Bacterial incorporation of leucine into protein down to -20 degrees C with evidence for potential activity in sub-eutectic saline ice formations

Direct evidence for metabolism in a variety of frozen environments has pushed temperature limits for bacterial activity to increasingly lower temperatures, so far to -20 degrees C. To date, the metabolic activities of marine psychrophilic bacteria, important components of sea-ice communities, have not been studied in laboratory culture, not in ice and not below -12 degrees C. We measured [3H]-leucine incorporation into macromolecules (further fractionated biochemically) by the marine psychrophilic bacterium Colwellia psychrerythraea strain 34H over a range of anticipated activity-permissive temperatures, from +13 to -20 degrees C, including expected negative controls at -80 and -196 degrees C. For incubation temperatures below -1 degrees C, the cell suspensions [all in artificial seawater (ASW)] were first quick-frozen in liquid nitrogen. We also examined the effect of added extracellular polymeric substances (EPS) on [3H]-leucine incorporation. Results showed that live cells of strain 34H incorporated substantial amounts of [3H]-leucine into TCA-precipitable material (primarily protein) down to -20 degrees C. At temperatures from -1 to -20 degrees C, rates were enhanced by EPS. No activity was detected in the killed controls for strain 34H (or in Escherichia coli controls), which included TCA-killed, heat-killed, and sodium azide- and chloramphenicol-treated samples. Surprisingly, evidence for low but significant rates of intracellular incorporation of [3H]-leucine into protein was observed for both ASW-only and EPS-amended (and live only) samples incubated at -80 and -196 degrees C. Mechanisms that could explain the latter results require further study, but the process of vitrification promoted by rapid freezing and the presence of salts and organic polymers may be relevant. Overall, distinguishing between intracellular and extracellular aspects of bacterial activity appears important to understanding behavior at sub-freezing temperatures.     

Thermophilic sulfate-reducing bacteria in cold marine sediment

Abstract Sulfate reduction was measured with the ³⁵SO₄²⁻ -tracer technique in slurries of sediment from Aarhus Bay, Denmark, where seasonal temperatures range from 0° to 15°C. The incubations were made at temperatures from 0°C to 80°C in temperature increments of 2°C to search for presence of psychrophilic, mesophilic and thermophilic sulfate-reducing bacteria. Detectable activity was initially only in the mesophilic range, but after a lag phase sulfate reduction by thermophilic sulfate-reducing bacteria were observed. No distinct activity of psychrophilic sulfate-reducing bacteria was detected. Time course experiments showed constant sulfate reduction rates at 4°C and 30°C, whereas the activity at 60°C increased exponentially after a lag period of one day. Thermophilic, endospore-forming sulfate-reducing bacteria, designated strain P60, were isolated and characterized as D esulfotomaculum kuznetsovii . The temperature response of growth and respiration of strain P60 agreed well with the measured sulfate reduction at 50°–70°C. Bacteria similar to strain P60 could thus be responsible for the measured thermophilic activity. The viable population of thermophilic sulfate-reducing bacteria and the density of their spores was determined in most probable number (MPN) dilutions. The density was 2.8·10⁴ cells·.g⁻¹ fresh sediment, and the enumerations suggested that they were all present as spores. This result agrees well with the observed lag period in sulfate reduction above 50°C. No environment with temperatures supporting the growth of these thermophiles is known in the region around Aarhus Bay.     

Temperature controls on aquatic bacterial production and community dynamics in arctic lakes and streams

