Pseudomonas nunensis sp. nov. isolated from a suppressive potato field in Greenland.

The bacterial strain In5T was previously isolated from a suppressive potato field in southern Greenland and has been characterized and described as Pseudomonas fluorescens. However, the results of new polyphasic analyses coupled with those of phenotypic, phylogenetic and genomic analyses reported here demonstrate that the affiliation to the species P. fluorescens was incorrect. The strain is Gram-stain-negative, rod-shaped, aerobic and displays growth at 4-28 °C (optimum temperature 20-25 °C) and at pH 5-9 (optimum pH 6-7). Major fatty acids were C16 : 0 (38.2 %), a summed feature consisting of C16 : 1ω6c and/or C16 : 1ω7c) (20.7 %), C17 : 0cyclo ω7c (14.3 %) and a summed feature consisting of C18 : 1ω6c and/or C18 : 1ω7c (11.7 %). The respiratory quinones were determined to be Q9 (95.5 %) and Q8 (4.5 %) and major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was determined to be 59.4 mol%. The results of phylogenetic analysis based on the 16S rRNA gene and multi-locus sequence analysis (MLSA; concatenated 16S rRNA, gyrB, rpoB and rpoD sequences) indicated that In5T was affiliated with the Pseudomonas mandelii subgroup within the genus Pseudomonas. Comparison of the genome sequence of In5T and those of related type strains of species of the genus Pseudomonas revealed an average nucleotide identity (ANI) of 87.7 % or less and digital DNA-DNA hybridization (dDDH) of less than 34.5 % relatedness, respectively. Two more strains, In614 and In655, isolated from the same suppressive soil were included in the genome analysis. The ANI and dDDH of In614 and In655 compared with In5T were ANI: 99.9 and 97.6 and dDDH (GGDC) 99.9 and 79.4, respectively, indicating that In5T, In614 and In655 are representatives of the same species. The results of the phenotypic, phylogenetic and genomic analyses support the hypothesis that strain In5T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nunensis sp. nov. is proposed. The type strain is In5T(=LMG 32653T=NCIMB 15428T).

Pseudomonas hormoni sp. nov., a plant hormone producing bacterium isolated from Arctic grass, Ellesmere Island, Canada.

Bacterial strain G20-18T was previously isolated from the rhizosphere of an Arctic grass on Ellesmere Island, Canada and was characterized and described as Pseudomonas fluorescens. However, new polyphasic analyses coupled with phenotypic, phylogenetic and genomic analyses reported here demonstrate that the affiliation to the species P. fluorescens was incorrect. The strain is Gram-stain-negative, rod-shaped, aerobic and displays growth at 5-25 °C (optimum, 20-25 °C), at pH 5-9 (optimum, pH 6-7) and with 0-4 % NaCl (optimum, 2 % NaCl). The major fatty acids are C16 : 0 (35.6 %), C17 : 0 cyclo ω7c (26.3 %) and summed feature C18 : 1/C18 : 1 ω7c (13.6 %). The respiratory quinones were determined to be Q9 (93.5 %) and Q8 (6.5 %) and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Strain G20-18T was shown to synthesize cytokinin and auxin plant hormones and to produce 1-aminocyclopropane-1-carboxylate deaminase. The DNA G+C content was determined to be 59.1 mol%. Phylogenetic analysis based on the 16S rRNA gene and multilocus sequence analysis (concatenated 16S rRNA, gyrB, rpoB and rpoD sequences) showed that G20-18T was affiliated with the Pseudomonas mandelii subgroup within the genus Pseudomonas. Comparisons of the G20-18T genome sequence and related Pseudomonas type strain sequences showed an average nucleotide identity value of ≤93.6 % and a digital DNA-DNA hybridization value of less than 54.4 % relatedness. The phenotypic, phylogenetic and genomic data support the hypothesis that strain G20-18T represents a novel species of the genus Pseudomonas. As strain G20-18T produces or modifies hormones, the name Pseudomonas hormoni sp. nov. is proposed. The type strain is G20-18T (=LMG 33086T=NCIMB 15469T).

Characterization of five marine family 29 glycoside hydrolases reveals an α-L-fucosidase targeting specifically Fuc(α1,4)GlcNAc.

$\text{L} $ -Fucose is the most widely distributed $\text{L} $-hexose in marine and terrestrial environments and presents a variety of functional roles. $\text{L} $-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae and is found in human milk oligosaccharides (HMOs) and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-$\text{L} $-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in Escherichia coli. All five enzymes (Fp231, Fp239, Fp240, Fp251 and Fp284) hydrolyzed the synthetic substrate CNP-α-$\text{L} $-fucose. Assayed against up to 17 fucose-containing oligosaccharides, Fp239 showed activity against the Lewis Y antigen, 2'- and 3-fucosyllactose, while Fp284 degraded 2'-fucosyllactose and Fuc(α1,6)GlcNAc. Furthermore, Fp231 displayed strict specificity against Fuc(α1,4)GlcNAc, a previously unreported specificity in GH29. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6-6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s-1 and Km = 660 µM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored.

A Novel Auxiliary Agarolytic Pathway Expands Metabolic Versatility in the Agar-Degrading Marine Bacterium Colwellia echini A3T.

Marine microorganisms encode a complex repertoire of carbohydrate-active enzymes (CAZymes) for the catabolism of algal cell wall polysaccharides. While the core enzyme cascade for degrading agar is conserved across agarolytic marine bacteria, gain of novel metabolic functions can lead to the evolutionary expansion of the gene repertoire. Here, we describe how two less-abundant GH96 α-agarases harbored in the agar-specific polysaccharide utilization locus (PUL) of Colwellia echini strain A3T facilitate the versatility of the agarolytic pathway. The cellular and molecular functions of the α-agarases examined by genomic, transcriptomic, and biochemical analyses revealed that α-agarases of C. echini A3T create a novel auxiliary pathway. α-Agarases convert even-numbered neoagarooligosaccharides to odd-numbered agaro- and neoagarooligosaccharides, providing an alternative route for the depolymerization process in the agarolytic pathway. Comparative genomic analysis of agarolytic bacteria implied that the agarolytic gene repertoire in marine bacteria has been diversified during evolution, while the essential core agarolytic gene set has been conserved. The expansion of the agarolytic gene repertoire and novel hydrolytic functions, including the elucidated molecular functionality of α-agarase, promote metabolic versatility by channeling agar metabolism through different routes. IMPORTANCEColwellia echini A3T is an example of how the gain of gene(s) can lead to the evolutionary expansion of agar-specific polysaccharide utilization loci (PUL). C. echini A3T encodes two α-agarases in addition to the core ß-agarolytic enzymes in its agarolytic PUL. Among the agar-degrading CAZymes identified so far, only a few α-agarases have been biochemically characterized. The molecular and biological functions of two α-agarases revealed that their unique hydrolytic pattern leads to the emergence of auxiliary agarolytic pathways. Through the combination of transcriptomic, genomic, and biochemical evidence, we elucidate the complete α-agarolytic pathway in C. echini A3T. The addition of α-agarases to the agarolytic enzyme repertoire might allow marine agarolytic bacteria to increase competitive abilities through metabolic versatility.

Fungal-Associated Molecules Induce Key Genes Involved in the Biosynthesis of the Antifungal Secondary Metabolites Nunamycin and Nunapeptin in the Biocontrol Strain Pseudomonas fluorescens In5.

Pseudomonas fluorescens In5 synthesizes the antifungal cyclic lipopeptides (CLPs) nunamycin and nunapeptin, which are similar in structure and genetic organization to the pseudomonas-derived phytotoxins syringomycin and syringopeptin. Regulation of syringomycin and syringopeptin is dependent on the two-component global regulatory system GacS-GacA and the SalA, SyrF, and SyrG transcription factors, which activate syringomycin synthesis in response to plant signal molecules. Previously, we demonstrated that a specific transcription factor, NunF, positively regulates the synthesis of nunamycin and nunapeptin in P. fluorescens In5 and that the nunF gene is upregulated by fungal-associated molecules. This study focused on further unravelling the complex regulation governing CLP synthesis in P. fluorescens In5. Promoter fusions were used to show that the specific activator NunF is dependent on the global regulator of secondary metabolism GacA and is regulated by fungal-associated molecules and low temperatures. In contrast, GacA is stimulated by plant signal molecules leading to the hypothesis that P. fluorescens is a hyphosphere-associated bacterium carrying transcription factor genes that respond to signals indicating the presence of fungi and oomycetes. Based on these findings, we present a model for how synthesis of nunamycin and nunapeptin is regulated by fungal- and oomycete-associated molecules.IMPORTANCE Cyclic lipopeptide (CLP) synthesis gene clusters in pseudomonads display a high degree of synteny, and the structures of the peptides synthesized are very similar. Accordingly, the genomic island encoding the synthesis of syringomycin and syringopeptin in P. syringae pv. syringae closely resembles that of P. fluorescens In5, which contains genes coding for synthesis of the antifungal and anti-oomycete peptides nunamycin and nunapeptin, respectively. However, the regulation of syringomycin and syringopeptin synthesis is different from that of nunamycin and nunapeptin synthesis. While CLP synthesis in the plant pathogen P. syringae pv. syringae is induced by plant signal molecules, such compounds do not significantly influence synthesis of nunamycin and nunapeptin in P. fluorescens In5. Instead, fungal-associated molecules positively regulate antifungal peptide synthesis in P. fluorescens In5, while the synthesis of the global regulator GacA in P. fluorescens In5 is positively regulated by plant signal molecules but not fungal-associated molecules.

Serratia inhibens sp. nov., a new antifungal species isolated from potato (Solanum tuberosum).

A novel bacterial strain, S40T, with strong antifungal activity was isolated from the rhizosphere of green potato collected from Zealand, Denmark. Polyphasic analysis with a combined phenotypic, phylogenetic and genomic approach was used to characterize S40T. Phylogenetic analysis based on the 16S rRNA gene and MLSA (concatenated gyrB, rpoD, infB and atpD sequences) showed that strain S40T was affiliated with the genus Serratia and with Serratia plymuthica PRI-2C as the closest related strain [average nucleotide identity (ANI), 99.26 %; DNA-DNA hybridization (dDDH), 99.20%]. However, whole genome sequence analyses revealed that S40T and S. plymuthica PRI-2C genomes displayed lower similarities when compared to all other S. plymuthica strains (ANI ≤94.34 %; dDDH ≤57.6 % relatedness). The DNA G+C content of strain S40T was determined to be 55.9 mol%. Cells of the strain were Gram-negative, rod-shaped, facultative anaerobic and displayed growth at 10-37 °C (optimum, 25-30 °C) and at pH 6-9 (optimum, pH 6-7). Major fatty acids were C16 : 0 (27.9 %), summed feature (C16 : 1 ω6c/C16 : 1 ω7c; 18.0 %) and C17 : 0 cyclo (15.1 %). The respiratory quinone was determined to be Q8 (94 %) and MK8 (95 %) and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The results of phenotypic, phylogenetic and genomic analyses support the hypothesis that strain S40T represents a novel species of the genus Serratia, for which the name Serratia inhibens sp. nov. is proposed. The type strain is S40T (=LMG 31467T=NCIMB 15235T). In addition, we propose that S. plymuthica PRI-2C is reclassified and transferred to the species S. inhibens as S. inhibens PRI-2C.

Identification and Characterization of a ß-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66T.

ß-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 ß-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied ß-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc ß-1,4 and ß-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-ß-Gal-1,4-ß-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM being 341 and 344 mM-1 s-1, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide ß-Gal-1,4-ß-Glc-1,1-ß-GlcNAc, and in low amounts the ß-1,2- or ß-1,3-linked trisaccharide ß-Gal-1,4(ß-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 ß-N-acetylhexosaminidases.

Colwellia echini sp. nov., an agar- and carrageenan-solubilizing bacterium isolated from sea urchin.

A novel bacterial strain, A3T, was isolated from the intestines of the sea urchin Strongylocentrotus droebachiensis collected in Øresund, Denmark. The strain was Gram-reaction-negative, rod-shaped and facultatively anaerobic, and displayed growth at 5-25 °C (optimum 20 °C), pH 7-9 (optimum at pH 7) and 1-6 % (w/v) NaCl (optimum 3 %). Furthermore, strain A3T grew on agar, agarose, κ-carrageenan, alginate and laminarin as sole carbon source. Complete liquefaction of agar and κ-carrageenan was observed on solid plate media as a result of enzymatic activities. Major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The respiratory quinones were determined to be ubiquinones Q-8 (92 %) and Q-7 (8 %), and polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was 36.9 mol%. Phylogenetical analyses based on the 16S rRNA gene showed that the bacterium was affiliated with the genus Colwellia within the Alteromonadaceae of the Gammaproteobacteria. The level of 16S rRNA gene sequence similarity between strain A3T and its closest relatives in the genus Colwellia (C. psychrerythraea ATCC 27364T and C. asteriadis KMD 002T) was 97.5 %. The average nucleotide identity between strain A3T and other members of Colwellia was 78.6-80.5 %, and DNA-DNA hybridization prediction revealed values of less than 23 % relatedness between strain A3T and other Colwellia species. The phenotypic, phylogenetic and genomic analyses support the hypothesis that strain A3T represents a novel species of the genus Colwellia, for which the name Colwellia echini sp. nov. is proposed. The type strain is A3T (=LMG 30125T=NCIMB 15095T).

