Comparative analysis of HiSeq3000 and BGISEQ-500 sequencing platform over whole genome sequencing metagenomics data.

Recent advances in sequencing technologies and platforms have enabled to generate metagenomics sequences using different sequencing platforms. In this study, we analyzed and compared shotgun metagenomic sequences generated by HiSeq3000 and BGISEQ-500 platforms from 12 sediment samples collected across the Norwegian coast. Metagenomics DNA sequences were normalized to an equal number of bases for both platforms and further evaluated by using different taxonomic classifiers, reference databases, and assemblers. Normalized BGISEQ-500 sequences retained more reads and base counts after preprocessing, while a slightly higher fraction of HiSeq3000 sequences were taxonomically classified. Kaiju classified a higher percentage of reads relative to Kraken2 for both platforms, and comparison of reference database for taxonomic classification showed that MAR database outperformed RefSeq. Assembly using MEGAHIT produced longer assemblies and higher total contigs count in majority of HiSeq3000 samples than using metaSPAdes, but the assembly statistics notably improved with unprocessed or normalized reads. Our results indicate that both platforms perform comparably in terms of the percentage of taxonomically classified reads and assembled contig statistics for metagenomics samples. This study provides valuable insights for researchers in selecting an appropriate sequencing platform and bioinformatics pipeline for their metagenomics studies.

Quorum Sensing Controls the CRISPR and Type VI Secretion Systems in Aliivibrio wodanis 06/09/139.

For bacteria to thrive in an environment with competitors, phages and environmental cues, they use different strategies, including Type VI Secretion Systems (T6SSs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to compete for space. Bacteria often use quorum sensing (QS), to coordinate their behavior as the cell density increases. Like other aliivibrios, Aliivibrio wodanis 06/09/139 harbors two QS systems, the main LuxS/LuxPQ system and an N-acyl homoserine lactone (AHL)-mediated AinS/AinR system and a master QS regulator, LitR. To explore the QS and survival strategies, we performed genome analysis and gene expression profiling on A. wodanis and two QS mutants (ΔainS and ΔlitR) at two cell densities (OD600 2.0 and 6.0) and temperatures (6 and 12°C). Genome analysis of A. wodanis revealed two CRISPR systems, one without a cas loci (CRISPR system 1) and a type I-F CRISPR system (CRISPR system 2). Our analysis also identified three main T6SS clusters (T6SS1, T6SS2, and T6SS3) and four auxiliary clusters, as well about 80 potential Type VI secretion effectors (T6SEs). When comparing the wildtype transcriptome data at different cell densities and temperatures, 13-18% of the genes were differentially expressed. The CRISPR system 2 was cell density and temperature-independent, whereas the CRISPR system 1 was temperature-dependent and cell density-independent. The primary and auxiliary clusters of T6SSs were both cell density and temperature-dependent. In the ΔlitR and ΔainS mutants, several CRISPR and T6SS related genes were differentially expressed. Deletion of litR resulted in decreased expression of CRISPR system 1 and increased expression of CRISPR system 2. The T6SS1 and T6SS2 gene clusters were less expressed while the T6SS3 cluster was highly expressed in ΔlitR. Moreover, in ΔlitR, the hcp1 gene was strongly activated at 6°C compared to 12°C. AinS positively affected the csy genes in the CRISPR system 2 but did not affect the CRISPR arrays. Although AinS did not significantly affect the expression of T6SSs, the hallmark genes of T6SS (hcp and vgrG) were AinS-dependent. The work demonstrates that T6SSs and CRISPR systems in A. wodanis are QS dependent and may play an essential role in survival in its natural environment.

Quorum sensing in Aliivibrio wodanis 06/09/139 and its role in controlling various phenotypic traits.

