Out of taxonomic crypsis: A new trans-arctic cryptic species pair corroborated by phylogenetics and molecular evidence.

Cryptic species are a common phenomenon in cosmopolitan marine species. The use of molecular tools has often uncovered cryptic species occupying a fraction of the geographic range of the original morphospecies. Shipworms (Teredinidae) are marine bivalves, living in drift and fixed wood, many of which have a conserved morphology across cosmopolitan distributions. Herein novel and GenBank mitochondrial (cytochrome c oxidase subunit I) and nuclear (18S rRNA) DNA sequences are employed to produce a phylogeny of the Teredinidae and delimit a cryptic species pair in the Psiloteredo megotara complex. The anatomy, biogeography, and ecology of P. megotara, Psiloteredo sp. and Nototeredo edax are compared based on private and historic museum collections and a thorough literature review. Morphological and anatomical characters of P. megotara from the North Atlantic and Psiloteredo sp. from Japan were morphologically indistinguishable, and differ in pallet architecture and soft tissue anatomy from N. edax. The two Psiloteredo species were then delimited as genetically distinct species using four molecular-based methods. Consequently, the Northwest Pacific species, Psiloteredo pentagonalis, first synonymized with N. edax and then with P. megotara, is resurrected. Nototeredo edax, P. megotara and P. pentagonalis are redescribed based upon morphological and molecular characters. Phylogenetic analysis further revealed cryptic species complexes within the cosmopolitan species Bankia carinata and possibly additional cryptic lineages within the cosmopolitan Lyrodus pedicellatus.

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant.

BACKGROUND: Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains. RESULTS: Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains. CONCLUSIONS: Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

Genomic Changes Associated with the Evolutionary Transitions of Nostoc to a Plant Symbiont.

Cyanobacteria belonging to the genus Nostoc comprise free-living strains and also facultative plant symbionts. Symbiotic strains can enter into symbiosis with taxonomically diverse range of host plants. Little is known about genomic changes associated with evolutionary transition of Nostoc from free-living to plant symbiont. Here, we compared the genomes derived from 11 symbiotic Nostoc strains isolated from different host plants and infer phylogenetic relationships between strains. Phylogenetic reconstructions of 89 Nostocales showed that symbiotic Nostoc strains with a broad host range, entering epiphytic and intracellular or extracellular endophytic interactions, form a monophyletic clade indicating a common evolutionary history. A polyphyletic origin was found for Nostoc strains which enter only extracellular symbioses, and inference of transfer events implied that this trait was likely acquired several times in the evolution of the Nostocales. Symbiotic Nostoc strains showed enriched functions in transport and metabolism of organic sulfur, chemotaxis and motility, as well as the uptake of phosphate, branched-chain amino acids, and ammonium. The genomes of the intracellular clade differ from that of other Nostoc strains, with a gain/enrichment of genes encoding proteins to generate l-methionine from sulfite and pathways for the degradation of the plant metabolites vanillin and vanillate, and of the macromolecule xylan present in plant cell walls. These compounds could function as C-sources for members of the intracellular clade. Molecular clock analysis indicated that the intracellular clade emerged ca. 600 Ma, suggesting that intracellular Nostoc symbioses predate the origin of land plants and the emergence of their extant hosts.

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.

Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.

High-Density Cultivation of Terrestrial Nostoc Strains Leads to Reprogramming of Secondary Metabolome.

Terrestrial symbiotic cyanobacteria of the genus Nostoc exhibit a large potential for the production of bioactive natural products of the nonribosomal peptide, polyketide, and ribosomal peptide classes, and yet most of the biosynthetic gene clusters are silent under conventional cultivation conditions. In the present study, we utilized a high-density cultivation approach recently developed for phototrophic bacteria to rapidly generate biomass of the filamentous bacteria up to a density of 400 g (wet weight)/liter. Unexpectedly, integrated transcriptional and metabolomics studies uncovered a major reprogramming of the secondary metabolome of two Nostoc strains at high culture density and a governing effect of extracellular signals in this process. The holistic approach enabled capturing and structural elucidation of novel variants of anabaenopeptin, including one congener with potent allelopathic activity against a strain isolated from the same habitat. The study provides a snapshot on the role of cell-type-specific expression for the formation of natural products in cyanobacteria.IMPORTANCE Terrestrial filamentous cyanobacteria are a largely untapped source of small-molecule natural products. Exploitation of the phototrophic organisms is hampered by their slow growth and the requirement of photobioreactors. The present study not only demonstrates the suitability of a recently developed two-tier vessel cultivation approach for the rapid generation of biomass of Nostoc strains but also demonstrates a pronounced upregulation of high value natural products at ultrahigh culture densities. The study provides new guidelines for high-throughput screening and exploitation of small-molecule natural products and can facilitate the discovery new bioactive products from terrestrial cyanobacteria.

Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries.

BACKGROUND: The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields. RESULTS: Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, ß-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl. CONCLUSIONS: The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.

Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium Thalassospira sp.

A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 °C and the thermal stability displayed a retention of 75 % relative activity at 40 °C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 Å revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.

Bioprospecting around Arctic islands: Marine bacteria as rich source of biocatalysts.

We have investigated the biotechnological potential of Arctic marine bacteria for their ability to produce a broad spectrum of cold-active enzymes. Marine bacteria exhibiting these features are of great interest for both fundamental research and industrial applications. Macrobiota, water and sediment samples have been collected during 2010 and 2011 expeditions around the Lofoten and Svalbard islands. Bacteria were isolated from this material and identified through 16S rRNA gene sequence analysis for the purpose of establishing a culture collection of marine Arctic bacteria. Herein, we present the functional screening for different extracellular enzymatic activities from 100 diversely chosen microbial isolates incubated at 4 and 20 °C. The production of esterase/lipase, DNase, and protease activities were revealed in 67, 53, and 56% of the strains, respectively, while 41, 23, 9, and 7% of the strains possessed amylase, chitinase, cellulase, and xylanase activities, respectively. Our findings show that phylogenetically diverse bacteria, including many new species, could be cultured from the marine arctic environment. The Arctic polar environment is still an untapped reservoir of biodiversity for bioprospecting.

Properties and distribution of a metallo-ß-lactamase (ALI-1) from the fish pathogen Aliivibrio salmonicida LFI1238.

OBJECTIVES: To characterize the chromosome-encoded metallo-ß-lactamase (MBL) from the psychrophilic, marine fish-pathogenic bacterium Aliivibrio salmonicida LFI1238 and check for the presence of the gene in other Aliivibrio isolates both connected to the fish-farming industry and from the environment. METHODS: The MBL gene was cloned and intracellularly expressed in Escherichia coli. Kinetic parameters, NaCl dependence, pH optimum and temperature optimum were determined using purified enzyme. The VIM-2 enzyme from a Pseudomonas aeruginosa hospital isolate was used as a counterpart in comparative analysis. PCRs with degenerate MBL primers were used to screen different A. salmonicida isolates for the presence of the gene. RESULTS: A. salmonicida MBL (ALI-1) is an Ambler class B ß-lactamase sharing 39% and 29% amino acid identity with IMP-1 and VIM-2, respectively. ALI-1 hydrolysed all ß-lactam antibiotics tested, except for the monobactam aztreonam and the penicillin piperacillin. A profound increase in activity was observed when adding NaCl to the assay mixture (60% active without addition of NaCl, increasing to 100% at 0.5 M NaCl). The increase was less noticeable for VIM-2 (100% active at 0.2 M NaCl). ALI-1 appears to be ubiquitous in nature as it is found in Aliivibrio isolates not affected by human activity. CONCLUSIONS: This work provides more data for the ever-expanding MBL group of enzymes. These periplasmic enzymes are activated by addition of NaCl, and the marine enzyme is highly salt tolerant and cold active. The observed enzyme properties very likely reflect the conditions that the enzymes face in situ.

Isolation and characterization of anti-adenoviral secondary metabolites from marine actinobacteria.

