Psychrotolerant Bacteria Research Articles

Characterization of cold-active dehydrogenases for secondary alcohols and glycerol in psychrotolerant bacteria isolated from Antarctic soil

A NAD(P) +-dependent secondary alcohol dehydrogenase (2°ADH) and a NAD +-dependent glycerol dehydrogenase (GDH) have been identified in a psychrotolerant Micrococcus sp. and Arthrobacter sp., respectively, both isolated from Antarctic soil. In partially purified cell-free extracts (enriched 84- and 78-fold for 2°ADH and GDH, respectively), the enzymes displayed cold activity: the 2°ADH had optimum for activity at 33 °C and retained 32% of its maximum activity at 6 °C, the corresponding values for the GDH were 35 °C and 43%. The enzymes were inactivated within 10 min at 50 and 60 °C, for 2°ADH and GDH, respectively. The 2°ADH was active against a wide range of medium chain-length alcohols, preferring ( R)-stereoisomers; the enzyme was reversible with a higher affinity for the reduction of ketones. The GDH was active against glycerol and diols containing a secondary alcohol group. These properties make the enzymes candidates for low temperature biotransformations.

Psychrophilic and Psychrotolerant Heterotrophic Microorganisms of Middle Siberian Karst Cavities

The natural microflora of Middle Siberian karst cavities, which comprises psychrotolerant bacteria and fungi capable of growing at 3–15 and 3...+28°C, respectively, has been studied. Bacteria are ubiquitous in caves, their count varying from 103 to 107 cells/g ground. The bacteria have been identified as Pseudomonas, Arthrobacter, Bacillus, and coryneform bacteria. Fungi have been found in places exposed to increased anthropogenic impact, their count being as large as 106 to 107 cells/g ground. Mucor, Penicillium, and Chrysosporium were dominant fungal genera.

Effect of isolation temperature on the characteristics of extracellular proteases produced by Antarctic bacteria

Protease-producing psychrotolerant bacteria were isolated from Antarctic biotopes on casein agar plates using different incubation temperatures. Most of the isolates were non-spore-forming Gram-negative motile rods with catalase activity, 30% were pigmented and none of them were glucose fermenters. All the strains were grown in liquid cultures at 20°C and protease secretion was tested using the azocasein method. Despite their capacity for production of a clear zone of hydrolysis in agar plates, some strains did not produce detectable levels of proteolytic activity in liquid cultures. The lowest apparent optimum temperature for protease activity found in culture supernatants was 40°C. Almost all the strains showed activation energy values about 10-20 kJ-mol?1 lower than that observed for a mesophilic Subtilisin. Most of the proteases showed optimal activity at neutral or alkaline pH values and developed a multiple-band profile on gelatine-SDS-PAGE. It was observed that the lower the strain isolation temperature was, the more stongly cold-adapted–in terms of optimal temperature and activation energy–were the proteases produced by them. This dependence of the characteristics of the proteases on the isolation temperature is an important factor to take into account in the design of screening programmes directed towards the isolation of psychrotolerant bacteria able to produce proteases strongly or weakly adapted to work in the cold. The Antarctic area explored proved to be a promising source of proteolytic bacteria with potential use in industrial processes to be carried out at low or moderate temperatures.

Psychrotolerant and microaerophilic bacteria in boreal groundwater

Abstract Growth temperature and microaerophily of 39 phylogenetically different isolates from boreal oxygen-deficient groundwater were studied. Based on growth temperatures, the isolates were mainly psychrotolerant bacteria as 35 grew at 2 degrees C and only three at 35 degrees C. Growth rates in the range of 4-35 degrees C fitted the Ratkowsky square root model well. Optimum temperatures of the groundwater isolates varied between 18 and 30 degrees C. In semisolid glucose and PYGV media, 59% and 28% of the isolates, respectively, preferred microaerophilic growth and 33% were catalase-negative. The microaerophilic isolates had the highest sensitivity to H(2)O(2) whereas sensitivity to the superoxide generator paraquat was similar among microaerophilic and aerobic isolates. The results show that the cold (6-8 degrees C) and oxygen-deficient groundwater harbors psychrotolerant and microaerophilic bacteria of different phylogenetic origins which are well adapted to their environment.

