Morphological adaptation of Pseudomonas nautica strain 617 to growth on eicosane and modes of eicosane uptake

The transfer of Pseudomonas nautica 617 from a hydrophilic growth substrate (acetate) to a solid hydrocarbon substrate (eicosane) induced major morphological changes. The cell surface was covered by extracellular vesicles, and filamentous structures were observed. Adherence and emulsification were the two main modes of transfer of hydrocarbon to the cell surface. Growth on eicosane was associated with biosurfactant production.     

Fatty acid and phospholipid composition of five psychrotrophic Pseudomonas spp. grown at different temperatures

The free fatty acid and phospholipid composition of 5 psychrotrophic marine Pseudomonas spp. have been determined in chemostat culture with glucose as the limiting substrate over the range 0–20°C. The predominant fatty acid present in all the isolates was hexadecenoic acid (C16:1) together with lesser quantities of octadecenoic acid (C 18:1) whilst none contained acids with chain lengths exceeding 18 carbon atoms. Decreasing the growth temperature from 20°C to 0°C resulted in little significant change in fatty acid composition. The principal phospholipid components of the five psychrotrophic pseudomonads have been identified as phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Decreasing the growth temperature did not elicit significant changes either in the total quantities of phospholipid synthesized or in the concentration of individual phospholipid components in any of the isolates. All the psychrotrophs showed maximum glucose uptake between 15°C and 20°C and the rate decreased rapidly as the temperature was decreased towards 0°C.Abbreviations PS Phosphatidylserine - PE phosphatidylethanolamine - PG phosphatidylglycerol - DPG diphosphatidylglycerol     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

Effect of bicarbonate and root zone temperature on uptake of Zn,Fe, Mn and Cu by different rice cultivars (Oryza sativa L.) grown in calcareous soil

Pot experiments were conducted with a calcareous soil (Inceptisol) to elucidate the effects of bicarbonate (0 and 20 mM) and root zone temperature (15° and 25°C) on the uptake of Zn, Fe, Mn and Cu by "Zn-efficient" and "Zn-inefficient" rice cultivars. Bicarbonate decreased concentrations and total uptake of Zn in shoots of "Zn-inefficient" cultivars, especially of IR 26 at 25°C, but not in Zn-efficient cultivars. Bicarbonate decreased concentrations and uptake of Fe in shoots of Zn inefficient cultivars, particularly in IR 26. Concentrations and total uptake of Mn were lower in bicarbonate treatment in the Zn-inefficient cultivars at 15°C, and in all cultivars at 25°C. However, concentration and uptake of Cu were not affected by bicarbonate in all cultivars. Compared to the 25°C root zone temperature, the concentrations and total uptake of both Zn and Cu in shoots at 15°C were lower in Zn-inefficient than in the Zn-efficient cultivars. The results indicate that Zn-efficiency in rice is causally related to high tolerance of plant to elavated bicarbonate concentrations in soil solution.     

Isolation of thermostable D-hydantoinase-producing thermophilicBacillus sp SD-1

Summary A thermophilic bacterium which showed highest thermostability and activity of the hydantoinase was isolated from 1m000 thermophiles and identified to beBacillus sp. SD-1 according to morphological and physiological characteristics. The optimal growth temperature of the bacterium was about 60°C. The hydantoinase ofBacillus sp. SD-1 was strictly D-specific, and optimal pH and temperature were determined to be about 8.0 and 70°C, respectively. The D-hydantoinase was stable upto 70°C, and half-life of the enzyme was about 20 min at 80°C.     