Adenovirus infections in immunocompromised patients are associated with high mortality rates. Currently, there are no effective anti-adenoviral therapies available. It is well known that actinobacteria can produce secondary metabolites that are attractive in drug discovery due to their structural diversity and their evolved interaction with biomolecules. Here, we have established an extract library derived from actinobacteria isolated from Vestfjorden, Norway, and performed a screening campaign to discover anti-adenoviral compounds. One extract with anti-adenoviral activity was found to contain a diastereomeric 1:1 mixture of the butenolide secondary alcohols 1a and 1b. By further cultivation and analysis, we could isolate 1a and 1b in different diastereomeric ratio. In addition, three more anti-adenoviral butenolides 2, 3 and 4 with differences in their side-chains were isolated. In this study, the anti-adenoviral activity of these compounds was characterized and substantial differences in the cytotoxic potential between the butenolide analogs were observed. The most potent butenolide analog 3 displayed an EC50 value of 91 µM and no prominent cytotoxicity at 2 mM. Furthermore, we propose a biosynthetic pathway for these compounds based on their relative time of appearance and structure.

Characterization of the sialic acid synthase from Aliivibrio salmonicida suggests a novel pathway for bacterial synthesis of 7-O-acetylated sialic acids.

Resolving the enzymatic pathways leading to sialic acids (Sias) in bacteria are vitally important for understanding their roles in pathogenesis and for subsequent development of tools to combat infections. A detailed characterization of the involved enzymes is also essential due to the highly applicable properties of Sias, i.e., as used in a wide range of medical applications and human nutrition. Bacterial strains that produce Sias display them mainly on their cell surface to mimic animal cells thereby evading the host's immune system. Despite several studies, little is known about the virulence mechanisms of the fish pathogen Aliivibrio salmonicida. The genome of A. salmonicida LFI1238 contains a gene cluster homologous to the Escherichia coli neuraminic acid (Neu) gene cluster involved in biosynthesis of Sias found in the E. coli capsule. This cluster is probably responsible for the biosynthesis of Neu found in A. salmonicida. In this work, we have produced and characterized the sialic acid (Sia) synthase NeuB1, the key enzyme in the pathway. The Sia synthase is an enzyme producing N-acetylneuraminic acid by the condensation of N-acetylmannosamine and phosphoenolpyruvate. Genome content, kinetic data obtained, together with structural considerations, have led us to the prediction that the substrate for NeuB1 from A. salmonicida, E. coli and Streptococcus agalactiae among others, is 4-O-acetyl-N-acetylmannosamine. This means that the product of its enzymatic reaction is 7-O-acetyl-N-acetylneuraminic acid. We propose a pathway for production of this Sia in A. salmonicida, and present evidence for the presence of diacetylated Neu in the bacterium.

Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara.

BACKGROUND: Cellulases play a key role in the enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. Thus, the discovery of novel cellulases from exotic biological niches is of great interest as they may present properties that are valuable in the biorefining of lignocellulosic biomass. RESULTS: We have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multi-domain enzyme from the unusual gill endosymbiont Teredinibacter waterburyi of the wood-digesting shipworm Psiloteredo megotara. The catalytic GH5 domain, was cloned and recombinantly produced with or without a C-terminal family 10 carbohydrate-binding module (CBM). Both variants showed hydrolytic endo-activity on soluble substrates such as ß-glucan, carboxymethylcellulose and konjac glucomannan, respectively. However, low activity was observed towards the crystalline form of cellulose. Interestingly, when co-incubated with a cellulose-active LPMO, a clear synergy was observed that boosted the overall hydrolysis of crystalline cellulose. The crystal structure of the GH5 catalytic domain was solved to 1.0 Å resolution and revealed a substrate binding cleft extension containing a putative + 3 subsite, which is uncommon in this enzyme family. The enzyme was active in a wide range of pH, temperatures and showed high tolerance for NaCl. CONCLUSIONS: This study provides significant knowledge in the discovery of new enzymes from shipworm gill endosymbionts and sheds new light on biochemical and structural characterization of cellulolytic cellulase. Study demonstrated a boost in the hydrolytic activity of cellulase on crystalline cellulose when co-incubated with cellulose-active LPMO. These findings will be relevant for the development of future enzyme cocktails that may be useful for the biotechnological conversion of lignocellulose.