BACKGROUND: Quorum Sensing (QS) is a cell-to-cell communication system that bacteria utilize to adapt to the external environment by synthesizing and responding to signalling molecules called autoinducers. The psychrotrophic bacterium Aliivibrio wodanis 06/09/139, originally isolated from a winter ulcer of a reared Atlantic salmon, produces the autoinducer N-3-hydroxy-decanoyl-homoserine-lactone (3OHC10-HSL) and encodes the QS systems AinS/R and LuxS/PQ, and the master regulator LitR. However, the role of QS in this bacterium has not been investigated yet. RESULTS: In the present work we show that 3OHC10-HSL production is cell density and temperature-dependent in A. wodanis 06/09/139 with the highest production occurring at a low temperature (6 °C). Gene inactivation demonstrates that AinS is responsible for 3OHC10-HSL production and positively regulated by LitR. Inactivation of ainS and litR further show that QS is involved in the regulation of growth, motility, hemolysis, protease activity and siderophore production. Of these QS regulated activities, only the protease activity was found to be independent of LitR. Lastly, supernatants harvested from the wild type and the ΔainS and ΔlitR mutants at high cell densities show that inactivation of QS leads to a decreased cytopathogenic effect (CPE) in a cell culture assay, and strongest attenuation of the CPE was observed with supernatants harvested from the ΔlitR mutant. CONCLUSION: A. wodanis 06/09/139 use QS to regulate a number of activities that may prove important for host colonization or interactions. The temperature of 6 °C that is in the temperature range at which winter ulcer occurs, plays a role in AHL production and development of CPE on a Chinook Salmon Embryo (CHSE) cell line.

Phylogenetic Revision of the Genus Aliivibrio: Intra- and Inter-Species Variance Among Clusters Suggest a Wider Diversity of Species.

Genus Aliivibrio is known to harbor species exhibiting bioluminescence as well as pathogenic behavior affecting the fish farming industry. Current phylogenetic understanding of Aliivibrio has largely remained dormant after reclassification disentangled it from the Vibrio genus in 2007. There is growing evidence of wider diversity, but until now the lack of genomes and selective use of type strains have limited the ability to compare and classify strains firmly. In this study, a total of 143 bacterial strains, including 51 novel sequenced strains, were used to strengthen phylogenetic relationships in Aliivibrio by exploring intra-species and inter-species relations. Multilocus sequence analysis (MLSA), applying the six housekeeping genes 16S ribosomal RNA (rRNA), gapA, gyrB, pyrH, recA, and rpoA, inferred 12 clades and a singular branch in Aliivibrio. Along with four new phylogenetic clades, the MLSA resolved prior inconsistencies circumscribing Aliivibrio wodanis and formed a unique clade we propose as the novel species Aliivibrio sp. "friggae." Furthermore, phylogenetic assessment of individual marker genes showed gyrB, pyrH, and recA superior to the 16S rRNA gene, resolving accurately for most species clades in Aliivibrio. In this study, we provide a robust phylogenetic groundwork for Aliivibrio as a reference point to classification of species.

Recommendations for connecting molecular sequence and biodiversity research infrastructures through ELIXIR.

Threats to global biodiversity are increasingly recognised by scientists and the public as a critical challenge. Molecular sequencing technologies offer means to catalogue, explore, and monitor the richness and biogeography of life on Earth. However, exploiting their full potential requires tools that connect biodiversity infrastructures and resources. As a research infrastructure developing services and technical solutions that help integrate and coordinate life science resources across Europe, ELIXIR is a key player. To identify opportunities, highlight priorities, and aid strategic thinking, here we survey approaches by which molecular technologies help inform understanding of biodiversity. We detail example use cases to highlight how DNA sequencing is: resolving taxonomic issues; Increasing knowledge of marine biodiversity; helping understand how agriculture and biodiversity are critically linked; and playing an essential role in ecological studies. Together with examples of national biodiversity programmes, the use cases show where progress is being made but also highlight common challenges and opportunities for future enhancement of underlying technologies and services that connect molecular and wider biodiversity domains. Based on emerging themes, we propose key recommendations to guide future funding for biodiversity research: biodiversity and bioinformatic infrastructures need to collaborate closely and strategically; taxonomic efforts need to be aligned and harmonised across domains; metadata needs to be standardised and common data management approaches widely adopted; current approaches need to be scaled up dramatically to address the anticipated explosion of molecular data; bioinformatics support for biodiversity research needs to be enabled and sustained; training for end users of biodiversity research infrastructures needs to be prioritised; and community initiatives need to be proactive and focused on enabling solutions. For sequencing data to deliver their full potential they must be connected to knowledge: together, molecular sequence data collection initiatives and biodiversity research infrastructures can advance global efforts to prevent further decline of Earth's biodiversity.

Full-length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis.