Elucidation of the mechanism of N-demethylation catalyzed by cytochrome P450 monooxygenase is facilitated by exploiting nitrogen-15 heavy isotope effects

Tryptophan indole-lyase (Trpase), PBPRA2532, from Photobacterium profundum SS9, a piezophilic marine bacterium, has been cloned, expressed in Escherichia coli, and purified. The P. profundum Trpase (PpTrpase) exhibits similar substrate specificity as the enzyme from E. coli (EcTrpase). PpTrpase has an optimum temperature for activity at about 30 °C, compared with 53 °C for EcTrpase, and loses activity rapidly (t_1/2 ∼ 30 min) when incubated at 50 °C, while EcTrpase is stable up to 65 °C. PpTrpase retains complete activity when incubated more than 3 h at 0 °C, while EcTrpase has only about 20% remaining activity. Under hydrostatic pressure, PpTrpase remains fully active up to 100 MPa (986 atm), while EcTrpase exhibits only about 10% activity at 100 MPa. PpTrpase forms external aldimine and quinonoid intermediates in stopped-flow experiments with l-Trp, S-Et-l-Cys, S-benzyl-l-Cys, oxindolyl-l-Ala, l-Ala and l-Met, similar to EcTrpase. However, with l-Trp a gem-diamine is observed that decays to a quinonoid complex. An aminoacrylate is observed with l-Trp in the presence of benzimidazole, as was seen previously with EcTrpase [28] but not with S-Et-l-Cys. The results show that PpTrpase is adapted for optimal activity in the low temperature, high pressure marine environment.     

Isolation and characterization of Methanoplanus endosymbiosus sp. nov., an endosymbiont of the marine sapropelic ciliate Metopus contortus quennerstedt

Epifluorescence microscopy revealed the presence of a methanogenic bacterium as an endosymbiont in the sapropelic marine ciliate Metopus contortus. The in situ methanogenic activity of the symbiont could be demonstrated. The isolated endosymbiont was an irregular, disc-shaped bacterium with a diameter of 1.6–3.4 m. It had a generation time of 7 or 12 hours on growth on H₂/CO₂ or formate, respectively. The temperature range for growth was between 16 and 36°C with an optimum at 32°C. The optimal pH range for growth was 6.8 to 7.3. Salts, with an optimum concentration of 0.25 M, and tungsten were required for growth. The mol% G+C was 38.7%. The cell envelope consisted of proteins and a glycoprotein with an apparent molecular weight of 110,000. Morphology, antigenic relationship and the G+C content established the isolate MC1 as a new species of the genus Methanoplanus, and the name Methanoplanus endosymbiosus is proposed.Abbreviations G+C Guanine+cytosine - SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2-ethane) sulfonic acid     

Crude biosurfactant from thermophilic Alcaligenes faecalis: Feasibility in petro-spill bioremediation

The thermophilic bacterium Alcaligenes faecalis isolated from the crude oil contaminated soil of Upper Assam, India. The isolated bacterium was first screened for the ability to produce biosurfactant. The strain growing at 42 °C could produce higher amount of biosurfactant in medium supplemented with 2% (v/v) diesel as sole source of carbon and energy. Biochemical characterizations including FT-IR and MS studies suggested the biosurfactant to be glycolipid. Tensiometric studies revealed that the biosurfactant produced by the bacterial strain could decrease the surface tension (??) at air-water interface from 71.6 to 32.3 mNm⁻¹ after 96 h of growth on hydrocarbon and possessed a low critical micelle concentration (CMC) value of approximately 38 mgl⁻¹, indicating high surface activity. The culture supernatant containing the biosurfactant was found to be functionally stable at varying pH (2-12), temperature (100 and 121 °C) and salinity (1-6% NaCl, w/v) conditions. Both the culture broth and the cell free supernatant exhibited high emulsifying activity against the different hydrocarbons and the crude oil components. The increase in cell surface hydrophobicity and glycolipid production by the strain suggested the existence of biosurfactant enhanced interfacial uptake of the hydrocarbons. Moreover, the partially purified biosurfactant exhibited antimicrobial activity by inhibiting the growth of several bacterial and fungal species. The strain represented a new class of biosurfactant producers and could be a potential candidate for the production of glycolipid biosurfactant which could be useful in a variety of biotechnological and industrial processes, particularly in the oil industry.     

Growth and biochemical composition with emphasis on the fatty acids of Tetraselmis sp.

