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See detailDiscovering novel enzymes y functional screening of plurigenomic libraries from alga-associated Flavobacteriia and Gammaproteobacteria
Martin, Marjolaine ULg; Vandermies, Marie ULg; Joyeux, Coline et al

in Microbiological Research (2016), 186

Alga-associated microorganisms, in the context of their numerous interactions with the host and thecomplexity of the marine environment, are known to produce diverse hydrolytic enzymes with ... [more ▼]

Alga-associated microorganisms, in the context of their numerous interactions with the host and thecomplexity of the marine environment, are known to produce diverse hydrolytic enzymes with originalbiochemistry. We recently isolated several macroalgal-polysaccharide-degrading bacteria from the sur-face of the brown alga Ascophyllum nodosum. These active isolates belong to two classes: the Flavobacteriiaand the Gammaproteobacteria. In the present study, we constructed two “plurigenomic” (with multi-ple bacterial genomes) libraries with the 5 most interesting isolates (regarding their phylogeny andtheir enzymatic activities) of each class (Fv and Gm libraries). Both libraries were screened for diversehydrolytic activities. Five activities, out of the 48 previously identified in the natural polysaccharolyticisolates, were recovered by functional screening: a xylanase (GmXyl7), a beta-glucosidase (GmBg1), anesterase (GmEst7) and two iota-carrageenases (Fvi2.5 and Gmi1.3). We discuss here the potential role ofthe used host-cell, the average DNA insert-sizes and the used restriction enzymes on the divergent screen-ing yields obtained for both libraries and get deeper inside the “great screen anomaly”. Interestingly, thediscovered esterase probably stands for a novel family of homoserine o-acetyltransferase-like-esterases,while the two iota-carrageenases represent new members of the poorly known GH82 family (contain-ing only 19 proteins since its description in 2000). These original results demonstrate the efficiencyof our uncommon “plurigenomic” library approach and the underexplored potential of alga-associatedcultivable microbiota for the identification of novel and algal-specific enzymes. [less ▲]

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See detailFunction-based Analyses of Bacterial Symbionts Associated with the Brown Alga Ascophyllum nodosum and Identification of Novel Bacterial Hydrolytic Enzyme Genes
Martin, Marjolaine ULg

Doctoral thesis (2016)

Marine macroalgae are highly colonized by microorganisms, with which they maintain a close relationship characterized by both beneficial and detrimental interactions. Alga-associated bacteria have notably ... [more ▼]

Marine macroalgae are highly colonized by microorganisms, with which they maintain a close relationship characterized by both beneficial and detrimental interactions. Alga-associated bacteria have notably developed a range of enzymes enabling them to colonize the host surface and to use algal biomass as a carbon source. The hydrolytic potential of these bacteria, however, has been investigated almost solely at individual scale. Studies have shown the ability of some seaweed-associated bacterial strains to hydrolyze lipids, algal-cell-wall polysaccharides, and other sugars. In this work we aimed to investigate the hydrolytic potential of the bacterial microbiota associated with the brown alga Ascophyllum nodosum. For this we employed two complementary function-based approaches: functional metagenomics applied to this microbiota and functional analysis of the cultivable fraction thereof. By functional metagenomics, we identified numerous esterase genes, a beta-glucosidase gene, and an endocellulase gene. The cellulase was purified and biochemically characterized, showing interesting biotechnological features such as halotolerance and activity at low temperature. Furthermore, we assigned tentative origins to the identified genes, thus getting a glimpse of the bacterial taxa associated with the studied alga. Secondly, we investigated the cultivable surface microbiota associated with three A. nodosum samples. More than 300 bacteria were isolated, assigned to a bacterial taxon and screened for algal-polysaccharide-degrading enzymes (agarase, iota-carrageenase, kappa-carrageenase, and alginate lyases). This allowed the identification of several polysaccharolytic isolates, some of them likely to be new strains or novel species, belonging to two classes: the Flavobacteriia and the Gammaproteobacteria. Subsequently, we constructed and screened two plurigenomic libraries, each produced with the genomes of five representative isolates of each class, and identified several functional genes. With this work we highlight the presence of A. nodosum-associated bacterial taxa likely to entertain a privileged relation with seaweeds and having developed a range of hydrolytic activities assumed to enable them to associate with algae. We also provide information (taxa, abundances, genomic potential) on macroalgal-polysaccharide-degrading bacteria, in which interest has grown over the last ten years. [less ▲]