Understanding fish-microbial relationships may be of great value for fish producers as fish growth, development and welfare are influenced by the microbial community associated with the rearing systems and fish surfaces. Accurate methods to generate and analyze these microbial communities would be an important tool to help improve understanding of microbial effects in the industry. In this study, we performed taxonomic classification and determination of operational taxonomic units on Atlantic salmon microbiota by taking advantage of full-length 16S rRNA gene sequences. Skin mucus was dominated by the genera Flavobacterium and Psychrobacter. Intestinal samples were dominated by the genera Carnobacterium, Aeromonas, Mycoplasma and by sequences assigned to the order Clostridiales. Applying Sanger sequencing on the full-length bacterial 16S rRNA gene from the pool of 46 isolates obtained in this study showed a clear assignment of the PacBio full-length bacterial 16S rRNA gene sequences down to the genus level. One of the bottlenecks in comparing microbial profiles is that different studies use different 16S rRNA gene regions. Comparisons of sequence assignments between full-length and in silico derived variable 16S rRNA gene regions showed different microbial profiles with variable effects between phylogenetic groups and taxonomic ranks.

Exploring the transcriptome of luxI- and ΔainS mutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation in Aliivibrio salmonicida.

BACKGROUND: Bacterial communication through quorum sensing (QS) systems has been reported to be important in coordinating several traits such as biofilm formation. In Aliivibrio salmonicida two QS systems the LuxI/R and AinS/R, have been shown to be responsible for the production of eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. We have previously demonstrated that inactivation of LitR, the master regulator of the QS system resulted in biofilm formation, similar to the biofilm formed by the AHL deficient mutant ΔainSluxI- . In this study, we aimed to investigate the global gene expression patterns of luxI and ainS autoinducer synthases mutants using transcriptomic profiling. In addition, we examined the influence of the different AHLs on biofilm formation. RESULTS: The transcriptome profiling of ΔainS and luxI- mutants allowed us to identify genes and gene clusters regulated by QS in A. salmonicida. Relative to the wild type, the ΔainS and luxI- mutants revealed 29 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation of luxI, but not ainS genes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI- ) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when the ΔainSluxI- mutant was supplemented with N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL) or N-3-hydroxy-decanoyl-L-homoserine lactone (3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and for repression of EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor. CONCLUSION: These findings imply that the LuxI and AinS autoinducer synthases play a critical role in the regulation of biofilm formation, EPS production, and motility.

Closely-related Photobacterium strains comprise the majority of bacteria in the gut of migrating Atlantic cod (Gadus morhua).

BACKGROUND: The population of Atlantic cod (Gadus morhua), also known as Northeast Arctic cod, migrating Atlantic cod, or simply "skrei," lives mainly in the Barents Sea and Svalbard waters and migrates in annual cycles to the Norwegian coast in order to spawn eggs during late winter. It is the world's largest population of Atlantic cod, and the population is distinct from the Norwegian coastal cod (or "fjord" cod). Despite the biological, economic, and cultural importance of migrating Atlantic cod, current knowledge on the associated microbiota is very limited. Using shotgun metagenomics and metaproteomics approaches, we present here the gut microbiota, metagenome-assembled genomes (MAGs) of the most abundant bacterial species, DNA-based functional profile, and the metaproteome of Atlantic cod specimens caught at a spawning area in an open ocean outside of Tromsø, Norway. RESULTS: Our analyses identified 268 bacterial families in DNA isolated from feces of 6 individual migrating Atlantic cod. The most abundant family was Vibrionaceae (52%; 83% if unclassified reads are excluded), with Photobacterium (genus) representing the vast majority. The recovery of metagenome-assembled genomes provided further details and suggests that several closely related Photobacterium strains from the Photobacterium phosphoreum clade are the most abundant. A genomic-based functional profiling showed that the most abundant functional subsystems are "Carbohydrates"; "Amino Acids and Derivatives"; "Protein Metabolism"; "Cofactors, Vitamins, Prosthetic, Groups, and Pigments"; and "DNA Metabolism," which is in agreement with other studies of gut microbiomes of marine organisms. Finally, the MS-based metaproteomic dataset revealed that the functional category "Protein Metabolism" is highly overrepresented (3×) when compared to the genome-based functional profile, which shows that ribosomal proteins are rich in the bacterial cytosol. CONCLUSION: We present here the first study of bacterial diversity of the gut of migrating Atlantic cod using shotgun sequencing and metagenome-assembled genomes (MAGs). The most abundant bacteria belong to the Photobacterium genus (Vibrionaceae family). We also constructed functional profiles of the gut microbiome. These may be used in future studies as a platform for mining of commercially interesting cold-active enzymes.