Summary The influence of temperature (15–32°C) and the ratio of nitrogen to phosphorus (N/P) in the culture medium (0.5–80) on the growth kinetics and protein, chlorophyll, lipid and fatty acid content of the marine microalga Tetraselmis sp. have been studied. Below an N/P of 20, growth was determined by N limitation and above 20 by P limitation. Protein increased with a rise in N content at any test temperature. The chlorophyll content increased with temperature, with maximum values at 25°C. The lipid content decreased with increasing N/P ratio above 20°C. The polyunsaturated fatty acid content tends to be inversely proportional to the growth rate within the N/P range 20–80. The quotient of the n ₃ and n ₆ polyunsaturated-fatty-acid fractions, an indicator of the nutritive value of microalgae, was found to be within the range 2–3. These values were obtained either between 25 and 28°C independent of the N/P ratio used at 20°C for N/P ratios higher than 40.0. Offsprint requests to: Emilio Molina     

Characterization of Chimeric Isocitrate Dehydrogenases of a Mesophilic Nitrogen-Fixing Bacterium, <Emphasis Type="Italic">Azotobacter vinelandii,</Emphasis> and a Psychrophilic Bacterium, <Emphasis Type="Italic">Colwellia maris</Emphasis>

Several properties of chimeric enzymes between a mesophilic isocitrate dehydrogenase (IDH) from a nitrogen-fixing bacterium, Azotobacter vinelandii, and a cold-adapted IDH isozyme (IDH-II) from a psychrophilic bacterium, Colwellia maris, were examined. Each of the genes encoding the IDHs was divided into four regions of almost equal lengths, and each region was ligated with different combinations to construct various chimeric genes. The resultant wild-type and chimeric genes were overexpressed in Escherichia coli. The wild-type and chimeric IDHs were classified into three groups based on optimum temperatures for activity of 20°, 30°, and 40°C. The IDHs with a lower optimum temperature were more thermolabile. The optimum temperature and thermostability of the chimeric enzymes decreased on increasing the proportion derived from the cold-adapted IDH-II of C. maris. Furthermore, the C-terminal region of the C. maris IDH-II was suggested to be responsible for its psychrophilic characteristics.     

Bionomics and population growth statistics of apterous virginoparae of woolly apple aphid, Eriosoma lanigerum, at constant temperatures

Development, survival, reproduction and population growth statistics of apterous virginoparae of woolly apple aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) at constant temperatures of 10, 13, 15, 20, 25, 30 and 32°C are reported. The developmental times of all life stages were inversely related to temperature ranging from 10 to 30°C. Span of total development (time from birth to adulthood) decreased from 57.8 days at 10°C to 11.7 days at 30°C and increased to 16.8 days at 32°C. A good linear model fit (R²>0.96) between developmental rate and temperature in the range 10–25°C was observed for all life stages. The lower developmental threshold was estimated at 5.8°C for instar I, 4.8°C for instar II, 4.9° for instar III and 4.4°C for instar IV. The lower temperature threshold for total development was estimated at 5.2°C. The upper developmental limit was found to be 32°C. Mean degree-day accumulations required for completion of instars I, II, III, IV and total development were: 125.6, 51.0, 47.7, 50.7 and 267.6, respectively. Fecundity, larviposition period and adult longevity were reduced with increasing temperature. Net reproductive rate was greatest at 15°C whereas intrinsic rate of increase peaked at 25°C. Optimal performance, as measured by fecundity, survival and intrinsic rate of increase, ocurred in the range 13–25°C.     

Rheological properties of polysaccharides produced by a Zoogloea sp.

A newly isolated marine bacterium, identified as Zoogloea sp., produced two different polysaccharides: one was water-soluble and the other was cell-bound. Both had non-Newtonian, pseudoplastic fluid behaviour and the solutions had low activation energies. The solutions of these polysaccharides showed rheological behaviour over a wide range of pH (2–12) and temperature (20–80 °C), and compatibility with NaCl.     