The impact of temperature on bacterial activity and community composition was investigated in arctic lakes and streams in northern Alaska. Aquatic bacterial communities incubated at different temperatures had different rates of production, as measured by ¹⁴C‐leucine uptake, indicating that populations within the communities had different temperature optima. Samples from Toolik Lake inlet and outlet were collected at water temperatures of 14.2°C and 15.9°C, respectively, and subsamples incubated at temperatures ranging from 6°C to 20°C. After 5 days, productivity rates varied from 0.5 to ～13.7 µg C l^?1 day^?1 and two distinct activity optima appeared at 12°C and 20°C. At these optima, activity was 2‐ to 11‐fold higher than at other incubation temperatures. The presence of two temperature optima indicates psychrophilic and psychrotolerant bacteria dominate under different conditions. Community fingerprinting via denaturant gradient gel electrophoresis (DGGE) of 16S rRNA genes showed strong shifts in the composition of communities driven more by temperature than by differences in dissolved organic matter source; e.g. four and seven unique operational taxonomic units (OTUs) were found only at 2°C and 25°C, respectively, and not found at other incubation temperatures after 5 days. The impact of temperature on bacteria is complex, influencing both bacterial productivity and community composition. Path analysis of measurements of 24 streams and lakes sampled across a catchment 12 times in 4 years indicates variable timing and strength of correlation between temperature and bacterial production, possibly due to bacterial community differences between sites. As indicated by both field and laboratory experiments, shifts in dominant community members can occur on ecologically relevant time scales (days), and have important implications for understanding the relationship of bacterial diversity and function.     

The impact of temperature change on the activity and community composition of sulfate-reducing bacteria in arctic versus temperate marine sediments

Arctic regions may be particularly sensitive to climate warming and, consequently, rates of carbon mineralization in warming marine sediment may also be affected. Using long-term (24 months) incubation experiments at 0°C, 10°C and 20°C, the temperature response of metabolic activity and community composition of sulfate-reducing bacteria were studied in the permanently cold sediment of north-western Svalbard (Arctic Ocean) and compared with a temperate habitat with seasonally varying temperature (German Bight, North Sea). Short-term ³⁵S-sulfate tracer incubations in a temperature-gradient block (between −3.5°C and +40°C) were used to assess variations in sulfate reduction rates during the course of the experiment. Warming of arctic sediment resulted in a gradual increase of the temperature optima ( T _opt) for sulfate reduction suggesting a positive selection of psychrotolerant/mesophilic sulfate-reducing bacteria (SRB). However, high rates at in situ temperatures compared with maximum rates showed the predominance of psychrophilic SRB even at high incubation temperatures. Changing apparent activation energies ( E _a) showed that increasing temperatures had an initial negative impact on sulfate reduction that was weaker after prolonged incubations, which could imply an acclimatization response rather than a selection process of the SRB community. The microbial community composition was analysed by targeting the 16S ribosomal RNA using catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH). The results showed the decline of specific groups of SRB and confirmed a strong impact of increasing temperatures on the microbial community composition of arctic sediment. Conversely, in seasonally changing sediment sulfate reduction rates and sulfate-reducing bacterial abundance changed little in response to changing temperature.     

Environmental factors affect the response of microbial extracellular enzyme activity in soils when determined as a function of water availability and temperature

Microorganisms govern soil carbon cycling with critical effects at local and global scales. The activity of microbial extracellular enzymes is generally the limiting step for soil organic matter mineralization. Nevertheless, the influence of soil characteristics and climate parameters on microbial extracellular enzyme activity (EEA) performance at different water availabilities and temperatures remains to be detailed. Different soils from the Iberian Peninsula presenting distinctive climatic scenarios were sampled for these analyses. Results showed that microbial EEA in the mesophilic temperature range presents optimal rates under wet conditions (high water availability) while activity at the thermophilic temperature range (60°C) could present maximum EEA rates under dry conditions if the soil is frequently exposed to high temperatures. Optimum water availability conditions for maximum soil microbial EEA were influenced mainly by soil texture. Soil properties and climatic parameters are major environmental components ruling soil water availability and temperature which were decisive factors regulating soil microbial EEA. This study contributes decisively to the understanding of environmental factors on the microbial EEA in soils, specifically on the decisive influence of water availability and temperature on EEA. Unlike previous belief, optimum EEA in high temperature exposed soil upper layers can occur at low water availability (i.e., dryness) and high temperatures. This study shows the potential for a significant response by soil microbial EEA under conditions of high temperature and dryness due to a progressive environmental warming which will influence organic carbon decomposition at local and global scenarios.     