Differential expression profiling of ΔlitR and ΔrpoQ mutants reveals insight into QS regulation of motility, adhesion and biofilm formation in Aliivibrio salmonicida.

BACKGROUND: The coordination of group behaviors in bacteria is achieved by a cell-cell signaling process called quorum sensing (QS). QS is an intercellular communication system, which synchronously controls expression of a vast range of genes in response to changes in cell density and is mediated by autoinducers that act as extracellular signals. Aliivibrio salmonicida, the causative agent of cold-water vibrosis in marine aquacultures, uses QS to regulate several activities such as motility, biofilm formation, adhesion and rugose colony morphology. However, little is known about either genes or detailed mechanisms involved in the regulation of these phenotypes. RESULTS: Differential expression profiling allowed us to define the genes involved in controlling phenotypes related to QS in A. salmonicida LFI1238. RNA sequencing data revealed that the number of expressed genes in A. salmonicida, ΔlitR and ΔrpoQ mutants were significantly altered due to changes in cell density. These included genes that were distributed among the 21 functional groups, mainly presented in cell envelope, cell processes, extrachromosomal/foreign DNA and transport-binding proteins functional groups. The comparative transcriptome of A. salmonicida wild-type at high cell density relative to low cell density revealed 1013 genes to be either up- or downregulated. Thirty-six downregulated genes were gene clusters encoding biosynthesis of the flagellar and chemotaxis genes. Additionally we identified significant expression for genes involved in acyl homoserine lactone (AHL) synthesis, adhesion and early colonization. The transcriptome profile of ΔrpoQ compared to the wild-type revealed 384 differensially expressed genes (DEGs) that allowed us to assign genes involved in regulating motility, adhesion and colony rugosity. Indicating the importance of RpoQ in controlling several QS related activities. Furthermore, the comparison of the transcriptome profiles of ΔlitR and ΔrpoQ mutants, exposed numerous overlapping DEGs that were essential for motility, exopolysaccharide production via syp operon and genes associated with tad operon. CONCLUSION: Our findings indicate previously unexplained functional roles for LitR and RpoQ in regulation of different phenotypes related to QS. Our transcriptome data provide a better understanding of the regulation cascade of motility, wrinkling colony morphology and biofilm formation and will offer a major source for further research and analysis on this important field.

Effects of Probiotic Supplementation on the Gut Microbiota and Antibiotic Resistome Development in Preterm Infants.

Objectives: In 2014 probiotic supplementation (Lactobacillus acidophilus and Bifidobacterium longum subspecies infantis; InfloranⓇ) was introduced as standard of care to prevent necrotizing enterocolitis (NEC) in extremely preterm infants in Norway. We aimed to evaluate the influence of probiotics and antibiotic therapy on the developing gut microbiota and antibiotic resistome in extremely preterm infants, and to compare with very preterm infants and term infants not given probiotics. Study design: A prospective, observational multicenter study in six tertiary-care neonatal units. We enrolled 76 infants; 31 probiotic-supplemented extremely preterm infants <28 weeks gestation, 35 very preterm infants 28-31 weeks gestation not given probiotics and 10 healthy full-term control infants. Taxonomic composition and collection of antibiotic resistance genes (resistome) in fecal samples, collected at 7 and 28 days and 4 months age, were analyzed using shotgun-metagenome sequencing. Results: Median (IQR) birth weight was 835 (680-945) g and 1,290 (1,150-1,445) g in preterm infants exposed and not exposed to probiotics, respectively. Two extremely preterm infants receiving probiotic developed NEC requiring surgery. At 7 days of age we found higher median relative abundance of Bifidobacterium in probiotic supplemented infants (64.7%) compared to non-supplemented preterm infants (0.0%) and term control infants (43.9%). Lactobacillus was only detected in small amounts in all groups, but the relative abundance increased up to 4 months. Extremely preterm infants receiving probiotics had also much higher antibiotic exposure, still overall microbial diversity and resistome was not different than in more mature infants at 4 weeks and 4 months. Conclusion: Probiotic supplementation may induce colonization resistance and alleviate harmful effects of antibiotics on the gut microbiota and antibiotic resistome. Clinical Trial Registration: Clinicaltrials.gov: NCT02197468. https://clinicaltrials.gov/ct2/show/NCT02197468.

