temperatures up to 110 degrees C, with an optimum rate at 103 degrees to 106 degrees C. This observation expands the upper temperature limit of this process in deep-ocean sediments by 20 degrees C and indicates the existence of an unknown group of hyperthermophilic bacteria with a potential importance for the biogeochemistry of sulfur above 100 degrees C.

A thermophilic green sulfur bacterium from New Zealand hot springs, Chlorobium tepidum sp. nov.

Thermophilic green sulfur bacteria of the genus Chlorobium were isolated from certain acidic high sulfide New Zealand hot springs. Cells were Gram-negative nonmotile rods of variable length and contained bacteriochlorophyll c and chlorosomes. Cultures of thermophilic chlorobia grew only under anaerobic, phototrophic conditions, either photoautotrophically or photoheterotrophically. The optimum growth temperature for the strains of thermophilic green sulfur bacteria isolated was 47–48°C with generation times of about 2 h being observed. The upper temperature limit for growth was about 52°C. Thiosulfate was a major electron donor for photoautotrophic growth while sulfide alone was only poorly used. N2 fixation was observed at 48°C and cell suspensions readily reduced acetylene to ethylene. The G+C content of DNA from strains of thermophilic chlorobia was 56.5–58.2 mol% and the organisms positioned phylogenetically within the green sulfur bacterial branch of the domain Bacteria. The new phototrophs are described as a new species of the genus Chlorobium, Chlorobium tepidum.

Autecological investigations on marine diatoms. 5:Coscinodiscus concinnus W. Smith andRhizosolenia setigera Brightwell

The cold oligo-eurytherm diatomsCoscinodiscus concinnus W. Smith andRhizosolenia setigera Brightwell were cultured to determine their best competitive position by growth. Comparison of their generation times with those of other diatoms indicate that they reach this position between 6°C and 12°C. Both species grew between −1.5°C and about 20°C. The experiments indicate thatC. concinnus flowerings are possible in a deep water column, during periods of high light intensities. The simultaneous death of species in the upper layer is also caused by high light intensities.C. concinnus appeared in two morphological forms; the normal voluminous form, and a flatter form with a few intercalary bands only, filled with large oil-droplets. The latter appeared at 0°C and below, and at the upper temperature limit for growth of about 19°C–20°C. The separation of nov. spec. fromC. concinnus based on the absence or presence of a hyaline area and intercalary bands as identification characteristics should be reconsidered.

Method to Isolate Escherichia coli O157:H7 from Food

A medium for direct overnight isolation of hemorrhagic colitis (HC) strains of Escherichia coli from foods has been developed. It is a direct plating medium incorporating tryptophan (as tryptone), sorbitol, an indicator dye and a fluorogenic substrate for diagnostic purposes. Sodium chloride is included to raise the upper temperature limit for growth and a bile salt concentration lower than that usual for E. coli media but still inhibitory to non-enteric organisms is used. Colonies of HC strains grown overnight at 44.5°C on membrane filters placed on the medium are blue. Subsequent indole staining of the membranes yields red positive colonies. Replicate colonies are confirmed serologically. In contrast, E. coli Type I colonies are yellow and give apparently negative indole reactions. Recovery of HC organisms from artificially contaminated ground beef is ≥90%.

Canopy Dynamics of Eucalyptus Maculata Hook. I. Distribution and Dynamics of Leaf Populations

The canopy dynamics of a regenerated 16-year-old stand of pole and sapling E. maculata were studied for 2½ years by repetitive non-destructive measurements in tree crowns accessed from a 20 m high scaffold tower. Average canopy leaf area density over a sample plot of 36 m2 was 0.23 m2 m-3 at a leaf area index of 4.3. Some 75% of leaf area was held in the canopies of overstorey eucalypts above 10 m in height. Average size of leaves increased gradually from top to bottom of tree canopies. Foliage production was usually concentrated in the upper crowns of trees where there was a higher proportion of active shoots, more frequent growth flushes and more rapid turnover of leaves than in lower canopy layers. Leaf area in the upper canopy fluctuated widely but increased in the long term, in mid canopy was more or less maintained and in lowest canopy declined. Crops of developing flower buds present on uppermost branches delayed and/or reduced shoot growth. Foliage production occurred in all months of the year. There was a unimodal annual rhythm of growth rate reaching a maximum in summer and a minimum in winter. Variable water supply, however, influenced production to peak in spring, summer or autumn. No shoot growth occurs in E. maculata at Kioloa when daily mean temperature (averaged for weekly intervals) falls below c. 10½C in winter. An upper temperature limit for growth could not be defined. The species apparently lacks dormancy mechanisms. Shoot growth is 'opportunistic' and occurs whenever environmental conditions are favourable. Patterns of leaf production and leaf fall were variable but peaks showed a general synchrony. Leaf fall, however, tended to lag behind leaf production. Leaves of all ages were shed but main losses were from older cohorts. Some 49% (s.d.±18%) of new leaves were lost while still small or immature, mainly during periods of vigorous shoot growth or low water supply. Browse of immature foliage was light. Normal senescence and leaf fall accounted for almost the entire loss of mature foliage.

