Metabolic activity of permafrost bacteria below the freezing point

Metabolic activity was measured in the laboratory at temperatures between 5 and -20 degrees C on the basis of incorporation of (14)C-labeled acetate into lipids by samples of a natural population of bacteria from Siberian permafrost (permanently frozen soil). Incorporation followed a sigmoidal pattern similar to growth curves. At all temperatures, the log phase was followed, within 200 to 350 days, by a stationary phase, which was monitored until the 550th day of activity. The minimum doubling times ranged from 1 day (5 degrees C) to 20 days (-10 degrees C) to ca. 160 days (-20 degrees C). The curves reached the stationary phase at different levels, depending on the incubation temperature. We suggest that the stationary phase, which is generally considered to be reached when the availability of nutrients becomes limiting, was brought on under our conditions by the formation of diffusion barriers in the thin layers of unfrozen water known to be present in permafrost soils, the thickness of which depends on temperature.     

Metabolic Injury in Frozen Bacteria

S ummary : Aerobacter aerogenes populations, partly killed by freezing and thawing, usually showed more viable bacteria when plated on a rich medium than on a poor one. This 'metabolic injury' was not due to osmotic sensitivity or to toxic materials in the agar. Freezing and thawing was not significantly mutagenic and mutation to nutritional exigence did not account for the phenomenon. The survivors of freezing and thawing showed long lag phases in the recovery media, during which lag periods further mortality occurred.     

The Effect of Freezing on the Radiation Sensitivity of Vegetative Bacteria

S ummary : Five strains of bacteria were irradiated, suspended in heart infusion broth or in phosphate buffer, in aerated or anoxic conditions, at temperatures of 10–13° or -79°. Survivors under the different conditions were enumerated by plate counts on heart infusion agar.
Exponential survivor-dose curves were obtained with a Pseudomonas strain and with Escherichia coli B/r when irradiated at room temperature with aeration, whereas an Alcaligenes strain and 2 strains of Streptococcus faecium gave sigmoid curves. The decreased radiosensitivity in the frozen state was measured by comparing the D₁₀ values for exponential curves, or for the exponential portion of sigmoid curves, with that observed for irradiation at room temperature with aeration. This 'D₁₀ ratio' svaried between 2°5 and 8·5. For the Alcaligenes strain it was about 4, whether the frozen irradiation took place in the presence or absence of oxygen. With the Pseudomonas irradiated in the frozen state in the absence of oxygen the 'D₁₀ ratio' was usually about 1·5 times higher than when oxygen was present. The highest ratio (8·5) was obtained for anoxic irradiation of the Pseudomonas strain.
In general, the shapes of survival curves for frozen irradiation differed from those obtained at room temperature. The sigmoid curves for the Alcaligenes strain irradiated under aerobic conditions when frozen showed a marked decrease in extrapolation numbers. E. coli B/r when frozen in heart infusion broth gave a double exponential curve with a shallow slope initially, followed by a steeper slope. The most radiation resistant strain, Strep. faecium R53, gave sigmoid curves with a D₁₀ value of 300 Krads when frozen, and was then of similar resistance to Clostridium botulinum spores.     

Pathogenicity of Salmonella gallinarum After Metabolic Injury by Freezing

Freezing (-75 C) and storage (-20 C) of a cell suspension of Salmonella gallinarum resulted in a heterogeneous population of dead, metabolically injured, and unharmed cells. Injured cells constituted as much as 40% of those surviving freezing and storage for 1 day. Replica plating of frozen and thawed cells indicated metabolic injury was repairable and not a stable mutation. Penicillin was used to increase the ratio of injured to uninjured cells from a frozen and thawed cell suspension. Pathogenicity was evaluated by observing per cent mortality after injecting injured or uninjured cells into separate sets of chicks. Mortality differences between wholly uninjured and predominantly injured populations were small and consistent (5% level) with a hypothesis of no difference.     