Persistence of microbial extracellular enzymes in soils under different temperatures and water availabilities

Microbial extracellular enzyme activity (EEA) is critical for the decomposition of organic matter in soils. Generally, EEA represents the limiting step governing soil organic matter mineralization. The high complexity of soil microbial communities and the heterogeneity of soils suggest potentially complex interactions between microorganisms (and their extracellular enzymes), organic matter, and physicochemical factors. Previous studies have reported the existence of maximum soil EEA at high temperatures although microorganisms thriving at high temperature represent a minority of soil microbial communities. To solve this paradox, we attempt to evaluate if soil extracellular enzymes from thermophiles could accumulate in soils. Methodology at this respect is scarce and an adapted protocol is proposed. Herein, the approach is to analyze the persistence of soil microbial extracellular enzymes at different temperatures and under a broad range of water availability. Results suggest that soil high‐temperature EEA presented longer persistence than enzymes with optimum activity at moderate temperature. Water availability influenced enzyme persistence, generally preserving for longer time the extracellular enzymes. These results suggest that high‐temperature extracellular enzymes could be naturally accumulated in soils. Thus, soils could contain a reservoir of enzymes allowing a quick response by soil microorganisms to changing conditions. This study suggests the existence of novel mechanisms of interaction among microorganisms, their enzymes and the soil environment with relevance at local and global levels.     

New Strains of Bacillus licheniformis and Bacillus coagulans producing Thermostable α-Amylase Active at Alkaline pH

Two species of Bacillus producing thermostable α-amylase with activity optima at alkaline pH are reported here. These organisms were isolated from soil and have been designated as Bacillus licheniformis CUMC 305 and B. coagulans CUMC 512. The enzymes released by these two species were partially purified up to about 81- and 72-fold respectively of the initial activity. The enzyme from B. licheniformis showed a wide temperature-range of activity, with optimum at 91°C. At this temperature it remained stable for 1 h. It retained 40–50% activity at 110°C and showed only 60% of its activity at 30°C. The enzyme showed a broad pH range of activity (4–10) retaining substantial activity on the alkaline side. The optimum pH was 9·5. The enzyme of B. coagulans showed activity up to 90°C, with optimum at 85°C and had a wide pH range with optimum at 7·5–8·5. The hydrolysis pattern of the substrate starch by these enzymes indicated that glucose, maltose, maltotriose and maltotetraose are the principal products rather than higher oligosaccharides.     

2-Aminoethylphosphonate Utilization by the Cold-Adapted Geomyces pannorum P11 Strain

Cold-adapted strain of Geomyces pannorum P11 was found to mineralize of phosphorus–carbon bond-containing compound—2-aminoethylphosphonic acid (2-AEP, ciliatine). The biodegradation process proceeded in the phosphate-independent manner. Ciliatine-metabolizing enzymes' activity was detectable in cell-free extracts prepared from psychrophilic G. pannorum pregrown on 4 mM 2-AEP. Phosphonoacetaldehyde hydrolase (phosphonatase) activity in a partially purified extract was demonstrated at 10 °C.     

Temperature profile of oxygen activation and defense against oxygen toxicity in a psychrophilic member of the Bacteroidaceae

Abstract The temperature profiles have been determined for O₂ reduction by activating substrates for whole cells and cell extracts of the psychrophilic, obligately anaerobic bacterium, strain B6, belonging to the Bacteroidaceae. The profiles were similar whether the cells were grown at 15 or 1°C, and also for cells harvested in the exponential or stationary phase. The H₂O producing pyruvate oxidase displayed in cell-free extracts a considerably higher activity than the H₂O₂ producing NADH and NADPH oxidases at all temperatures in the range 30–1°C, and characteristically makes up a larger proportion of the total O₂ reduction capacity the lower the temperature. It thus seems that the O₂ scavenging property of the pyruvate oxidase, postulated to be utilized in a defense mechanism against the detrimental effects of the H₂O₂ producing pyridine nucleotide oxidases, is particularly well adapted to function at the low temperatures of the Barents Sea, from which this obligately anaerobic organism originates.     