A unique class I polyhydroxyalkanoate synthase (PhaC) from Brevundimonas sp. KH11J01 exists as a functional trimer: A comparative study with PhaC from Cupriavidus necator H16.

Polyhydroxyalkanoates (PHAs) are natural biodegradable polyesters that are produced by numerous prokaryotic microorganisms primarily as a carbon- and energy reserve. The PhaC enzyme catalyzes the last step in the PHA biosynthesis pathway and synthesizes PHA polymers from hydroxyalkanoic acids. A type I PhaC from a PHA-producing marine bacterium Brevundimonas sp. KH11J01 (BrPhaC) was identified, produced recombinantly and characterized. Its properties were compared with its homolog from C. necator H16 (RePhaC). Unlike other PhaCs, it was found that BrPhaC is a lag-phase free enzyme organized as a trimer, even without the presence of a substrate. The enzymatic reaction is initiated instantly irrespective of temperature, in contrast to RePhaC in which the duration of the lag-phase was highly affected by temperature. At 10 °C BrPhaC was 40% active whereas RePhaC was barely active. The significance of using marine microorganisms, harboring cold-active PHA biosynthesis enzymes, for energy efficient PHA production, is also discussed briefly. The unique trimeric organization of BrPhaC challenges our understanding of the PhaC reaction mechanisms, which is mainly based on the crystal structures of the inactive forms of the enzyme.

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation.

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics.

The crystal structure of the tetrameric DABA-aminotransferase EctB, a rate-limiting enzyme in the ectoine biosynthesis pathway.

l-2,4-diaminobutyric acid (DABA) aminotransferases can catalyze the formation of amines at the distal ω-position of substrates, and is the intial and rate-limiting enzyme in the biosynthesis pathway of the cytoprotecting molecule (S)-2-methyl-1,4,5,6-tetrahydro-4-pyrimidine carboxylic acid (ectoine). Although there is an industrial interest in the biosynthesis of ectoine, the DABA aminotransferases remain poorly characterized. Herein, we present the crystal structure of EctB (2.45 Å), a DABA aminotransferase from Chromohalobacter salexigens DSM 3043, a well-studied organism with respect to osmoadaptation by ectoine biosynthesis. We investigate the enzyme's oligomeric state to show that EctB from C. salexigens is a tetramer of two functional dimers, and suggest conserved recognition sites for dimerization that also includes the characteristic gating loop that helps shape the active site of the neighboring monomer. Although ω-transaminases are known to have two binding pockets to accommodate for their dual substrate specificity, we herein provide the first description of two binding pockets in the active site that may account for the catalytic character of DABA aminotransferases. Furthermore, our biochemical data reveal that the EctB enzyme from C. salexigens is a thermostable, halotolerant enzyme with a broad pH tolerance which may be linked to its tetrameric state. Put together, this study creates a solid foundation for a deeper structural understanding of DABA aminotransferases and opening up for future downstream studies of EctB's catalytic character and its redesign as a better catalyst for ectoine biosynthesis. In summary, we believe that the EctB enzyme from C. salexigens can serve as a benchmark enzyme for characterization of DABA aminotransferases. DATABASE: Structural data are available in PDB database under the accession number 6RL5.

Sequence analysis of nonulosonic acid biosynthetic gene clusters in Vibrionaceae and Moritella viscosa.

Nonulosonic acid (NulO) biosynthesis in bacteria is directed by nab gene clusters that can lead to neuraminic, legionaminic or pseudaminic acids. Analysis of the gene content from a set mainly composed of Aliivibrio salmonicida and Moritella viscosa strains reveals the existence of several unique nab clusters, for which the NulO products were predicted. This prediction method can be used to guide tandem mass spectrometry studies in order to verify the products of previously undescribed nab clusters and identify new members of the NulOs family.

Cultivable marine fungi from the Arctic Archipelago of Svalbard and their antibacterial activity.