Metabolic Features of Methane- and Methanol-Utilizing Bacteria

Investigations of a wide range of methane- and methanol-utilizers showed a striking versatility of their metabolism dependent on the genotype and growth conditions. A correlation between pathways of carbon and nitrogen metabolism was found. It was most stringent in obligate methane-utilizers: the hexulosephosphate pathway bacteria assimilated NH₃ by the reductive amination of α-ketoglutarate or pyruvate whereas the serine pathway bacteria used the glutamate cycle (glutamine synthetase + glutamate-oxoglutarate aminotransferase). Multiple enzymic lesions were found in central metabolism of obligate methylotrophs, i.e. the absence of the enzymes of glycolytic and pentosephosphate pathways, gluconeogenesis, citric acid cycle and glyoxylate shunt. These metabolic blocks were not so profound and could be compensated in restricted and facultative methylotrophs during heterotrophic growth. The average levels of exogenous CO₂ fixation in methylotrophic bacteria with the hexulosephosphate, serine and ribulosebisphosphate pathways were found to be 10, 30 and 80% of their total cell carbon, respectively. These results served as a basis for biotechnological applications of metabolic potential of methylotrophs (production of biomass, polysaccharides and enzymes as well as for microbiological treatment of industrial waters containing toxic C₁- and C_n-compounds).     

Characterization of Viable Bacteria from Siberian Permafrost by 16S rDNA Sequencing

Abstract Viable bacteria were found in permafrost core samples from the Kolyma-Indigirka lowland of northeast Siberia. The samples were obtained at different depths; the deepest was about 3 million years old. The average temperature of the permafrost is −10°C. Twenty-nine bacterial isolates were characterized by 16S rDNA sequencing and phylogenetic analysis, cell morphology, Gram staining, endospore formation, and growth at 30°C. The majority of the bacterial isolates were rod shaped and grew well at 30°C; but two of them did not grow at or above 28°C, and had optimum growth temperatures around 20°C. Thirty percent of the isolates could form endospores. Phylogenetic analysis revealed that the isolates fell into four categories: high-GC Gram-positive bacteria, β-proteobacteria, γ-proteobacteria, and low-GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and β-proteobacteria, and all γ-proteobacteria, came from samples with an estimated age of 1.8–3.0 million years (Olyor suite). Most low-GC Gram-positive bacteria came from samples with an estimated age of 5,000–8,000 years (Alas suite). Received: 11 April 1996 Accepted: 8 May 1996     

Plant Water Potential Gradients Measured in the Field by Freezing Point

A portable freezing point meter was used in the field to measure the water potential gradients in sunflower (Helianthus annuus), beans (Phaseolus vulgaris), corn (Zea mays), wheat (Triticum aestivum), pumpkin (Cucurbita pepo), potato (Solanum tuberosum), alfalfa (Medicago sativa), and sugarbeets (Beta vulgaris). The measurements were made between daybreak and sunrise, and again during the middle of the afternoon on days when the potential evapotranspiration varied between 6.5 and 8.0 mm of water. The gradients varied from a maximum of 0.2 bar per cm in a wheat, down to an undetectable value for pumpkin. Although most of the soil in the root zone was kept at potentials above –1 bar, the bulk of the root tissue had water potentials of –5 to –10 bars. Differences in water potential between shaded and unshaded leaves, and between leaf tissue and guttation fluid suggested a similar drop of several bars between xylem elements and the surrounding leaf tissue in some plant species. The implications of such drops are discussed with respect to plant water transport equations and pressure cell potential measurements.     

Studies on the Metabolic Activity of Muscle Proteins

The activities of glutamic oxaloacetic transaminase and Ca⁺⁺ ion-activated ATPase of muscle in the adult rats fed a protein-free diet for 8, 16 and 24 days were measured in order to clarify their metabolic responses with respect to reserve proteins. It was found that these enzyme activities, or presumably their enzyme proteins, decreased at the stage as early as the 8th day of protein depletion following the same pattern as seen in reserve proteins. Their responses, particularly those in unit activity, were somewhat different from each other. The metabolic significance of those responses was discussed in relation to protein nutrition.     