Pararhizobium antarcticum sp. nov., isolated from Antarctic water samples.

Two bacterial strains were isolated from sediments and microbial mats of Kingfisher Pond, Antarctica and characterized in a taxonomic study using a polyphasic approach. Cells were strictly aerobic, Gram-stain-negative, rod-shaped, motile (+50 flagellum-specific genes present in the genome sequence; motility observed under microscope) and formed creamy white, half-transparent colonies. Growth occurred at 4 to 28 °C with an optimum at 20 °C, with 0-5.0 % (w/v) NaCl (optimum at 0-1.0 %) and at pH 4.0-11.0 (optimum pH 7.0-9.0). The major fatty acid was C18 : 1ω7c. The respiratory quinone was ubiquinone 10 (Q-10). The DNA G+C content was 60.7 mol %. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmethanolamine in addition to three unidentified lipids, one unknown glycolipid and five unidentified phospholipids. Comparative analysis of 16S rRNA gene sequences showed highest sequence similarity (98.1 %) to Pararhizobium giardinii H152T, Pararhizobium herbae CCBAU 83011T, and 'Pararhizobium polonicum' F5.1. In silico average nucleotide identity (ANI) and genome-to-genome distance calculator (GGDC) showed 81.1 % identity (ANI) and 22.6 % identity (GGDC) to the closest relative, 'P. polonicum' F5.1. On the basis of phenotypic, phylogenetic, genomic and chemotaxonomic data, the two strains represent a novel species of the genus Pararhizobium, for which the name Pararhizobium antarcticum sp. nov. is proposed. The type strain is NAQVI 59T(=DSM 103442T=LMG 29675T).

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).

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.

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.

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.

Metagenomic exploration of cold-active enzymes for detergent applications: Characterization of a novel, cold-active and alkali-stable GH8 endoglucanase from ikaite columns in SW Greenland.

Microbial communities from extreme environments are largely understudied, but are essential as producers of metabolites, including enzymes, for industrial processes. As cultivation of most microorganisms remains a challenge, culture-independent approaches for enzyme discovery in the form of metagenomics to analyse the genetic potential of a community are rapidly becoming the way forward. This study focused on analysing a metagenome from the cold and alkaline ikaite columns in Greenland, identifying 282 open reading frames (ORFs) that encoded putative carbohydrate-modifying enzymes with potential applications in, for example detergents and other processes where activity at low temperature and high pH is desired. Seventeen selected ORFs, representing eight enzyme families were synthesized and expressed in two host organisms, Escherichia coli and Aliivibrio wodanis. Aliivibrio wodanis demonstrated expression of a more diverse range of enzyme classes compared to E. coli, emphasizing the importance of alternative expression systems for enzymes from extremophilic microorganisms. To demonstrate the validity of the screening strategy, we chose a recombinantly expressed cellulolytic enzyme from the metagenome for further characterization. The enzyme, Cel240, exhibited close to 40% of its relative activity at low temperatures (4°C) and demonstrated endoglucanase characteristics, with a preference for cellulose substrates. Despite low sequence similarity with known enzymes, computational analysis and structural modelling confirmed its cellulase-family affiliation. Cel240 displayed activity at low temperatures and good stability at 25°C, activity at alkaline pH and increased activity in the presence of CaCl2, making it a promising candidate for detergent and washing industry applications.

Investigating eukaryotic and prokaryotic diversity and functional potential in the cold and alkaline ikaite columns in Greenland.

The ikaite columns in the Ikka Fjord, SW Greenland, represent a permanently cold and alkaline environment known to contain a rich bacterial diversity. 16S and 18S rRNA gene amplicon and metagenomic sequencing was used to investigate the microbial diversity in the columns and for the first time, the eukaryotic and archaeal diversity in ikaite columns were analyzed. The results showed a rich prokaryotic diversity that varied across columns as well as within each column. Seven different archaeal phyla were documented in multiple locations inside the columns. The columns also contained a rich eukaryotic diversity with 27 phyla representing microalgae, protists, fungi, and small animals. Based on metagenomic sequencing, 25 high-quality MAGs were assembled and analyzed for the presence of genes involved in cycling of nitrogen, sulfur, and phosphorous as well as genes encoding carbohydrate-active enzymes (CAZymes), showing a potentially very bioactive microbial community.

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.

Enzymatic Process for the Carrageenolytic Bioconversion of Sulfated Polygalactans into ß-Neocarrabiose and 3,6-Anhydro-d-galactose.