Psychrotolerant and microaerophilic bacteria in boreal groundwater

Growth temperature and microaerophily of 39 phylogenetically different isolates from boreal oxygen-deficient groundwater were studied. Based on growth temperatures, the isolates were mainly psychrotolerant bacteria as 35 grew at 2°C and only three at 35°C. Growth rates in the range of 4–35°C fitted the Ratkowsky square root model well. Optimum temperatures of the groundwater isolates varied between 18 and 30°C. In semisolid glucose and PYGV media, 59% and 28% of the isolates, respectively, preferred microaerophilic growth and 33% were catalase-negative. The microaerophilic isolates had the highest sensitivity to H 2O 2 whereas sensitivity to the superoxide generator paraquat was similar among microaerophilic and aerobic isolates. The results show that the cold (6–8°C) and oxygen-deficient groundwater harbors psychrotolerant and microaerophilic bacteria of different phylogenetic origins which are well adapted to their environment.

Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7.

A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8. Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.

Cloning of two pectate lyase genes from the marine Antarctic bacterium Pseudoalteromonas haloplanktis strain ANT/505 and characterization of the enzymes.

A marine Antarctic psychrotolerant bacterium (strain ANT/505), isolated from sea ice-covered surface water from the Southern Ocean, showed pectinolytic activity on citrus pectin agar. The sequencing of the 16S rRNA of isolate ANT/505 indicates a taxonomic affiliation to Pseudoalteromonas haloplanktis. The supernatant of this strain showed three different pectinolytic activities after growth on citrus pectin. By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated. Subcloning and sequencing revealed two open reading frames (ORF) of 1,671 and 1,968 nt, corresponding to proteins of 68 and 75 kDa, respectively. The deduced amino acid sequence of the two ORFs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans. The pectate lyases contain signal peptides of 17 and 26 amino acids that were correctly processed after overexpression in E. coli BL21. Both enzymes were purified by anionic exchange chromatography. Maximal enzymatic activities for both pectate lyases were observed at 30 degrees C and a pH range of 9 to 10. The Km values of both lyases for pectate and citrus pectin were 1 g l(-1) and 5 g l(-1), respectively. Calcium was required for activity on pectic substrates, whereas the addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These two enzymes represent the first pectate lyases isolated and characterized from a cold-adapted marine bacterium.

Apparent contradiction: psychrotolerant bacteria from hydrocarbon-contaminated arctic tundra soils that degrade diterpenoids synthesized by trees.

Resin acids are tricyclic terpenoids occurring naturally in trees. We investigated the occurrence of resin acid-degrading bacteria on the Arctic tundra near the northern coast of Ellesmere Island (82 degrees N, 62 degrees W). According to most-probable-number assays, resin acid degraders were abundant (10(3) to 10(4) propagules/g of soil) in hydrocarbon-contaminated soils, but they were undetectable (<3 propagules/g of soil) in pristine soils from the nearby tundra. Plate counts indicated that the contaminated and the pristine soils had similar populations of heterotrophs (10(6) to 10(7) propagules/g of soil). Eleven resin acid-degrading bacteria belonging to four phylogenetically distinct groups were enriched and isolated from the contaminated soils, and representative isolates of each group were further characterized. Strains DhA-91, IpA-92, and IpA-93 are members of the genus Pseudomonas. Strain DhA-95 is a member of the genus Sphingomonas. All four strains are psychrotolerant, with growth temperature ranges of 4 degrees C to 30 degrees C (DhA-91 and DhA-95) or 4 degrees C to 22 degrees C (IpA-92 and IpA-93) and with optimum temperatures of 15 to 22 degrees C. Strains DhA-91 and DhA-95 grew on the abietanes, dehydroabietic and abietic acids, but not on the pimaranes, isopimaric and pimaric acids. Strains IpA-92 and IpA-93 grew on the pimaranes but not the abietanes. All four strains grew on either aliphatic or aromatic hydrocarbons, which is unusual for described resin acid degraders. Eleven mesophilic resin acid degraders did not use hydrocarbons, with the exception of two Mycobacterium sp. strains that used aliphatic hydrocarbons. We conclude that hydrocarbon contamination in Arctic tundra soil indirectly selected for resin acid degraders, selecting for hydrocarbon degraders that coincidentally use resin acids. Psychrotolerant resin acid degraders are likely important in the global carbon cycle and may have applications in biotreatment of pulp and paper mill effluents.