Life Tables of Phytoseiulus Macropilis (Banks) (Gamasida: Phytoseiidae) at Different Temperatures

The biology of Phytoseiulus macropilis (Banks) fed on Tetranychus urticae Koch was studied at different temperatures. The total development times averaged 7.5, 5.7, 4.2, 4.2 and 5.6 days at 20, 25, 28, 30 and 32°C, respectively at 78 ± 2% RH and 16 h photoperiod daily. The intrinsic rate of natural increase (r _m) and the net reproduction (R _o) reached maximum values 0.47 and 88.9, respectively, at 28°C. The mean generation time decreased (20.0-8.8 days) with increasing temperature 20-28°C.     

Respiratory energy losses in the protozoan predator Didinium nasutum Müller (Ciliophora)

Summary Respiration in Didinium nasutum, an active protozoan predator, was investigated in relation to cell weight at 10, 15, and 20° C by means of cartesian diver microrespirometry. Oxygen uptake increased progressively over the 10–20° C temperature range; a table of Q ₁₀ related to weight is presented. Regression coefficients of log weight versus log oxygen uptake (b) were 0.96 at 10° C, 0.98 at 15° C and 1.00 at 20° C. D. nasutum was shown to expend very much higher levels of respiratory energy than a mainly sedentary carnivorous ciliate of comparable weight.     

Production of extracellular emulsifying agent byPseudomonas aeruginosa UG1

Summary Twenty-three bacterial strains were isolated from oil-contaminated soil samples. Of these, 20 displayed some ability to effect oil dispersion and they were screened quantitatively for the ability to emulsify 0.5% (v/v) reference oil. One strain, identified asPseudomonas aeruginosa UG1, produced extracellular material that emulsified reference oil, hexadecane and 2-methylnaphthalene at concentrations as high as 6% (v/v) in nutrient broth. Emulsification activity increased during a 10 day incubation period at 30°C. The activity was not influenced by pH over the range 5 to 9. The emulsifying agent was precipitated by cold ethanol. The highest emulsifying activity was detected in the extracellular fraction precipitated between 30 and 50% (v/v) ethanol. A linear relationship was observed between emulsifier concentration (mg/ml) and emulsifying activity. Genetic analysis showed that thePseudomonas aeruginosa UG1 strain did not carry extrachromosomal plasmids, suggesting that the gene(s) coding for emulsifying activity was carried on the chromosome.     

Production of N-acetyl-β-d-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme

A bacterium, GJ-18, having strong chitinolytic activity was isolated from coastal soil. The isolated strain was identified as Aeromonas sp. by morphological and biochemical properties along with 16S rRNA gene sequence. The crude chitinolytic activity of culture supernatants was maximal on the 5th day of culture. Below 45°C, chitin was effectively hydrolyzed to N-acetyl--d-glucosamine (GlcNAc) by Aeromonas sp. GJ-18 crude enzymes, but hydrolysis decreased markedly above 50°C. The optimum pH for enzyme activity was 5.0. TLC and HPLC analysis revealed that, below 45°C, the major reaction product was GlcNAc with a small amount of (GlcNAc)₂ and (GlcNAc)₃, whereas above 50°C the major product was (GlcNAc)₂. When swollen chitin (100 mg) was incubated with crude enzyme preparations (10 U) at 40°C, chitin was hydrolyzed to 83.0 and 94.9% yield of GlcNAc within 5 and 9 days, respectively.     

A cytochrome cd1-type nitrite reductase isolated from the marine denitrifier Pseudomonas nautica 617: purification and characterization

Nitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments.     

Effects of water temperature change on immune function in surf clams, Mactra veneriformis (Bivalvia: Mactridae)

Surf clam, Mactra veneriformis is one of the crucial fishery resources in Korea. This study was performed to examine the immune functions of the surf clam under the stress of water temperature changes at 10 °C, 20 °C or 30 °C for 24 h. Viable bacterial counts (VBC), total haemocyte count (THC), phagocytic activity, lysozyme activity, NRR times and SOD activity were assessed in three different water temperature groups. Clams held at 10 °C decreased in THC, lysozyme activity and NRR times, but phagocytic activity was increased. The highest temperature (30 °C) significantly increased in THC, whereas it decreased in phagocytic activity, lysozyme activity and NRR times. In clams maintained at 20 °C, phagocytic activity, lysozyme activity and NRR times were increased whereas THC was somewhat decreased with respect to clams held at 30 °C. However, water temperature changes did not elicit any alteration of VBC and SOD activity. The present study demonstrates that acute water temperature change affects the haemocytic and haemolymphatic functions, reducing immunosurveillance in stressed surf clam, M. veneriformis.     