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See detailThe cultivable surface microbiota of the brown alga Ascophyllum nodosum is enriched in macroalgal-polysaccharide-degrading bacteria
Martin, Marjolaine ULg; Barbeyron, Tristan; Martin, Renée ULg et al

in Frontiers in Microbiology (2015), 6(december),

Bacteria degrading algal polysaccharides are key players in the global carbon cycle and in algal biomass recycling. Yet the water column, which has been studied largely by metagenomic approaches, is poor ... [more ▼]

Bacteria degrading algal polysaccharides are key players in the global carbon cycle and in algal biomass recycling. Yet the water column, which has been studied largely by metagenomic approaches, is poor in such bacteria and their algal-polysaccharide-degrading enzymes. Even more surprisingly, the few published studies on seaweed-associated microbiomes have revealed low abundances of such bacteria and their specific enzymes. However, as macroalgal cell-wall polysaccharides do not accumulate in nature, these bacteria and their unique polysaccharidases must not be that uncommon. We, therefore, looked at the polysaccharide-degrading activity of the cultivable bacterial subpopulation associated with Ascophyllum nodosum. From A. nodosum triplicates, 324 bacteria were isolated and taxonomically identified. Out of these isolates, 78 (∼25%) were found to act on at least one tested algal polysaccharide (agar, ι- or κ-carrageenan, or alginate). The isolates “active” on algal-polysaccharides belong to 11 genera: Cellulophaga, Maribacter, Algibacter, and Zobellia in the class Flavobacteriia (41) and Pseudoalteromonas, Vibrio, Cobetia, Shewanella, Colwellia, Marinomonas, and Paraglaceciola in the class Gammaproteobacteria (37). A major part represents likely novel species. Different proportions of bacterial phyla and classes were observed between the isolated cultivable subpopulation and the total microbial community previously identified on other brown algae. Here, Bacteroidetes and Gammaproteobacteria were found to be the most abundant and some phyla (as Planctomycetes and Cyanobacteria) frequently encountered on brown algae weren’t identified. At a lower taxonomic level, twelve genera, well-known to be associated with algae (with the exception for Colwellia), were consistently found on all three A. nosodum samples. Even more interesting, 9 of the 11 above mentioned genera containing polysaccharolytic isolates were predominant in this common core. The cultivable fraction of the bacterial community associated with A. nodosum is, thus, significantly enriched in macroalgal-polysaccharide-degrading bacteria and these bacteria seem important for the seaweed holobiont even though they are under-represented in alga-associated microbiome studies. [less ▲]

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See detailBiotechnological potential of the microflora associated with the brown alga Ascophyllum nodosum
Martin, Marjolaine ULg; Martin, Renée ULg; Barbeyron, Tristan et al

in Journal of Microbial and Biochemical Technology (2015, August 18), 7(4), 179

Bacteria associated with algae are underexplored despite their huge biodiversity and the fact that they differ markedly from those living freely in seawater. These bacterial communities are known to ... [more ▼]

Bacteria associated with algae are underexplored despite their huge biodiversity and the fact that they differ markedly from those living freely in seawater. These bacterial communities are known to represent great potential for the production of diverse bioactive compounds, such as specific glycoside hydrolases, as they interact in multiple complex ways with their host. Furthermore, enzymes from marine bacteria have original properties, like cold-adapted, halotolerant and highly stable, which are constantly searched out by bio-industries. The aim of our study was to identify bacteria, associated with the brown alga Ascophyllum nodosum, showing diverse polysaccharolytic activities. To isolate cultivable microorganisms, algal thalli of Ascophyllum nodosum were swabbed with sterile cotton tips and marine agar plates were inoculated. Three-hundred isolated bacteria were screened for agarase, kappa- and iota-carrageenase, and sulfatase activities on specific marine media. Thirty-two bacteria with polysaccharolytic activities were isolated and a part of their 16S rDNA (8F-1492R) were amplified and sequenced. Twenty-seven were classified as Flavobacteriia and five as Gammaproteobacteria. Putative new strains and species of Zobellia, Maribacter, Cellulophaga, Shewanella, Glaciecola, Pseudoalteromonas and Colwellia were identified by phylogenetic analysis. All those genera are well-known to colonize algal surface but only some of them are famous to degrade algal polysaccharides (Zobellia, Maribacter, Cellulophaga, and Pseudoalteromonas). However, all those novel bacterial strains/species showed multiple and diverse enzymatic activities (agarase, iota-and kappa-carrageenase, cellulase, beta-glucosidase, sulfatase and/or amylase activities). Genomics libraries with their DNA were constructed in Escherichia coli and Bacillus subtilis and are screened to identify the genes coding for the observed enzymatic activities. Those novel glycoside hydrolases from unknown marine bacteria should have original and innovative properties with great biotechnological potential. [less ▲]