During a research cruise in 2016, we isolated fungi from sediments, seawater, driftwood, fruiting bodies, and macroalgae using three different media to assess species richness and potential bioactivity of cultivable marine fungi in the High Arctic region. Ten stations from the Svalbard archipelago (73-80 °N, 18-31 °E) were investigated and 33 fungal isolates were obtained. These grouped into 22 operational taxonomic units (OTUs) using nuc rDNA internal transcribed spacer regions (ITS1-5.8S-ITS2 = ITS) with acut-off set at 98% similarity. The taxonomic analysis showed that 17 OTUs belonged to Ascomycota, one to Basidiomycota, two to Mucoromycota and two were fungal-like organisms. The nuc rDNA V1-V5 regions of 18S (18S) and D1-D3 regions of 28S (28S) were sequenced from representative isolates of each OTU for comparison to GenBank sequences. Isolates of Lulworthiales and Eurotiales were the most abundant, with seven isolates each. Among the 22 OTUs, nine represent potentially undescribed species based on low similarity to GenBank sequences and 10 isolates showed inhibitory activity against Gram-positive bacteria in an agar diffusion plug assay. These results show promise for the Arctic region as asource of novel marine fungi with the ability to produce bioactive secondary metabolites with antibacterial properties.

The crystal structure of the N-acetylglucosamine 2-epimerase from Nostoc sp. KVJ10 reveals the true dimer.

N-Acetylglucosamine 2-epimerases (AGEs) catalyze the interconversion of N-acetylglucosamine and N-acetylmannosamine. They can be used to perform the first step in the synthesis of sialic acid from N-acetylglucosamine, which makes the need for efficient AGEs a priority. This study presents the structure of the AGE from Nostoc sp. KVJ10 collected in northern Norway, referred to as nAGE10. It is the third AGE structure to be published to date, and the first one in space group P42212. The nAGE10 monomer folds as an (α/α)6 barrel in a similar manner to that of the previously published AGEs, but the crystal did not contain the dimers that have previously been reported. The previously proposed `back-to-back' assembly involved the face of the AGE monomer where the barrel helices are connected by small loops. Instead, a `front-to-front' dimer was found in nAGE10 involving the long loops that connect the barrel helices at this end. This assembly is also present in the other AGE structures, but was attributed to crystal packing, even though the `front' interface areas are larger and are more conserved than the `back' interface areas. In addition, the front-to-front association allows a better explanation of the previously reported observations considering surface cysteines. Together, these results indicate that the `front-to-front' dimer is the most probable biological assembly for AGEs.

Features and structure of a cold active N-acetylneuraminate lyase.

N-acetylneuraminate lyases (NALs) are enzymes that catalyze the reversible cleavage and synthesis of sialic acids. They are therefore commonly used for the production of these high-value sugars. This study presents the recombinant production, together with biochemical and structural data, of the NAL from the psychrophilic bacterium Aliivibrio salmonicida LFI1238 (AsNAL). Our characterization shows that AsNAL possesses high activity and stability at alkaline pH. We confirm that these properties allow for the use in a one-pot reaction at alkaline pH for the synthesis of N-acetylneuraminic acid (Neu5Ac, the most common sialic acid) from the inexpensive precursor N-acetylglucosamine. We also show that the enzyme has a cold active nature with an optimum temperature for Neu5Ac synthesis at 20°C. The equilibrium constant for the reaction was calculated at different temperatures, and the formation of Neu5Ac acid is favored at low temperatures, making the cold active enzyme a well-suited candidate for use in such exothermic reactions. The specific activity is high compared to the homologue from Escherichia coli at three tested temperatures, and the enzyme shows a higher catalytic efficiency and turnover number for cleavage at 37°C. Mutational studies reveal that amino acid residue Asn 168 is important for the high kcat. The crystal structure of AsNAL was solved to 1.65 Å resolution and reveals a compact, tetrameric protein similar to other NAL structures. The data presented provides a framework to guide further optimization of its application in sialic acid production and opens the possibility for further design of the enzyme.

Draft Genome Sequence of the Symbiotically Competent Cyanobacterium Nostoc sp. Strain KVJ20.

Nostoc sp. strain KVJ20 was isolated from the symbiotic organs of the liverwort Blasia pusilla This cyanobacterium has been shown to have broad symbiotic competence, and bacterial extracts have inhibitory effects on cancer cell lines and microbes. An array of genes for the production of secondary metabolites is present.