Lipase and esterase formation by psychrophilic and mesophilic Acinetobacter species.

Acinetobacter O16, a psychrophilic species, produced extracellular lipase (measured by hydrolysis of olive oil, tributyrin, or beta-naphthyl laurate) when grown on a complex medium (peptone plus yeast extract). Most lipase was produced during the logarithmic phase of growth. Very little cell-bound lipase was formed. These cells also produced an esterase (measured by the hydrolysis of beta-naphthyl acetate). At first, all esterase was cell bound; significant amounts appeared in the external medium late in growth. Breaking the cells did not increase cell-bound lipase activity. After breaking of the cells, most of the cell-bound lipase and esterase activity was solubilized, even after very high speed centrifugation. No appreciable amounts of these enzymes were released by osmotic shock. Lipase formation was greatly affected by nutrient conditions. Lowering either the yeast extract of the peptone content of the normal complex medium lowered or abolished lipase formation. Esterase activity was lowered to a lesser extent. Cells growing in synthetic amino acid plus vitamin medium or in acid-hydrolyzed casein produced substantial amounts of esterase but no cell-free or cell-bound lipase. However, if sodium taurocholate was added to these media, lipase was produced. Greatest production occurred if a mixture of di- and poly-peptides was also present. Taurocholate also stimulated lipase production in the normal complex medium. Adding Tween 80 or ethanol to the normal complex medium inhibited lipase production. Sodium acetate, oleic acid, olive oil, or Tween 20 added to synthetic media did not affect lipase production. The psychrophile grew more quickly at 30 degrees C than at 15 or 20 degrees C but produced more lipase at the lower temperatures. Esterase production was about the same at 20 and 30 degrees C. A mesophilic Acinetobacter species produced the same amount of lipase and esterase at 20 and 30 degrees C. The best production of lipase by the psychrophile occurred in standing cultures.     

Production of β-glucosidase (cellobiase) by Curvularia sp.

A Curvularia sp. isolated from soil was found to produce extracellular β-glucosidase activity when grown in yeast extract, peptone, carboxymethylcellulose (YPC) medium. An initial medium pH of 6·5 and cultivation temperature of 30°C were found to be most suitable for high enzyme productivity. The pH and temperature optima for the enzyme were 4·0 and 70°C, respectively. Under these conditions, the enzyme exhibited a K_m (0-nitrophenyl-β- d -glucoside) value of 0.20 mmol/l. Several divalent metal ions inhibited enzyme activity at high concentration. EDTA. also inhibited β-glucosidase activity.     

A novel cold-tolerant Clostridium strain PXYL1 isolated from a psychrophilic cattle manure digester that secretes thermolabile xylanase and cellulase

A Clostridium strain PXYL1 was isolated from a cold-adapted cattle manure biogas digester at 15 degrees C. It could grow at temperatures as low as 5 degrees C up to 50 degrees C with highest specific growth rate at 20 degrees C and is a psychrotroph. It produced extracellular hydrolytic enzymes namely xylanase, endoglucanase, beta-xylosidase, beta-glucosidase and filter paper cellulase, all of which had maximal activity at 20 degrees C. The induction of xylanase was highest on birch wood xylan (37 IU(mg protein)(-1)) compared with xylose (1.11 IU(mg protein)(-1)), cellobiose (1.43 IU(mg protein)(-1)) and glucose (no activity). The xylanase was thermolabile with a half-life of 30 min at 40 degrees C and 8 min at 50 degrees C but stable for over 2 h at 20 degrees C. The crude enzyme released reducing sugars (1.25 g l(-1)) from finger millet flour at 20 degrees C, while commercial food-grade xylanases showed no hydrolysis at this temperature. This is the first report of a Clostridium strain growing at 20 degrees C and producing an array of xylanolytic and cellulolytic enzymes, possessing low temperature optima of 20 degrees C, which may facilitate degradation of plant fibre under low-temperature conditions.     