Studies on the Metabolic Activity of Muscle Proteins

The time-course of changes in total amount of proteins of sarcoplasmic, myofibrillar and stromal fractions in muscle of the rats fed a protein-free diet for 8, 16, 24 and 32 days, together with the referential data of those changes in the rats fed a protein-free diet up to time of death and a 60% casein diet for 12 days was determined respectively. The results were as follows: (1) The sarcoplasmic and the myofibrillar fractions decreased much more than the stromal fraction in the earlier stages of protein depletion following the same pattern as seen in reserve proteins. (2) The sarcoplasmic fraction decreased slightly more than the myofibrillar fraction as early as 8 days of the depletion, but the relative proportion between these two fractions was thereafter almost the same as that of the standard diet group. (3) In rats fed a 60% casein diet, the sarcoplasmic fraction increased markedly than the others.     

Phylogenetic Analysis of Bacteria Preserved in a Permafrost Ice Wedge for 25,000 Years

Phylogenetic analysis of bacteria preserved within an ice wedge from the Fox permafrost tunnel was undertaken by cultivation and molecular techniques. The radiocarbon age of the ice wedge was determined. Our results suggest that the bacteria in the ice wedge adapted to the frozen conditions have survived for 25,000 years.     

Physiological Activity of Campylobacter jejuni Far below the Minimal Growth Temperature

The behavior of Campylobacter jejuni at environmental temperatures was examined by determining the physiological activities of this human pathogen. The minimal growth temperatures were found to be 32 and 31°C for strains 104 and ATCC 33560, respectively. Both strains exhibited a sudden decrease in growth rate from the maximum to zero within a few degrees not only near the maximal growth temperature but also near the minimal growth temperature. This could be an indication that a temperature-dependent transition in the structure of a key enzyme(s) or regulatory compound(s) determines the minimal growth temperature. Oxygen consumption, catalase activity, ATP generation, and protein synthesis were observed at temperatures as low as 4°C, indicating that vital cellular processes were still functioning. PCR analysis showed that cold shock protein genes, which play a role in low-temperature adaptation in many bacteria, are not present in C. jejuni. The fact that chemotaxis and aerotaxis could be observed at all temperatures shows that the pathogen is able to move to favorable places at environmental temperatures, which may have significant implications for the survival of C. jejuni in the environment.     

Heterotrophic Activity of Deep-Sea Sediment Bacteria

Sediment samples, containing mixed microbial populations that were decompressed during retrieval from 7,750 and 8,130 m in the Puerto Rican Trench, were recompressed and incubated at the approximate in situ temperature (3 C) and pressure (775 or 815 atm) in the presence of 14C-labeled amino acids. Heterotrophic activity (total uptake, CO2 respiration, and cellular assimilation) and cellular-associated "pool" concentrations were measured. Compared with atmospheric controls held at 3 C, the total uptake at elevated pressure at 3 C was reduced, on an average, 55 times, CO2 respiration was reduced 45 times, and cellular assimilation was reduced 69 times. Rate of total uptake at elevated pressure was found to range from 4.0 X 10(-11) mug/cell per h for leucine to 2.61 X 10(-10) mug/cell per h for an amino acid mixture. Also, the percentage of total uptake at elevated pressures, respired as CO2, increased at the expense of cellular assimilation (ca. 22% increase). Two cellular-associated amino acid pools were detected, a large, loosely bound, outer pool and a small, tightly bound internal pool. The loosely bound outer pool was removed by a change in the pH of the incubation medium. Even though heterotrophic uptake and the outer, cellular-associated pool were markedly reduced at an elevated pressure, the percentage of total uptake calculated for the unincorporated, tightly bound, intracellular pool was 2 to 19 times that obtained for cultures held at 1 atm. The results were interpreted as indicating that bacterial metabolism and biosynthesis in the deep sea are markedly reduced, with a greater proportion of metabolic activity devoted to cellular maintenance.     

Collagenolytic Activity of Some Marine Bacteria

Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources.