Seasonal Incidence of Bacterial Temperature Types in Louisiana Soil and Water

Psychrophilic bacteria were not detected in soil, mud, and water in the summer. In winter, they were present in water and mud and constituted a significant portion of the bacterial flora in lake water.     

A meta-analysis of the activity,stability, and mutational characteristics of temperature-adapted enzymes

Understanding the characteristics that define temperature-adapted enzymes has been a major goal of extremophile enzymology in recent decades. In the present study, we explore these characteristics by comparing psychrophilic, mesophilic, and thermophilic enzymes. Through a meta-analysis of existing data, we show that psychrophilic enzymes exhibit a significantly larger gap (T_g) between their optimum and melting temperatures compared with mesophilic and thermophilic enzymes. These results suggest that T_g may be a useful indicator as to whether an enzyme is psychrophilic or not and that models of psychrophilic enzyme catalysis need to account for this gap. Additionally, by using predictive protein stability software, HoTMuSiC and PoPMuSiC, we show that the deleterious nature of amino acid substitutions to protein stability increases from psychrophiles to thermophiles. How this ultimately affects the mutational tolerance and evolutionary rate of temperature adapted organisms is currently unknown.     

Psychrophilic and Mesophilic Fungi in Frozen Food Products

The mold flora of certain frozen pastries and chicken pies was investigated. Molds were determined qualitatively or quantitatively, or both, by preparing pour plates of the blended product and incubating the plates at various temperatures. The mesophilic fungal flora developed on plates incubated at 10 and 20 C, whereas psychrophilic fungi were obtained on plates incubated at 0 and 5 C. About 2,000 cultures of fungi, representing about 100 different species, were isolated from various products. Four different brands of blueberry, two brands of cherry pastries, two brands of apple, and one brand of raspberry pastries were examined. In addition, two brands of chicken pies were studied. Blueberry pastries had a much higher total fungal population than the other products, although different brands of blueberry pastries varied considerably. Blueberry pastries had from 347 to 1,586 psychrophilic fungi per g. Cherry pastries had about 70 to 110 psychrophiles per g, and apple pastries had 19 to 92 psychrophiles per g. Chicken pies contained very few psychrophilic fungi, about 15 per g. Aureobasidium pullulans was recovered most frequently. About 90% of the psychrophilic fungi found in blueberry products was A. pullulans. Depending upon the brand of cherry pastry, either Phoma spp. or A. pullulans was the most common fungus present. Apple pastries also displayed brand variation, but were unique in having many mesophilic aspergilli. This genus was generally absent from other products. The Penicillium content of apple pastries was also rather high; 50% of the psychrophilic flora was represented by this genus. The psychrophilic fungal flora of chicken pies was composed primarily of penicillia (50%) and Chrysosporium pannorum (46%).     

Structural prediction and comparative docking studies of psychrophilic ��- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.     

Psychrophilic versus psychrotolerant bacteria--occurrence and significance in polar and temperate marine habitats.

The numerical dominance and ecological role of psychrophilic bacteria in bottom sediments, sea ice, surface water and melt pools of the polar oceans were investigated using isolates, colony forming units (CFU) and metabolic activities. All sediment samples of the Southern Ocean studied showed a clear numerical dominance of cold-loving bacteria. In Arctic sediments underlying the influence of cold polar water bodies psychrophiles prevailed also but they were less dominant in sediments influenced by the warm Atlantic Water. A predominance of psychrophiles was further found in consolidated Antarctic sea ice as well as in multiyear Arctic sea ice and in melt pools on top of Arctic ice floes. A less uniform adaptation response was, however, met in polar surface waters. In the very northern part of the Fram Strait (Arctic Ocean) we found bacterial counts and activities at 1 degree C exceeding those at 22 degrees C. In surface water of the Weddell Sea (Southern Ocean) psychrophiles also dominated numerically in early autumn but the dominance declined obviously with the onset of winter-water and a decrease of chlorphyll a. Otherwise in surface water of the Southern Ocean CFUs were higher at 22 degrees C than at 1 degree C while activities were vice versa indicating at least a functional dominance of psychrophiles. Even in the temperate sediments of the German Bight true psychrophiles were present and a clear shift towards cold adapted communities in winter observed. Among the polar bacteria a more pronounced cold adaptation of Antarctic in comparison with Arctic isolates was obtained. The results and literature data indicate that stenothermic cold adapted bacteria play a significant role in the global marine environment. On the basis of the temperature response of our isolates from different habitats it is suggested to expand the definition of Morita in order to meet the cold adaptation strategies of the bacteria in the various cold habitats.     