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See detailStudying the great potential of cultivable bacteria associated with the brown alga Ascophyllum nodosum
Martin, Marjolaine ULg; Martin, Renée ULg; Barbeyron, Tristan et al

Poster (2015, June 09)

Bacteria associated with algae differ markedly from those living freely in seawater and represent great potential for the production of diverse bioactive compounds as they interact in multiple complex ... [more ▼]

Bacteria associated with algae differ markedly from those living freely in seawater and represent great potential for the production of diverse bioactive compounds as they interact in multiple complex ways with their host. Here we identified new bacterial species, and their polysaccharolytic activities, associated with the brown alga Ascophyllum nodosum. To isolate cultivable microorganisms, algal thalli of Ascophyllum nodosum were swabbed with sterile cotton tips and marine agar plates were inoculated. Three-hundred isolated bacteria were screened for agarase, kappa- or iota-carrageenase activities on specific marine media. Thirty-two bacteria with polysaccharolytic activities were isolated and a part of their 16S rDNA (8F-1492R) were amplified and sequenced. Twenty-seven were classified as Flavobacteriia and five as Gammaproteobacteria. Putative new strains and species of Zobellia, Maribacter, Cellulophaga, Shewanella, Glaciecola, Pseudoalteromonas and Colwellia were identified by phylogenetic analysis. Genomics libraries with their DNA were constructed in Escherichia coli and Bacillus subtilis and are currently screened for diverse enzymatic activities (agarases, iota-and kappa-carrageenases, cellulases, beta-glucosidases, sulfatases and amylases). In an era where high throughput sequencing is mostly used to study bacterial communities, cultivation methods are underestimated. Here, we revealed that only ten percent of the cultivable bacteria on this brown alga could degrade algal polysaccharides, which lead to asking us; who and what are the 90 other percents doing there? Furthermore, by this cultivation method we could also identify putative new bacterial strains/species, which are screened for polysaccharidases. Novel glycoside hydrolases from unknown marine bacteria represent great biotechnological potential as they should have original industrial properties. [less ▲]

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See detailIdentification and Characterization of a Halotolerant, Cold-Active Marine Endo-β-1,4-Glucanase by Using Functional Metagenomics of Seaweed-Associated Microbiota
Martin, Marjolaine ULg; Biver, Sophie ULg; Steels, Sébastien ULg et al

in Applied and Environmental Microbiology (2014), 80(16), 4958-4967

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two ... [more ▼]

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-β-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions. [less ▲]

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See detailMicroorganisms living on macroalgae: diversity, interactions, and biotechnological applications
Martin, Marjolaine ULg; Portetelle, Daniel ULg; Michel, Gurvan et al

in Applied Microbiology & Biotechnology (2014)

Marine microorganisms play key roles in every marine ecological process, hence the growing interest in studying their populations and functions. Microbial communities on algae remain underexplored ... [more ▼]

Marine microorganisms play key roles in every marine ecological process, hence the growing interest in studying their populations and functions. Microbial communities on algae remain underexplored, however, despite their huge biodiversity and the fact that they differ markedly from those living freely in seawater. The study of this microbiota and of its relationships with algal hosts should provide crucial information for ecological investigations on algae and aquatic ecosystems. Furthermore, because these microorganisms interact with algae in multiple, complex ways, they constitute an interesting source of novel bioactive compounds with biotechnological potential, such as dehalogenases, antimicrobials, and alga-specific polysaccharidases (e.g., agarases, carrageenases, and alginate lyases). Here, to demonstrate the huge potential of alga-associated organisms and their metabolites in developing future biotechnological applications, we first describe the immense diversity and density of these microbial biofilms. We further describe their complex interactions with algae, leading to the production of specific bioactive compounds and hydrolytic enzymes of biotechnological interest. We end with a glance at their potential use in medical and industrial applications. [less ▲]