Patterns of cold adaptation in Antarctica: glutamate dehydrogenase from a psychrotolerant bacterium and a teleost

Patterns of cold adaptation in Antarctica: glutamate dehydrogenase from a psychrotolerant bacterium and a teleost

Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum.

The major carotenoid pigments of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum, were identified as zeaxanthin, beta-cryptoxanthin, and beta-carotene. Analysis was based on ultraviolet-visible spectroscopy, mass spectroscopy, and reversed-phase HPLC. Photoacoustic spectroscopy of intact bacterial cells revealed that the bulk of the pigments in S. antarcticus and S. multivorum was associated with the cell membrane. In vitro studies with synthetic membranes of phosphatidylcholine demonstrated that the major pigment was bound to the membranes and decreased their fluidity. The relative amounts of polar pigments were higher in cells grown at 5 degrees C than in cells grown at 25 degrees C. In the mesophilic strain, the synthesis of polar carotenoids was quantitatively less than that of the psychrotolerant strain.

Structural analysis of the elongation factor G protein from the low-temperature-adapted bacterium Arthrobacter globiformis SI55.

The first structural analysis of elongation factor G (EF-G) from a cold-adapted bacterium is presented. EF-G is an essential protein involved in the elongation process during protein synthesis and is therefore thought to play a crucial role in the low-temperature adaptation of cold-adapted microorganisms. To define its importance, the EF-G gene (fus) from the psychrotolerant bacterium Arthrobacter globiformis SI55 was cloned and sequenced. The deduced primary structure of the elongation factor is composed of 700 amino acids with a predicted molecular mass of 77.4 kDa. A three-dimensional model of the protein was constructed based on the known crystal structures of structurally homologous proteins. Structural features that might potentially be important for activity and flexibility at low temperature were deduced by comparisons with models of the EF-G proteins from the closely related mesophiles Micrococcus luteus and Mycobacterium tuberculosis. These features include a loss in the number of salt bridges in intradomain and interdomain positions, increased solvent interactions mediated by greater charge and polarity on domain surfaces, loop insertions, loss of proline residues in loop structures, and an increase of hydrophobicity in core regions. Specific changes have also been identified in the catalytic domain (G domain) and sites of potential ribosome interaction, which may directly affect guanosine triphosphate (GTP) hydrolysis and elongation rates at low temperature.

NADP+-dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1. Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases.

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+-dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five-residue insertion in a loop between two alpha-helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold-active enzymes.

Shewanella pealeana sp. nov., a member of the microbial community associated with the accessory nidamental gland of the squid Loligo pealei.

A new, mesophillic, facultatively anaerobic, psychrotolerant bacterium, strain ANG-SQ1T (T = type strain), was isolated from a microbial community colonizing the accessory nidamental gland of the squid Loligo pealei. It was selected from the community on the basis of its ability to reduce elemental sulfur. The cells are motile, Gram-negative rods (2.0-3.0 microns long, 0.4-0.6 micron wide). ANG-SQ1T grows optimally over the temperature range of 25-30 degrees C and a pH range of 6.5-7.5 degrees C in media containing 0.5 M NaCl. 16S rRNA sequence analysis revealed that this organism belongs to the gamma-3 subclass of the Proteobacteria. The closest relative of ANG-SQ1T is Shewanella gelidimarina, with a 16S rRNA sequence similarity of 97.0%. Growth occurs with glucose, lactate, acetate, pyruvate, glutamate, citrate, succinate, Casamino acids, yeast extract or peptone as sole energy source under aerobic conditions. The isolate grows anaerobically by the reduction of iron, manganese, nitrate, fumarate, trimethylamine-N-oxide, thiosulfate or elemental sulfur as terminal electron acceptor with lactate. Growth of ANG-SQ1T was enhanced by the addition of choline chloride to growth media lacking Casamino acids. The addition of leucine or valine also enhanced growth in minimal growth media supplemented with choline. The results of both phenotypic and genetic characterization indicate that ANG-SQ1T is a Shewanella species. Thus it is proposed that this new isolate be assigned to the genus Shewanella and that it should be named Shewanella pealeana sp. nov., in recognition of its association with L. pealei.

Clostridium frigidicarnis sp. nov., a psychrotolerant bacterium associated with 'blown pack' spoilage of vacuum-packed meats.