Studies on psychrophilic bacteria in two lakes of different trophy

The number of bacteria capable of growth at low temperatures in two lakes was found to be subject to considerable variation throughout the year although the changes were not correlated with changes in ambient temperature. A correlation was, however, observed between temperature and ratio of number of psychrophiles to "total" count. All of the psychrophilic bacteria isolated from the studied lakes were able to grow at temperatures below 0 degrees C. Microorganisms isolated from eutrophic lakes were characterized by more pronounced stenothermy than those isolated from lake with low tropic level. These bacteria represented a type resembling a type resembling obligatory or ,,true" psychrophiles. Most of the isolates belonged to the genus Pseudomonas and to the Flavobacterium--Cytophaga group.     

Actinomycetes and Fungi in Surface Waters and in Potable Water

In Finnish lakes and rivers used as water supplies, mesophilic fungi and actinomycetes were common, whereas thermophilic fungi and actinomycetes were present only in low concentrations. Fungi and actinomycetes were more abundant in eutrophic and mesotrophic lakes than in oligotrophic lakes. River water contained more thermophilic actinomycetes and fungi and mesophilic actinomycetes than did lake water. Runoff from soil seemed to be an important factor contributing to the incidence of these microbes in water. Chemical coagulation removed actinomycetes and fungi efficiently, but sand filtration allowed their passage. Disinfection could not prevent actinomycetes and fungi from reaching the distribution system. During infiltration in the production of recharged groundwater, mesophilic actinomycetes could even multiply appreciably.     

Competition of marine psychrophilic bacteria at low temperatures

The occurrence of obligately and facultatively psychrophilic bacteria in the marine environment suggests that environmental conditions exist which can favour each of these groups in competitive processes. Differences were found in the way in which temperature affected the growth rates of obligate and facultative psychrophiles. Maximum specific growth rates of a number of obligately and facultatively psychrophilic bacteria were determined in batch culture and competition experiments were carried out in a chemostat at growth-limiting substrate concentrations. From the results the relation between the specific growth rate and the concentration of the growth-limiting substrate for both types of organisms at different temperatures was deduced. Both at low and high substrate concentrations obligate psychrophiles grew faster than facultative psychrophiles at the lower temperature extreme (? 4 C). These results suggest that obligately psychrophilic chemoorganotrophs are responsible for mineralization processes in cold natural environments such as ocean waters and the arctic and antarctic regions. In these environments they can successfully compete with facultative psychrophiles because they can grow faster.     

Effects of temperature on the growth of psychrophilic bacteria from glaciers.

Growth of five strains of psychrophilic bacteria (four Arthrobacter and one Pseudomonas) isolated from glacial deposits was studied at different temperatures. Three strains were facultative psychrophiles, having an optimum temperature for growth at about 25-28 degrees C and a maximum at about 32-34 degrees C. The two Arthrobacter glacialis strains were found to be obligate psychrophiles with an optimum at 13-15 degrees C and a maximum at 18 degrees C. Arrhenius plots showed that A. glacialis could compete with the facultative psychrophilic bacteria only at 0 degrees C, that is, the temperature of its natural environment. The psychrophilic Arthrobacter species studied here are more resistant to thermal stress than are marine psychrophilic bacteria. For Arthrobacter, in contrast to Pseudomonas, temperatures above the optimum induced formation of filaments and abnormal cells. The culture turbidity increased 10 to 30 times, whereas viable count tended to decrease. The thermal block seems to prevent cell wall synthesis and septation, but at a different step for each species.     

Life at low temperatures: is disorder the driving force?

The thermodynamic characterization of various biological systems from psychrophiles points to a larger entropic contribution when compared to the corresponding mesophilic or (hyper) thermophilic counterparts, either at the level of the macromolecules (thermodynamic and kinetic stabilities) or of their function (ligand binding, catalytic activity). It is suggested here that in an environment characterized by a low heat content (enthalpy) and at temperatures that strongly slowdown molecular motions, the cold-adapted biological systems rely on a larger disorder to maintain macromolecular dynamics and function. Such pre-eminent involvement of entropy is observed in the experimental results and, from a macroscopic point of view, is also reflected for instance by the steric hindrances introduced by cis-unsaturated and branched lipids to maintain membrane fluidity, by the loose conformation of psychrophilic proteins or by the local destabilization of tRNA by dihydrouridine in psychrophilic bacteria.     