Endo-(1 → 3)β-D-glucanase activity secreted by Bacillus sp. no. 215

The production of an extracellular endo-(1 → 3)-β-D-glucanase by Bacillus sp. no. 215 was induced during growth with (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU as carbon and energy source. Maximum levels of activity (0.26 U ml^-1 resp. 1.40 U mg^-1) were detected in cell-free culture supernatant fluid after 25 h of aerobic growth at 55°C. The cells secreted an endo-(1 → 3)-β-D-glucanase with low electrophoretic mobility that used curdlan from C. flavigena strain KU and from Agrobacterium sp. (formerly Alcaligenes faecalis var. myxogenes ) as substrates. The enzyme activity was highest at pH 7.0 and 55°C. It exhibited a remarkably low thermal stability with a half-life of 14 min at 55°C in the presence of substrate. Divalent metal cations were required for enzyme activity.     

Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments

Five psychrophilic sulphate-reducing bacteria (strains ASv26, LSv21, PSv29, LSv54 and LSv514) isolated from Arctic sediments were examined for their adaptation to permanently low temperatures. All strains grew at −1.8°C, the freezing point of sea water, but their optimum temperature for growth ( T _opt) were 7°C (PSv29), 10°C (ASv26, LSv54) and 18°C (LSv21, LSv514). Although T _opt was considerably above the in situ temperatures of their habitats (−1.7°C and 2.6°C), relative growth rates were still high at 0°C, accounting for 25–41% of those at T _opt. Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2–9°C above T _opt. In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between −1.8°C and T _opt. For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0°C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields at in situ conditions.     

Production of thermostable acid protease by Aspergillus niger

Aspergillus niger F2078 produces high levels of extracellular thermostable acid protease within 96 h. Although glucose and peptone were the best carbon and nitrogen sources, respectively, sucrose and a cheap nitrogen source, corn steep liquor, also gave satisfactory enzyme yields. Supplementation of groundnut meal to the basal medium enhanced enzyme production. Temperature and pH optima of the enzyme activity were 60°C and 3.0–4.0, respectively. The enzyme was stable between pH 3.0 and 6.0 and at temperatures up to 60°C.     

Effect of Some Environmental Factors on Psychrophilic Microbacteria

S ummary . The growth of three psychrophilic strains of Microbacterium isolated from meat was studied at 10 temperatures between 0° and 35° and at 6 water activity (a_w) levels between 0.99 and 0.94. The temperature and water relations of the three strains were similar. For all strains the rate of growth was fastest at 25° and a_w 0.99, and growth did not occur at 35°.
Further experiments on one strain showed that under aerobic conditions the range of water activities permitting growth was independent of temperature, but under anaerobic conditions the minimum water activity increased from about 0.94—0.96 as the temperature was reduced from 25° to 0°. Growth was inhibited by low concentrations of undissociated nitrous acid, and inhibition was greater at 0° than at 10° or 25°.     

Characterization of alanine and malate dehydrogenases from a marine psychrophile strain PA-43

Alanine dehydrogenase (AlaDH: EC 1.4.1.1), malate dehydrogenase (MDH: EC 1.1.1.37), and glutamate dehydrogenase (EC 1.4.1.2), all NAD+ dependent, were detected in extracts from a psychrophilic bacterium, strain PA-43, isolated from a sea urchin off the Icelandic coast. Characterization tests suggested that the strain had a close relationship to Vibrio, but sequencing of part of the 16S rDNA gene placed the bacterium among Shewanella species in a constructed phylogenetic tree. The bacterium had an optimum growth temperature of 16.5 degrees C, and maximum dehydrogenase expression was obtained in a rich medium supplemented with NaCl. Both AlaDH and MDH were purified to homogeneity. AlaDH is a hexamer, with an approximate relative molecular mass of 260,000, whereas MDH is dimeric, with an apparent relative molecular mass of approximately 70,000. Both enzymes were thermolabile, and the optimum temperatures for activity were shifted toward lower temperatures than those found in the same enzymes from mesophiles, 37 degrees C for MDH and approximately 47 degrees C for AlaDH. The pH optima for AlaDH in the forward and reverse reactions were 10.5 and 9, respectively, whereas those for MDH were 10-10.2 and 8.8, respectively. Partial amino acid sequences, comprising approximately 30% of the total sequences from each enzyme, were determined for N-terminal, tryptic, and chymotryptic fragments of the enzymes. The AlaDH showed the highest similarity to AlaDHs from the psychrotroph Shewanella Ac10 and the mesophile Vibrio proteolyticus, whereas MDH was most similar to the MDHs from the mesophiles Escherichia coli and Haemophilus influenzae, with lower identity to the psychrophilic malate dehydrogenases from Vibrio 5710 and Photobacterium SS9.     