Two strains of a psychrotolerant Clostridium, isolated from vacuum-packed, temperature-abused beef, were characterized using a multiphasic approach. The strains were Gram-positive motile rods producing elliptical subterminal spores during early stationary growth phase. The strains were psychrotolerant. At pH 7.0, they grew between 3.8 and 40.5 degrees C; their optimum growth temperature was 30.0-38.5 degrees C. At 30 degrees C, the pH range for growth was between 4.7 and 9.5; the optimum pH for growth was 6.4-7.2. The organisms were proteolytic and saccharolytic, lecithinase-positive and hydrolysed gelatin. The fermentation products formed in peptone/yeast extract/glucose/starch broth were acetate, ethanol, butyrate, isovalerate, butanol, isobutyrate, oxalacetate, lactate, hydrogen and carbon dioxide. The DNA G + C compositions of the two meat strains were 27.3 and 28.4 mol%. Phylogenetic analyses indicated that the strains belong to Cluster I of the genus Clostridium (sensu Collins et al., 1994). The new strains differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strains were assigned to a new species for which the name Clostridium frigidicarnis is proposed; strain SPL77AT (= DSM 12271T) is the type strain.

Physicochemical parameters for growth of the sea ice bacteria Glaciecola punicea ACAM 611(T) and Gelidibacter sp. strain IC158.

The water activity and pH ranges for growth of Glaciecola punicea (a psychrophile) were extended when this organism was grown at suboptimal rather than optimal temperatures. No such extension was observed for Gelidibacter sp. strain IC158 (a psychrotolerant bacterium) at analogous temperatures. Salinity and pH may be primary physicochemical parameters controlling bacterial community development in sea ice.

Preliminary crystallographic studies of citrate synthase from an Antarctic psychrotolerant bacterium.

Recombinant citrate synthase from a psychrotolerant bacterium, DS2-3R, recently isolated in Antarctica, has been crystallized. The crystals belong to space group P6122 or P6522, with cell dimensions a = b = 70.8, c = 307.8 A. Diffraction data collected on a synchrotron from a cryoprotected crystal extends to at least 2.0 A. Knowledge of the structure of this enzyme will add to the understanding of cold activity and thermolability, and will be of biotechnological interest. Previously, the structure of citrate synthase from Archaea inhabiting environments at 328 and 373 K, has been reported. This present study will extend our understanding of the structural integrity and activity of proteins at the temperature extremes of life.

Biodiversity and ecophysiology of bacteria associated with Antarctic sea ice

A total of 135 bacterial strains were isolated from congelation (land fast) sea ice samples and ice algae biomass samples obtained from the coastal areas of the Vestfold Hills in East Antarctica (68°S, 78°E) during the summers of 1992–95. The sea ice isolates, along with reference strains. were analysed by numerical taxonomy and for DNA base composition in order to determine the biodiversity of sea ice bacteria. From these analyses 22 clusters of strains (phena) were obtained with most phena apparently representing novel bacterial taxa. The sea ice isolates could be categorized into three groups based on their ecophysiology: 1) slightly halophilic, psychrophilic bacteria often possessing fastidious growth requirements and which were predominantly isolated from sea ice algae biomass or from algae-rich ice samples; 2) halotolerant and psychrotolerant bacteria; and 3) non-halophilic bacteria isolated primarily from upper sections of congelation ice and other ice samples with low levcls of algal biomass.

Influence of changing temperature on growth rate and competition between two psychrotolerant Antarctic bacteria: competition and survival in non-steady-state temperature environments