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See detailBiomass hydrolyzing enzymes identified by functional screening of a metagenomic library from algal biofilms.
Martin, Marjolaine ULg; Biver, Sophie ULg; Barbeyron, Tristan et al

Poster (2013, June)

Biomass hydrolyzing enzymes are increasingly searched for the production of biofuels and renewable chemical compounds using biomass. Microorganisms living on algaes are an interesting reservoir of biomass ... [more ▼]

Biomass hydrolyzing enzymes are increasingly searched for the production of biofuels and renewable chemical compounds using biomass. Microorganisms living on algaes are an interesting reservoir of biomass hydrolyzing enzymes, as they are in constant interaction with algal biomass. Therefore, we are interested in looking for some of those enzymes synthesized by the microflora living on the surface of the brown algae Ascophyllum nodosum. Algae samples were collected in the winter 2012 and a microbial DNA extraction method was developed. The whole extracted microbial genomes of the microorganisms living on the algae were restricted, inserted in a cloning vector and ligated products were used for transformation of cultivable Escherichia coli host cells. This metagenomic library was then screened for diverse enzymatic activities (lipolytic enzymes, cellulases, beta-glucosidases, alpha-amylases, arabinanases, xylanases and proteases) on agar plates with specific substrates. Five putative lipolytic enzymes, one cellulase and one beta-glucosidase were identified. Sequence analysis revealed low (<50%) sequence identities with known enzymes sequences, meaning new enzymes from unknown genomes have been discovered. To our knowledge this is the first functional screening that was realized with a metagenomic library from algal biofilms and this is the first cellulase identified by marine metagenomics. A second library has been constructed from algae sample from summer 2012 and is currently being screened. New enzymatic tests are being developed for the identification of enzymes degrading specific algal polysaccharides like agarases, carrageenases, alginate lyases, laminarinases,… Those very specific enzymes aren’t well known yet, and our metagenomic approach will probably help us to identify new families and structures of those algal biomass hydrolyzing enzymes. [less ▲]

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See detailMicroorganisms living on algae : An interesting reservoir of enzymes hydrolyzing algal biomass
Martin, Marjolaine ULg; Biver, Sophie ULg; Barbeyron, Tristan et al

Poster (2013, April 18)

Algal polysaccharides are increasingly used in food industry for their gelling properties and in pharmacology for their therapeutic properties. Furthermore, increasingly interest is taken on algae for ... [more ▼]

Algal polysaccharides are increasingly used in food industry for their gelling properties and in pharmacology for their therapeutic properties. Furthermore, increasingly interest is taken on algae for their use in the production of biofuels and bioenergies. To purify algal polysaccharides and degrade algal biomass, specific microbial enzymes are needed. Microorganisms living on algae are an interesting source of those enzymes, as they are in constant interaction with algal biomass. The aim of our study is to identify new enzymes degrading algae, produced by microorganisms living on the surface of algae. Therefore we developed a method for microbial DNA extraction from biofilms living on brown algae (Ascophyllum nodosum). Microbial DNA was extracted, restricted and inserted in cultivable host cells of Echerichia coli, for the construction of our metagenomic DNA library. This metagenomic library was first screened, on solid media with specific substrates, for enzymes generally used in the degradation of biomass (lipases, cellulases, proteases, beta-glucosidases, alpha-amylases, arabinanases and xylanases). Five lipolytic enzymes, one beta-glucosidase and one cellulase were identified. Those enzymes show very low percentages of sequences identities with known enzymes, meaning we identified new and unknown enzymes. Those enzymes and their activity are being characterized. Preliminary tests show interesting results, like a cellulase active at low temperature. Screening tests are now being developed to identify enzymes hydrolyzing algal polysaccharides like agarases, carrageenases, alginate lyases, laminarinases,… Those enzymes aren’t well known yet and we hope to identify new enzymes (families) with our rich DNA library by our approach. [less ▲]