Effect of growth substrates on fatty acid composition of Corynebacterium cyclohexanicum

Fatty acids occurring in lipids of Corynebacterium cyclohexanicum are 12-mefhyltridecanoic, myristic, 13-methyltetradecanoic, 12-methyltetradecanoic, n-pentadecanoic, 14-methylpentadecanoic, palmitic, 15-methylhcxadecanoic, and 14-methylhexadecanoic (in order of increasing retention times) identified by gas–liquid chromatographic retention characteristics and mass spectrometry. In glucose-grown cells, 12-methyltetradecanoic (35%) and 14-methylpentadecanoic (35%) acids were major acid components, whereas in the cells grown on cyclohexanecarboxylic, m-hydroxybenzoic, butyric, and acetic acids, 12-methyltetradecanoic acid was always the major cellular fatty acid, constituting 65–81% of the total fatty acids. The phase transition temperature of phospholipid sample from cells grown on glucose (22 °C) was higher than the corresponding values with butyrate (10 °C) and acetate (13 °C). The upper limit of growth temperature, however, was no different between cells grown on butyrate or glucose.

Salt extends the upper temperature limit for growth of food-poisoning bacteria

Inclusion of NaCl into the growth medium raised the upper temperature limit of growth of the following organisms: Staphylococcus aureus (two strains), Salmonella senftenberg, S. typhimurium, Escherichia coli, Streptococcus faecalis, Bacillus cereus, Clostridium sporogenes, C. perfringens (two strains). The magnitude of the response varied with the culture, the largest being 3.5 degrees with B. cereus cells. The spores of B. cereus were not protected by salt but clostridial spores behaved as the vegetative cells (response of 2.5 degrees). The optimal salt concentration for the protective effect varied with the organism ranging from 0.2 M for the Gram-negative organisms to 1.0 M for S. aureus.

The effect of NaCl on the upper temperature limit for growth of and enterotoxin synthesis by Staphylococcus aureus.

The upper temperature limit for growth of an enterotoxin A producing strain (MF31) and of an enterotoxin B producing strain (S6) was about 44-45 degrees C in either a peptone-based medium or heart infusion broth. In the presence of NaCl, growth and production of enterotoxin, coagulase, and thermostable nuclease occurred up to 2 degrees C higher. The optimal concentration of NaCl for the temperature elevation effect was 1 M.

OBSERVATIONS ON THE PHYSIOLOGY OF PSYCHROPHILISM IN A YEAST

In a psychrophilic Candida species, survival, respiration of intact cells, activity of alcohol dehydrogenase, and entry of glucosamine into the cell were more sensitive to temperatures above 10 °C than in a mesophilic species, Candida lipolytica. However, growth was more sensitive to temperature than any of these other properties. Respiration of the psychrophile was more rapid than that of the mesophile at all temperatures up to 30°, the difference being most striking at 0°–10 °C. It is suggested that the upper temperature limit for growth of the psychrophile is set by the reversible inactivation of some vital reaction not yet identified.Uptake of glucosamine in the psychrophile was rapid and the rate only slightly influenced by temperature between 0° and 30°. In contrast there was scarcely any uptake by the mesophile at 0° and 10°, and the rate at 20° was much lower than that at 30°. It is suggested that the lower temperature limit of growth for the mesophile is set by the temperature dependence of the mechanisms for transporting substrates into the cell, and that an increased permeability at low temperatures may be an important factor in permitting some organisms to thrive in the cold.