Competition between two psychrotolerant bacteria was examined in glycerol-limited chemostat experiments subjected to non-steady-state conditions of temperature. One bacterium, a Brevibacterium sp. strain designated CR3/1/15, responded rapidly to temperature change, while a second, Hydrogenophaga pseudoflava, designated CR3/2/10, exhibited a lag in growth after a shift-down during a square-wave temperature cycle but not after a shift-up. The effects on competition and survival by these bacteria of both sine-wave and square-wave temperature changes between 2 and 16 degrees C over a 24-h cycle time were examined, as well as square-wave cycles over 12 and 96 h. The changing proportion of each bacterium in the chemostat was determined by plate counting at regular intervals. Under a sine-wave temperature cycle H. psedoflava outcompeted the Brevibacterium sp., but under square-wave temperature cycles the two bacteria coexisted because the lag by H. pseudoflava after the temperature shift-down favored the faster-responding Brevibacterium sp. The two bacteria thus exhibited different survival strategies, with H. pseudoflava adapted to effective competition under steady-state conditions and the Brevibacterium sp. adapted to rapid adaptation and survival in a changing environment. The degree of perturbation of the bacteria, expressed as a temperature challenge index (delta temp/delta time), was greater under a square-wave temperature cycle than under a sine-wave cycle of equivalent amplitude and frequency, and higher-temperature challenge favored the Brevibacterium sp. A computer model was developed to examine competition between the bacteria in transient environments. The frequency of the temperature cycle influenced competition, as with a longer cycle (96 h) the significance of the lag by H. pseudoflava decreased compared with that of a 24-h cycle, and H. pseudoflava predominated in a mixed culture with a 96-h cycle. The shift-down lag by H. pseudoflava, during which it adapted to low temperature, disadvantaged it in a changing temperature environment, but at a short cycle time (12 h) this disadvantage was countered by the incomplete loss of low-temperature adaptation between cycles and thus the carryover of some low-temperature adaptation. Also, it was demonstrated that, as well as consideration of the effect of temperature changes on inducing lags in growth, the loss of adaptation to low temperature between cycles had to be taken into account in the computer model if it was to reproduce the trends in the experimental data.

The role of microorganisms in biosorption of heavy metals and radionuclides G.M. Gadd

Psychrotolerant bacteria play a particularly important role in the remediation of heavy metal in contamination sites at low temperatures. In the current study, a psychrotolerant Ni-resistant bacterial strain, identified as Bacillus cereus D2, was isolated from a nickel mining area in China. The isolated strain could produce a large amount of urease enzyme (194.6 U/mL), grow well in harsh environmental conditions at a temperature of 10 °C, and at a Ni (II) concentration up to 400 mg/L. Also, under the low temperature (10 °C), this strain has been revealed to induce carbonate precipitation (Ni2CO3(OH)2·H2O) through biomineralization for removing the high efficiency of Ni ions (73.47%) from the culture solution. Furthermore, strain D2 could immobilize the DTPA-Ni in contaminated soil under the case of the laboratory condition at 10 °C. These data support that the psychrotolerant bacterial strain D2 may play an important role in remediation technology by eliminating Ni ions from the contaminated soil at low temperatures.

Membrane lipid composition and glucose uptake in two psychrotolerant bacteria from Antarctica

The thermal responses of membrane lipid composition and function in two bacterial species isolated recently from Heywood Lake sediment in Antarctica have been investigated. Both isolates are Gram-negative psychrotolerant (psychrotrophic) species growing well at 0 and 25 °C, but having very different optimum growth temperatures, of 97 °C for strain CR3/F/w/1/15 and 20·9 °C for strain CR3/F/w/2/10. The acyl lipids in strain 1/15 contain predominantly branched-chain fatty acids, mainly anteiso-15:0. The fatty acid composition and its response to temperature depends on the culture medium used. In nutrient-poor medium a wider range is found, including unsaturated fatty acids, which are absent when a nutrient-rich medium is used. A decrease in growth temperature produced a shortening of the average fatty acid chain length in rich medium, whereas in poor medium there was a decrease in straight chain, saturated and unsaturated relative to branched-chain fatty acids. The acyl lipids of strain 2/10 contain 16:0 and 16:1 as the major fatty acids, particularly in rich medium, when they comprise &gt;90% of the total. Increasing proportions of 16:1 were converted to cyclopropane-17:0 at higher temperatures during growth in poor medium. A decrease in growth temperature in rich or poor medium resulted in an increase in the unsaturation index. When the bacteria were grown in rich or poor medium at 5 °C the strain with more ‘psychrophilic’ characteristics (1/15) took up glucose at a faster rate than did the strain with more ‘psychrotrophic’ characteristics (2/10), whereas when they were grown at 20 °C in either medium the rate of glucose uptake by strain 2/10 was generally faster than that of strain 1/15. Thus, when the bacteria were grown at a low temperature the glucose uptake system of the more ‘psychrophilic’ strain was better adapted to function at zero, compared with that of the more ‘psychrotrophic’ strain. The basis of this difference is not due to the lack of temperature-dependent fatty acid changes, as reported previously for some other cold-adapted bacteria.