Norwegian e-Infrastructure for Life Sciences (NeLS).

The Norwegian e-Infrastructure for Life Sciences (NeLS) has been developed by ELIXIR Norway to provide its users with a system enabling data storage, sharing, and analysis in a project-oriented fashion. The system is available through easy-to-use web interfaces, including the Galaxy workbench for data analysis and workflow execution. Users confident with a command-line interface and programming may also access it through Secure Shell (SSH) and application programming interfaces (APIs).  NeLS has been in production since 2015, with training and support provided by the help desk of ELIXIR Norway. Through collaboration with NorSeq, the national consortium for high-throughput sequencing, an integrated service is offered so that sequencing data generated in a research project is provided to the involved researchers through NeLS. Sensitive data, such as individual genomic sequencing data, are handled using the TSD (Services for Sensitive Data) platform provided by Sigma2 and the University of Oslo. NeLS integrates national e-infrastructure storage and computing resources, and is also integrated with the SEEK platform in order to store large data files produced by experiments described in SEEK.   In this article, we outline the architecture of NeLS and discuss possible directions for further development.

The alternative sigma factor RpoQ regulates colony morphology, biofilm formation and motility in the fish pathogen Aliivibrio salmonicida.

BACKGROUND: Quorum sensing (QS) is a cell-to cell communication system that bacteria use to synchronize activities as a group. LitR, the master regulator of QS in Aliivibrio salmonicida, was recently shown to regulate activities such as motility, rugosity and biofilm formation in a temperature dependent manner. LitR was also found to be a positive regulator of rpoQ. RpoQ is an alternative sigma factor belonging to the sigma -70 family. Alternative sigma factors direct gene transcription in response to environmental signals. In this work we have studied the role of RpoQ in biofilm formation, colony morphology and motility of A. salmonicida LFI1238. RESULTS: The rpoQ gene in A. salmonicida LFI1238 was deleted using allelic exchange. We found that RpoQ is a strong repressor of rugose colony morphology and biofilm formation, and that it controls motility of the bacteria. We also show that overexpression of rpoQ in a ΔlitR mutant of A. salmonicida disrupts the biofilm produced by the ΔlitR mutant and decreases its motility, whereas rpoQ overexpression in the wild-type completely eliminates the motility. CONCLUSION: The present work demonstrates that the RpoQ sigma factor is a novel regulatory component involved in modulating motility, colony morphology and biofilm formation in the fish pathogen A. salmonicida. The findings also confirm that RpoQ functions downstream of the QS master regulator LitR. However further studies are needed to elucidate how LitR and RpoQ work together in controlling phenotypes related to QS in A. salmonicida.

The MAR databases: development and implementation of databases specific for marine metagenomics.

We introduce the marine databases; MarRef, MarDB and MarCat (https://mmp.sfb.uit.no/databases/), which are publicly available resources that promote marine research and innovation. These data resources, which have been implemented in the Marine Metagenomics Portal (MMP) (https://mmp.sfb.uit.no/), are collections of richly annotated and manually curated contextual (metadata) and sequence databases representing three tiers of accuracy. While MarRef is a database for completely sequenced marine prokaryotic genomes, which represent a marine prokaryote reference genome database, MarDB includes all incomplete sequenced prokaryotic genomes regardless level of completeness. The last database, MarCat, represents a gene (protein) catalog of uncultivable (and cultivable) marine genes and proteins derived from marine metagenomics samples. The first versions of MarRef and MarDB contain 612 and 3726 records, respectively. Each record is built up of 106 metadata fields including attributes for sampling, sequencing, assembly and annotation in addition to the organism and taxonomic information. Currently, MarCat contains 1227 records with 55 metadata fields. Ontologies and controlled vocabularies are used in the contextual databases to enhance consistency. The user-friendly web interface lets the visitors browse, filter and search in the contextual databases and perform BLAST searches against the corresponding sequence databases. All contextual and sequence databases are freely accessible and downloadable from https://s1.sfb.uit.no/public/mar/.