Compositional changes in RNA,DNA and proteins for bacterial adaptation to higher and lower temperatures

It is known that in thermophiles the G+C content of ribosomal RNA linearly correlates with growth temperature, while that of genomic DNA does not. Although the G+C contents (singlet) of the genomic DNAs of thermophiles and methophiles do not differ significantly, the dinucleotide (doublet) compositions of the two bacterial groups clearly do. The average amino acid compositions of proteins of the two groups are also distinct. Based on these facts, we here analyzed the DNA and protein compositions of various bacteria in terms of the optimal growth temperature (OGT). Regression analyses of the sequence data for thermophilic, mesophilic and psychrophilic bacteria revealed good linear relationships between OGT and the dinucleotide compositions of DNA, and between OGT and the amino acid compositions of proteins. Together with the above-mentioned linear relationship between ribosomal RNA and OGT, the DNA and protein compositions can be regarded as thermostability measures for RNA, DNA and proteins, covering a wide range of temperatures. Both the DNA and proteins of psychrophiles apparently exhibit characteristics diametrically opposite to those of thermophiles. The physicochemical parameters of dinucleotides suggested that supercoiling of DNA is relevant to its thermostability. Protein stability in thermophiles is realized primarily through global changes that increase charged residues (i.e., Glu, Arg, and Lys) on the molecular surface of all proteins. This kind of global change is attainable through a change in the amino acid composition coupled with alterations in the DNA base composition. The general strategies of thermophiles and psychrophiles for adaptation to higher and lower temperatures, respectively, that are suggested by the present study are discussed.     

Psychrophilic Microorganisms from Areas Associated with the Viking Spacecraft

Microorganisms capable of growth at 7 C were enumerated and isolated from soil samples from the manufacture and assembly areas of the Viking spacecraft. Populations ranging from 4.2 X 10(3) to 7.7 X 10(6)/g of soil were isolated from the 15 soil samples examined. Temperature requirements were determined, and those growing at 3 C, but not at 32 C, were designated as obligate psychrophiles in this investigation. Populations of soil bacteria, including aerobic sporeformers, ranging from 1.5 X 10(2) to 9.8 X 10(5)/g were capable of growth at 3 C, but not at 32 C. Bacterial isolates were identified to major generic groups. No psychrophilic sporeformers were isolated from soil from the manufacture area, but psychrophilic sporeformers ranged from 0 to 6.1 X 10(3)/g from soil from the assembly area.     

The Bacteria in an Antarctic Peat

S ummary : Of a total of 119 strains of bacteria from 3 depths in an acid peat on Signy Island, S. Orkney, 52% belonged to the genus Brevibacterium. Twelve other genera were recorded of which numerically the most important were Arthrobacter, Cellulomonas, Kurthia and Micrococcus. 62% of the collection were psychrophilic, but only 4 strains were obligate psychrophiles. No pattern could be established for the various genera from different depths. The fine structure of an obligately psychrophilic pleomorphic rod from the peat is illustrated and discussed.     

Extracellular enzymes produced by microorganisms isolated from maritime Antarctica

Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Antártico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.     

Structural adaptation to low temperatures--analysis of the subunit interface of oligomeric psychrophilic enzymes

Enzymes from psychrophiles show higher catalytic efficiency in the 0-20 degrees C temperature range and often lower thermostability in comparison with meso/thermophilic homologs. Physical and chemical characterization of these enzymes is currently underway in order to understand the molecular basis of cold adaptation. Psychrophilic enzymes are often characterized by higher flexibility, which allows for better interaction with substrates, and by a lower activation energy requirement in comparison with meso/thermophilic counterparts. In their tertiary structure, psychrophilic enzymes present fewer stabilizing interactions, longer and more hydrophilic loops, higher glycine content, and lower proline and arginine content. In this study, a comparative analysis of the structural characteristics of the interfaces between oligomeric psychrophilic enzyme subunits was carried out. Crystallographic structures of oligomeric psychrophilic enzymes, and their meso/thermophilic homologs belonging to five different protein families, were retrieved from the Protein Data Bank. The following structural parameters were calculated: overall and core interface area, characterization of polar/apolar contributions to the interface, hydrophobic contact area, quantity of ion pairs and hydrogen bonds between monomers, internal area and total volume of non-solvent-exposed cavities at the interface, and average packing of interface residues. These properties were compared to those of meso/thermophilic enzymes. The results were analyzed using Student's t-test. The most significant differences between psychrophilic and mesophilic proteins were found in the number of ion pairs and hydrogen bonds, and in the apolarity of their subunit interface. Interestingly, the number of ion pairs at the interface shows an opposite adaptation to those occurring at the monomer core and surface.     

