Paraglaciecola hydrolytica sp. nov., a bacterium with hydrolytic activity against multiple seaweed-derived polysaccharides.

A novel bacterial strain, S66T, was isolated from eelgrass collected on the coastline of Zealand, Denmark. Polyphasic analyses involving phenotypic, phylogenetic and genomic methods were used to characterize strain S66T. The strain was Gram-reaction-negative, rod-shaped, aerobic, and displayed growth at 10-25 °C (optimum 20-25 °C) and at pH 7-9 (optimum pH 7.5). Furthermore, strain S66T grew on seaweed polysaccharides agar, agarose, porphyran, κ-carrageenan, alginate and laminarin as sole carbon sources. Major fatty acids were C16 : 0, C16 : 1ω7c and C18 : 1ω7c. The respiratory quinone was determined to be Q-8, and major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content was determined to be 42.2 mol%. Phylogenetic analyses based on the 16S rRNA gene and GyrB sequence comparisons showed that the bacterium was affiliated with the genus Paraglaciecola within the family Alteromonadaceae of the class Gammaproteobacteria. The percentage similarity between the 16S rRNA gene and GyrB sequences of strain S66T and other members of the genus Paraglaciecola were 94-95 % and 84-85 %, respectively. Based on the genome sequence of S66T, the average nucleotide identity (ANI) between strain S66T and other members of the genus Paraglaciecola was 77-80 %, and DNA-DNA hybridization prediction showed values of less than 24 % relatedness, respectively, between S66T and other species of the genus Paraglaciecola. The phenotypic, phylogenetic and genomic analyses support the hypothesis that strain S66T represents a novel species of the genus Paraglaciecola, for which the name Paraglaciecola hydrolytica sp. nov. is proposed. The type strain is S66T (=LMG 29457T=NCIMB 15060T=DSM 102834T).

An exceptionally cold-adapted alpha-amylase from a metagenomic library of a cold and alkaline environment.

A cold-active α-amylase, AmyI3C6, identified by a functional metagenomics approach was expressed in Escherichia coli and purified to homogeneity. Sequence analysis showed that the AmyI3C6 amylase was similar to α-amylases from the class Clostridia and revealed classical characteristics of cold-adapted enzymes, as did comparison of the kinetic parameters K m and k cat to a mesophilic α-amylase. AmyI3C6 was shown to be heat-labile. Temperature optimum was at 10-15 °C, and more than 70 % of the relative activity was retained at 1 °C. The pH optimum of AmyI3C6 was at pH 8-9, and the enzyme displayed activity in two commercial detergents tested, suggesting that the AmyI3C6 α-amylase may be useful as a detergent enzyme in environmentally friendly, low-temperature laundry processes.

Improved cultivation and metagenomics as new tools for bioprospecting in cold environments.

Only a small minority of microorganisms from an environmental sample can be cultured in the laboratory leaving the enormous bioprospecting potential of the uncultured diversity unexplored. This resource can be accessed by improved cultivation methods in which the natural environment is brought into the laboratory or through metagenomic approaches where culture-independent DNA sequence information can be combined with functional screening. The coupling of these two approaches circumvents the need for pure, cultured isolates and can be used to generate targeted information on communities enriched for specific activities or properties. Bioprospecting in extreme environments is often associated with additional challenges such as low biomass, slow cell growth, complex sample matrices, restricted access, and problematic in situ analyses. In addition, the choice of vector system and expression host may be limited as few hosts are available for expression of genes with extremophilic properties. This review summarizes the methods developed for improved cultivation as well as the metagenomic approaches for bioprospecting with focus on the challenges faced by bioprospecting in cold environments.

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing.

BACKGROUND: The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. RESULTS: A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and ß-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the ß-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities. CONCLUSIONS: Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (ß-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The ß-galactosidase BGalI17E2 was able to hydrolyze lactose at low temperature, suggesting a possibly use in the dairy industry for this enzyme. The two different approaches complemented each other by targeting different microbial communities, highlighting the usefulness of combining methods for bioprospecting. Finally, we document here that ikaite columns constitute an important source of cold- and/or alkaline-active enzymes with industrial application potential.

Improving diversity in cultures of bacteria from an extreme environment.

The ikaite columns in the Ikka Fjord in Greenland represent one of the few permanently cold and alkaline environments on Earth, and the interior of the columns is home to a bacterial community adapted to these extreme conditions. The community is characterized by low cell numbers imbedded in a calcium carbonate matrix, making extraction of bacterial cells and DNA a challenge and limiting molecular and genomic studies of this environment. To utilize this genetic resource, cultivation at high pH and low temperature was studied as a method for obtaining biomass and DNA from the fraction of this community that would not otherwise be amenable to genetic analyses. The diversity and community dynamics in mixed cultures of bacteria from ikaite columns was investigated using denaturing gradient gel electrophoresis and pyrosequencing of 16S rDNA. Both medium composition and incubation time influenced the diversity of the culture and many hitherto uncharacterized genera could be brought into culture by extended incubation time. Extended incubation time also gave rise to a more diverse community with a significant number of rare species not detected in the initial community.