Dichlorophenolindophenol-linked formate dehydrogenase of the methanol-utilizing Mycobacterium gastri MB19

Abstract 2,6-Dichlorophenolindophenol (DCIP)-linked formate dehydrogenase activity has been demonstrated for the first time in the cell-free extract of a methylotrophic mycobacterium, Mycobacterium gastri MB19. The enzyme was produced when the strain was cultivated with methanol, glucose or mannitol as a carbon source, whereas no enzyme production occurred with other multi-carbon compounds. The enzyme was located in the particulate fraction. Although the enzyme was unstable on preservation at 4° C in potassium phosphate buffer (pH 7.0), it was stabilized under acidic conditions (pH 5.0). Glycerol and EDTA were also effective for the enzyme's stability. The optimum pH and temperature for the enzyme's activity were 7.0° and 55° C, respectively.     

Significance of temperature and preincubation temperature on survival of Listeriamonocytogenes at pH 4·8

Listeria monocytogenes is a food-borne pathogenic bacterium that can be found in softcheese. At the beginning of cheese ripening, the pH is about 4·85–4·90. The aimof this work was to study the influence of temperature, preincubation temperature (temperature atwhich the inoculum was cultivated) and initial bacterial concentration on the survival of L.monocytogenes (strain Scott A) at pH 4·8. It was demonstrated in an earlier study thatthese factors did influence growth kinetics. Survival studies of L. monocytogenes weredone in a laboratory broth simulating cheese composition. Four test temperatures (2, 6, 10 and14°C) and two preincubation temperatures were studied (30°C or the test temperature). Listeria monocytogenes (strain Scott A) was unable to grow at pH 4·8 under allconditions tested. The time for 10% survival was about 11 and 2 d, at 2°C with preincubationat 2°C and 30°C, respectively; 9 d at 6°C with preincubation at 6°C; 4 d at 6°Cwith preincubation at 30°C; and 1 d at 14°C with preincubation at 14°C or at 30°C.The results show that survival of L. monocytogenes (strain Scott A) at pH 4·8 is notdependent on initial bacterial concentration but on both the test and preincubation temperatures.     

Homeostatic regulation of acetazolamide-sensitive esterase activity in the bluegill sunfish, Lepomis macrochirus, following acute cold shock

Acetazolamide-sensitive esterase activity was elevated in branchial homogenates of control juvenile bluegill sunfish, Lepomis macrochirus , acclimated at 20° C but decreased rapidly within 9 h following an acute hypothermal shock to 8° C. After 2 weeks at 8° C, shocked-fish enzyme activity was similar to control fish. At 20° C acclimation temperature, specific activity of bluegills was similar in swimbladder, liver, kidney, gill, spleen, and gonad homogenates and was significantly higher (α=0.05 level) in whole blood homogenates. The pH optima for enzymes extracted from fish acclimated at 20° and 8° C were 7.29 and 8.00, respectively. Polyacrylamide gel electrophoresis (PAGE) demonstrated two distinct forms of acetazolamide-sensitive esterase activity present in both 20° and 8° C acclimated fish. Specific activity for homogenates from both 20° and 8° C acclimated fish differed significantly when assayed at 20° C, suggesting both qualitative and quantitative changes in acetazolamide-sensitive esterase. It is postulated that relatively rapid alterations in esterase activity promote survival in bluegill following acute cold shock through the central role of enzymes in the regulation of plasma ion concentrations and acid/base equilibria.