Effects of temperature and salinity on survival of toxigenicVibrio cholerae O1 in seawater

In 1991 and 1992, the Latin American epidemic strain of Vibrio cholerae O1 was isolated from ballast water, bilge water, and sewage taken from cargo ships docked in Mobile Bay, Alabama. The findings raised questions regarding the organism's ability to survive long-term aboard ships and to withstand the exchange of ballast at sea. The effects of temperature (6, 18, and 30°C) and salinity (8, 16, and 32 ppt) on survival of V. cholerae O1 strains C6706 and C6707 and a ballast water isolate in sterile seawater were determined. The ballast water isolate, which had a D-value (number of days required to produce a 1 log₁₀ reduction in colony-forming units per milliliter) of 240 days at 18°C, 32 ppt salinity, had the longest survival time. The range of D-values was 36–240 days at 18°C, 60–120 days at 30°C, and 5–20 days at 6°C. In sterile seawater short-term survival was temperature dependent, whereas long-term survival was salinity dependent. In raw seawater, survival time of the ballast water isolate was reduced to 12–27 days, implying the existence of biological influences. As also shown in our previous work, the organism appeared to be able to survive for several months under relatively stable conditions in ballast water aboard ships; however, viability may be reduced to only a few weeks after the organism is introduced into estuarine or marine environments. Correspondence to: Susan A. McCarthy.     

Effect of temperature on osmotic and ionic regulation in goldfish,Carassius auratus L.

Summary Urine flow increased with acute temperature increases and showed temperature acclimation. When measured at 20 °C the urine flow of 10 °C acclimated fish was 3.2 times greater than the urine flow of 30 °C acclimated fish. In fish acclimated to 24 °C renal reabsorption of Na and Cl was independent of temperature over an intermediate range of temperatures (14–24 °C) but near the lower lethal temperature (6.5 °C) renal Na and Cl reabsorption was inhibited. Water permeability of the renal tubules was not affected by acute temperature change between 6.5 and 24 °C. Urine osmolality and urine Na, K and Cl concentrations showed nearly perfect temperature compensation in fish acclimated to 10 °C and 30 °C. The rate of renal excretion of Na and Cl showed temperature acclimation in that Na and Cl ecxretion measured at 20 °C was 7 to 8 times greater in 10 °C acclimated fish than in 30 °C acclimated fish. The rate of excretion of Na and Cl measured at 30 °C in 30 °C acclimated fish was approximately 1.7 times the rate of excretion measured at 10 °C in 10 °C acclimated fish.The branchial uptake of Na, measured in tap water, of fish acclimated to 10, 20 and 30 °C in demineralized water increased with acute increases in temperature. When the three acclimation groups were compared at an intermediate temperature (20 °C), the 10 °C acclimated group showed the highest rate of net uptake, and the 30 °C group the lowest rate of uptake. This apparent temperature acclimation of Na uptake was correlated with differences in the plasma Na concentration of the three acclimation groups. Plasma Cl concentrations were also correlated with acclimation temperature in fish acclimated in demineralized water, but the rate of net Cl uptake was considerably less than that for Na. Sodium and Cl uptake in fish which had been acclimated in tap water was very variable and was not clearly affected by acute changes in temperature. Uptake of Na and Cl by fish held in tap water did not show temperature acclimation. The difference between uptake and excretion of fish acclimated in tap water was not significantly different from zero, indicating that the fish were in salt balance.The study was supported by National Institutes of Health Grant GM 16932-02 to Dr. Bodil Schmidt-Nielsen. I am grateful to Dr. Schmidt-Nielsen for many useful discussions during the course of this work.