Microorganisms isolated from the water phase of tropospheric clouds at the Puy de Dôme: major groups and growth abilities at low temperatures

This work constitutes the first large report on aerobic cultivable microorganisms present in cloud water. Seven cloud-event samples were collected at the Puy de D?me summit, and cultivation was performed leading to the isolation of 71 bacterial, 42 fungal and 15 yeast strains. Most of the fungi isolated were of Cladosporium or Trametes affiliation, and yeasts were of Cryptococcus affiliation. Bacteria, identified on the basis of their 16S rRNA gene sequence, were found to belong to Actinobacteria, Firmicutes, Proteobacteria (Alpha, Beta and Gamma subclasses) and Bacteroidetes phyla, and mainly to the genera Pseudomonas, Sphingomonas, Staphylococcus, Streptomyces, and Arthrobacter. These strains appear to be closely related to some bacteria described from cold environments, water (sea and freshwater), soil or vegetation. Comparison of the distribution of Gram-negative vs. Gram-positive bacteria shows that the number of Gram-negative bacteria is greater in summer than in winter. Finally, a very important result of this study concerns the ability of half of the tested strains to grow at low temperatures (5 degrees C): most of these are Gram-negative bacteria, and a few are even shown to be psychrophiles. On the whole, these results give a good picture of the microbial content of cloud water in terms of classification, and suggest that a large proportion of bacteria present in clouds have the capacity to be metabolically active there. This is of special interest with respect to the potential role of these microorganisms in atmospheric chemistry.     

Staphylococci in Competition: II. Effect of Total Numbers and Proportion of Staphylococci in Mixed Cultures on Growth in Artificial Culture Medium

In studies carried on in bacteriological media with selected cultures, definite repressive effects were noted on the growth of the Staphylococcus population by a mixture of saprophytic, psychrophilic bacterial species. This repressive effect became more pronounced as the relative proportion of the bacterial population which was staphylococcal became smaller. A varied saprophytic bacterial flora of some numbers apparently would offer definite protection to foods through repression of staphylococcal growth and by rendering the food inedible before the rise of appreciable numbers of staphylococci. It would appear that at the optimal temperature for staphylococcal growth, staphylococci could multiply rapidly in the mixed population due to the comparative shortness of the generation time of this species and because of the lengthened lag phase of the saprophytic bacterial species at this elevated temperature, especially when only cultures having psychrophilic characteristics were present. This temperature is substantially above that encountered in practical experience. With the passage of time, the staphylococcal population was completely overgrown by the saprohytes present. This effect might be eliminated in the presence of psychrophilic and mesophilic, saprophytic species. The repressive effect of competition by saprophytic, psychrophilic organisms is extremely effective up to room temperature on the staphylococcal population. Even when significant staphylococcal populations were achieved in the artificial media, such tremendous numbers of saprophytes were obtained either earlier or at the same time so that a frozen food containing this population would be organoleptically unacceptable due to the degradative action of enzymes from the saprophytic psychrophile population.     

Explanation for the decline of bacteria introduced into lake water

The sizes of the populations of individual bacterial species diminished following their addition to water from lakes with different trophic levels at temperatures of 5, 10, 15, and 30°C. Some species persisted after their initial reduction in cell numbers, but others were undetectable after 3 to 15 days. The decline of these introduced bacteria was not a result of their inoculation at higher densities than are found in nature. The death of most of the test species was not the result of starvation, abiotic factors, bdellovibrios, or bacteriophages. Despite the presence of lytic bacteria, the lake water did not have lytic activity against the test species. Protozoan predation was a significant factor in the fall in bacterial population sizes because protozoa increased in numbers as the bacterial density fell, the suppression of protozoa led to the elimination or delay of the decline of the bacteria, and the addition of protozoa to lake water in which indigenous protozoa were suppressed produced the same pattern of bacterial elimination as in untreated lake water.     

Distinctions in adenylate metabolism among organisms inhabiting temperature extremes

Microbiota from multiple kingdoms (e.g., Eubacteria, Fungi, Protista) thrive at temperature optima ranging from 0–20°C (psychrophiles) to 40–85°C (thermophiles). In this study, we have monitored changes in adenylate levels and growth rate as a function of temperature in disparate thermally adapted organisms. Our data indicate that growth rate and adenylate levels increase with temperature in mesophilic and thermophilic species, but rapid losses of adenosine 5-triphosphate (ATP) occur upon cold or heat shock. By contrast, psychrophilic species decrease adenylate levels but increase growth rate as temperatures rise within their viable range. Moreover, psychrophilic ATP levels fell rapidly upon heat shock, but dramatic gains in ATP (~20–50%) were observed upon cold shock, even at sub-zero temperatures. These results suggest that energy metabolism in thermophiles resembles that in mesophiles, but that elevated adenylate nucleotides in psychrophiles may constitute a compensatory strategy for maintaining biochemical processes at low temperature.     

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.