Oligosaccharides and anhydro-sugars derived from carrageenan have great potential as functional foods and drugs showing various bioactivities, including antioxidant, anti-inflammatory, antiviral, antitumor, and cytotoxic activities. Although preparation of sulfated carrageenan oligosaccharides by chemical and enzymatic processes has been widely reported, preparation of nonsulfated ß-neocarrabiose (ß-NC2) and the rare sugar 3,6-anhydro-d-galactose (d-AHG) was not reported in the literature. Based on the carrageenan catabolic pathway in marine heterotrophic bacteria, an enzymatic process was designed and constructed with recombinant κ-carrageenase, GH127/GH129 α-1,3 anhydrogalactosidase, and cell-free extract from marine carrageenolytic bacteria Colwellia echini A3T. The process consisted of three successive steps, namely, (i) depolymerization, (ii) desulfation, and (iii) monomerization, by which carrageenan oligosaccharides, ß-NC2, and d-AHG were obtained from κ-carrageenan. Unlike the chemical process, enzymatic hydrolysis yields oligosaccharides with the desired degree of polymerization facilitates specific removal of sulfated groups, free of toxic byproducts, and avoids chemical modifications. The final optimized enzymatic process produced 0.52 g of ß-NC2 and 0.24 g of d-AHG from 1 g of κ-carrageenan. The carrageenolytic process designed for the enzymatic hydrolysis of κ-carrageenan can be scaled up for the mass production of bioactive carrageeno-oligosaccharides.

Plant-Root Exudate Analogues Influence Activity of the 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Gene in Pseudomonas hormoni G20-18T.

Plants exposed to abiotic stress such as drought and salinity produce 1-aminocyclopropane-1-carboxylic acid (ACC) that is converted into the stress hormone ethylene. However, plant growth-promoting bacteria (PGPB), which synthesize the enzyme ACC deaminase, may lower the ACC concentration thereby reducing the concentration of ethylene and alleviating the abiotic stress. The PGPB Pseudomonas hormoni G20-18T (previously named P. fluorescens G20-18) harbors the genes acdR and acdS that encode regulation and synthesis of ACC deaminase, respectively. Regulation of the acdS gene has been investigated in several studies, but so far, it has been an open question whether plants can regulate microbial synthesis of ACC deaminase. In this study, small molecules in wheat root exudates were identified using untargeted metabolomics, and compounds belonging to amino acids, organic acids, and sugars were selected for evaluation of their influence on the expression of the acdS and acdR genes in P. hormoni G20-18T. acdS and acdR promoters were fused to the fluorescence reporter gene mCherry enabling the study of acdS and acdR promoter activity. In planta studies in wheat seedlings indicated an induced expression of acdS in association with the roots. Exudate molecules such as aspartate, alanine, arginine, and fumarate as well as glucose, fructose, and mannitol actively induced the acdS promoter, whereas the plant hormone indole-3-acetic acid (IAA) inhibited expression. Here, we present a model for how stimulatory and inhibitory root exudate molecules influence acdS promoter activity in P. hormoni G20-18T.

Constructing Marine Bacterial Metabolic Chassis for Potential Biorefinery of Red Algal Biomass and Agaropectin Wastes.

Marine red algal biomass is a promising feedstock for sustainable production of value-added chemicals. However, the major constituents of red algal biomass, such as agar and carrageenan, are not easily assimilated by most industrial metabolic chassis developed to date. Synthetic biology offers a solution by utilizing nonmodel organisms as metabolic chassis for consolidated biological processes. In this study, the marine heterotrophic bacterium Pseudoalteromonas atlantica T6c was harnessed as a metabolic chassis to produce value-added chemicals from the affordable red algal galactans or agaropectin, a byproduct of industrial agarose production. To construct a heterologous gene expression device in P. atlantica T6c, promoters related to agar metabolism were screened from the differentially expressed genes using RNA-Seq analysis. The expression device was built and tested with selected promoters fused to a reporter gene and tuned by incorporation of a cognate repressor predicted from the agar-specific polysaccharide utilization locus. The feasibility of the marine bacterial metabolic chassis was examined by introducing the biosynthetic gene clusters of ß-carotene and violacein. Our results demonstrate that the metabolic chassis platform enables direct conversion of low-cost red algal galactans or industrial waste agaropectin into valuable bioactive pigments without any pretreatment of biomass. The developed marine bacterial chassis could potentially be used in a biorefinery framework to produce value-added chemicals from marine algal galactans.

Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1.

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to -25°C. P355's stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (k cat ) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.