ELIXIR pilot action: Marine metagenomics - towards a domain specific set of sustainable services.

Metagenomics, the study of genetic material recovered directly from environmental samples, has the potential to provide insight into the structure and function of heterogeneous microbial communities.  There has been an increased use of metagenomics to discover and understand the diverse biosynthetic capacities of marine microbes, thereby allowing them to be exploited for industrial, food, and health care products. This ELIXIR pilot action was motivated by the need to establish dedicated data resources and harmonized metagenomics pipelines for the marine domain, in order to enhance the exploration and exploitation of marine genetic resources. In this paper, we summarize some of the results from the ELIXIR pilot action "Marine metagenomics - towards user centric services".

The metagenomic data life-cycle: standards and best practices.

Metagenomics data analyses from independent studies can only be compared if the analysis workflows are described in a harmonized way. In this overview, we have mapped the landscape of data standards available for the description of essential steps in metagenomics: (i) material sampling, (ii) material sequencing, (iii) data analysis, and (iv) data archiving and publishing. Taking examples from marine research, we summarize essential variables used to describe material sampling processes and sequencing procedures in a metagenomics experiment. These aspects of metagenomics dataset generation have been to some extent addressed by the scientific community, but greater awareness and adoption is still needed. We emphasize the lack of standards relating to reporting how metagenomics datasets are analysed and how the metagenomics data analysis outputs should be archived and published. We propose best practice as a foundation for a community standard to enable reproducibility and better sharing of metagenomics datasets, leading ultimately to greater metagenomics data reuse and repurposing.

Pan genome and CRISPR analyses of the bacterial fish pathogen Moritella viscosa.

BACKGROUND: Winter-ulcer Moritella viscosa infections continue to be a significant burden in Atlantic salmon (Salmo salar L.) farming. M. viscosa comprises two main clusters that differ in genetic variation and phenotypes including virulence. Horizontal gene transfer through acquisition and loss of mobile genetic elements (MGEs) is a major driving force of bacterial diversification. To gain insight into genomic traits that could affect sublineage evolution within this bacterium we examined the genome sequences of twelve M. viscosa strains. Matches between M. viscosa clustered, regularly interspaced, short palindromic, repeats and associated cas genes (CRISPR-Cas) were analysed to correlate CRISPR-Cas with adaptive immunity against MGEs. RESULTS: The comparative genomic analysis of M. viscosa isolates from across the North Atlantic region and from different fish species support delineation of M. viscosa into four phylogenetic lineages. The results showed that M. viscosa carries two distinct variants of the CRISPR-Cas subtype I-F systems and that CRISPR features follow the phylogenetic lineages. A subset of the spacer content match prophage and plasmid genes dispersed among the M. viscosa strains. Further analysis revealed that prophage and plasmid-like element distribution were reflected in the content of the CRISPR-spacer profiles. CONCLUSIONS: Our data suggests that CRISPR-Cas mediated interactions with MGEs impact genome properties among M. viscosa, and that patterns in spacer and MGE distributions are linked to strain relationships.

Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

BACKGROUND: The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. RESULTS: MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

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.

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.

Biological surface-active compounds from marine bacteria.

Surface-active compounds (SACs) are widely used in different industries as well as in many daily consumption products. However, with the increasing concern for their environmental acceptability, attention has turned towards biological SACs which are biodegradable, less toxic and more environmentally friendly. In this work, 176 marine hydrocarbon-degrading bacterial isolates from petroleum-contaminated sites along the Norwegian coastline were isolated and screened for their capacity to produce biological SACs. Among them, 18 isolates were capable of reducing the surface tension of the culture medium by at least 20 mN m(-1) and/or capable of maintaining more than 40% of the emulsion volume after 24 h when growing on glucose or kerosene as carbon and energy source. These isolates were members of the genera Pseudomonas, Pseudoalteromonas, Rhodococcus, Catenovulum, Cobetia, Glaciecola, Serratia, Marinomonas and Psychromonas. Two isolates, Rhodococcus sp. LF-13 and Rhodococcus sp. LF-22, reduced surface tension of culture medium by more than 40 mN m(-1) when growing on kerosene, n-hexadecane or rapeseed oil. The biosurfactants were produced by resting cells of the two Rhodococcus strains suggesting the biosynthesis of the biosurfactants was not necessarily associated with their growth on hydrocarbons.