The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4 degrees C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses alpha and gamma of Proteobacteria, the Bacillus-Clostridium group, the order Actinomycetales, and the Cytophaga-Flexibacter-Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria gamma subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25 degrees C. Only a few strains were psychrophilic, with an optimal growth at 10-15 degrees C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4 degrees C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, alpha-amylase and beta-galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10 degrees C, and almost no production was detected at higher temperatures (20-30 degrees C). Catalytic activity was detected even below the freezing point of water (at -5 degrees C), demonstrating the unique properties of these enzymes.

Using starch as a carbon source at a cultivation temperature of 4˚C, a number of Gram-negative, aerobic strains was isolated from sea-ice and sea-water samples collected at Spitzbergen in the Arctic. Analysis of the genetic diversity of the novel isolates by random amplification of polymorphic DNA (RAPD) and ERIC fingerprinting revealed a homogenic group of biofilm-forming bacteria that contained small extrachromosomal elements. As a representative of the group, strain Pull 5.3 T ,isolated from a sea-water sample, was used for detailed characterization. In recent years, increasing attention in research has been directed to cold-adapted micro-organisms that are able to grow at or close to the freezing point of water, namely psychrophiles. They are defined by an optimal temperature for growth of about 15˚C or below, a maximal growth temperature of about 20˚C and the ability to grow at 0˚C (Morita, 1975). In comparison, psychrotolerant microorganisms, sometimes also referred to as 'psychrotrophs', generally have optimum and maximum growth temperatures of 20˚C or above (Ingraham & Stokes, 1959).Cold-adapted micro-organisms are found in both permanently and temporarily cold habitats, which comprise more than 80 % of the Earth's biosphere. Oceans, covering threequarters of the Earth, polar regions (14 % of the Earth's surface), high mountains and deep lakes provide various aquatic and terrestrial cold environments where the temperature seldom or never reaches 5˚C (Gounot, 1999). The ability of micro-organisms to grow at low temperatures is not restricted to prokaryotes. A wide variety of micro-organisms, including bacteria, archaea, yeast, fungi and algae, is found in cold environments. These microorganisms are free-living in soil and fresh and saline waters or are associated with plants and cold-blooded animals such as fish or crustaceans. Among the bacteria, almost all types have been identified either after isolation (Ravenschlag et al., 1999; Bowman et al., 1997) or by detection in their natural habitats using a 16S rRNA approach (Fuhrman et al., 1993;DeLong et al., 1994;Vetriani et al., 1998). Unlike hyperthermophiles, they do not belong to new phyla. The majority of psychrophiles studied to date belong to the Gram-negative Proteobacteria. This is not surprising, since Gram-negatives are predominant in marine waters, where most investigations have been performed.Psychrophiles and psychrotolerant micro-organisms have a wide range of adaptations, including alterations in the protein and lipids of their membrane, energy-generation systems, protein synthesis and hydrolytic enzymes (Russell, 1998). Higher specific activity at low temperatures and thermosensitivity of cold-active enzymes provide a valuable source for exploration of novel biotechnological processes (Feller & Gerday, 1997). Since it has become clear that psychrophiles also represent a naturally occurring model for investigating protein adaptation to cold (Aghajari et al., 1998), an increasing number of novel bacterial strains from different Arctic and A...

The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hypothetical membrane linker region was found to be cleaved during processing in E. coli. The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography. Optimal activity was detected at 95 degrees C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5. More than 35% of enzymatic activity was detected even at 120 degrees C. The enzyme was stable at 90 degrees C for several hours and exhibited a half-life of 2.5 h at 100 degrees C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack alpha-1,6- as well as alpha-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose. The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products.

Genomic DNA amplification from 51 species of the family Chironomidae shows that most contain relatives of NLRCth1 LINE and CTRT1 SINE retrotransposons first found in Chironomus thummi. More than 300 cloned PCR products were sequenced. The amplified region of the reverse transcriptase gene in the LINEs is intact and highly conserved, suggesting active elements. The SINEs are less conserved, consistent with minimal/no selection after transposition. A mitochondrial gene phylogeny resolves the Chironomus genus into six lineages (Guryev et al. 2001). LINE and SINE phylogenies resolve five of these lineages, indicating their monophyletic origin and vertical inheritance. However, both the LINE and the SINE tree topologies differ from the species phylogeny, resolving the elements into "clusters I-IV" and "cluster V" families. The data suggest a descent of all LINE and SINE subfamilies from two major families. Based on the species phylogeny, a few LINEs and a larger number of SINEs are cladisitically misplaced. Most misbranch with LINEs or SINEs from species with the same families of elements. From sequence comparisons, cladistically misplaced LINEs and several misplaced SINEs arose by convergent base substitutions. More diverged SINEs result from early transposition and some are derived from multiple source SINEs in the same species. SINEs from two species (C. dorsalis, C. pallidivittatus), expected to belong to the clusters I-IV family, branch instead with cluster V family SINEs; apparently both families predate separation of cluster V from clusters I-IV species. Correlation of the distribution of active SINEs and LINEs, as well as similar 3' sequence motifs in CTRT1 and NLRCth1, suggests coevolving retrotransposon pairs in which CTRT1 transposition depends on enzymes active during NLRCth1 LINE mobility.

A high proportion of microorganisms that colonise cold environments originate from marine sites; hence, they must combine adaptation to low temperature with osmoregulation. However, little or nothing is known about the nature of compatible solutes used by cold-adapted organisms to balance the osmotic pressure of the external medium. We studied the intracellular accumulation of small organic solutes in the Arctic isolate Carnobacterium strain 17-4 as a function of the growth temperature and the NaCl concentration in the medium. Data on 16S rDNA sequence and DNA-DNA hybridisation tests corroborate the assignment of this isolate as a new species of the bacterial genus Carnobacterium. The growth profiles displayed maximal specific growth rate at 30°C in medium without NaCl, and maximal values of final biomass at growth temperatures between 10 and 20°C. Therefore, Carnobacterium strain 17-4 exhibits halotolerant and psychrotolerant behaviours. The solute pool contained glycine-betaine, the main solute used for osmoregulation, and an unknown compound whose structure was identified as α-glucopyranosyl-(1-3)-β-glucopyranosyl-(1-1)-α-glucopyranose (abbreviated as gluconeotrehalose), using nuclear magnetic resonance and mass spectrometry. This unusual solute consistently accumulated to high levels (0.35 ± 0.05 mg/mg cell protein) regardless of the growth temperature or salinity. The efficiency of gluconeotrehalose in the stabilisation of four model enzymes against heat damage was also assessed, and the effects were highly protein dependent. The lack of variation in the gluconeotrehalose content observed under heat stress, osmotic stress, and starvation provides no clue for the physiological role of this rare solute.