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See detailFunctional screening of a metagenomic library from algal biofilms
Martin, Marjolaine ULg

Conference (2013, February 08)

Macroalgae, and particularly their lignin-free polysaccharides, are increasingly used for their gelling and therapeutic properties and for the production of biofuels and renewable chemical compounds. To ... [more ▼]

Macroalgae, and particularly their lignin-free polysaccharides, are increasingly used for their gelling and therapeutic properties and for the production of biofuels and renewable chemical compounds. To extract, hydrolyze and purify this biomass, algae hydrolyzing enzymes are needed. Our work aims to identify and characterize algal biomass hydrolyzing enzymes expressed by microorganisms living on the surface of algae, by functional metagenomics. Therefore, a microbial DNA extraction method was developed to isolate the gDNA from the microorganisms of the brown algae Ascophyllum nodosum and a metagenomic library was constructed in Escherichia coli. The library was screened for diverse enzymatic activities (esterases, xylanases, cellulases, α-amylases, arabinanases, caseinases and β-glucosidases) on agar plates with specific enzymes substrates. Several new microbial enzymes (esterases, β-glucosidases, α-amylases and cellulases) were identified revealing the wealth of our library. Furthermore, those enzymes had less than 50% sequence identity with known protein sequences; meaning that our approach allows to identify new microbial enzymes expressed by uncultured microorganisms. Plate tests for medium-throughput screening of specific enzymes hydrolyzing algal polysaccharides (agarases, carrageenases and alginate lyases) are currently being developed. Our approach will probably allow us to identify new families of those ill-known enzymes, with particular enzymatic activities. [less ▲]

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See detailFunctional screening of a metagenomic library from algal biofilms
Martin, Marjolaine ULg; Barbeyron, Tristan; Michel, Gurvan et al

in Smagghe, Guy; Boeckx, Pascal; Bossier, Peter (Eds.) et al Communications in Agricultural and Applied Biological Sciences (2013, February 08)

Macroalgae, and particularly their lignin-free polysaccharides, are increasingly used for their gelling and therapeutic properties and for the production of biofuels and renewable chemical compounds. To ... [more ▼]

Macroalgae, and particularly their lignin-free polysaccharides, are increasingly used for their gelling and therapeutic properties and for the production of biofuels and renewable chemical compounds. To extract, hydrolyze and purify this biomass, algae hydrolyzing enzymes are needed. Our work aims to identify and characterize algal biomass hydrolyzing enzymes expressed by microorganisms living on the surface of algae, by functional metagenomics. Therefore, a microbial DNA extraction method was developed to isolate the gDNA from the microorganisms of the brown algae Ascophyllum nodosum and a metagenomic library was constructed in Escherichia coli. The library was screened for diverse enzymatic activities (esterases, xylanases, cellulases, α-amylases, arabinanases, caseinases and β-glucosidases) on agar plates with specific enzymes substrates. Several new microbial enzymes (esterases, β-glucosidases, α-amylases and cellulases) were identified revealing the wealth of our library. Furthermore, those enzymes had less than 50% sequence identity with known protein sequences; meaning that our approach allows to identify new microbial enzymes expressed by uncultured microorganisms. Plate tests for medium-throughput screening of specific enzymes hydrolyzing algal polysaccharides (agarases, carrageenases and alginate lyases) are currently being developed. Our approach will probably allow us to identify new families of those ill-known enzymes, with particular enzymatic activities. [less ▲]

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See detailIdentification of new microbial enzymes from forest and marine ecosystems by functional metagenomics
Martin, Marjolaine ULg; Biver, Sophie ULg; Barbeyron, Tristan et al

Poster (2012, August 21)

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See detailImplémentation d'une méthode de détection du virus de la diarrhée virale bovine au sein de la fondation de la promotion des productions andines Proinpa (Bolivie)
Martin, Marjolaine ULg

Master's dissertation (2010)

An animal infected by the Bovine Viral Diarrhea Virus will have diarrheas, which can lead to milk production reduction, reproduction problems (and particularly abortion problems) and general weakness ... [more ▼]

An animal infected by the Bovine Viral Diarrhea Virus will have diarrheas, which can lead to milk production reduction, reproduction problems (and particularly abortion problems) and general weakness. This all leads to an economic loss. This is why the virus detection and the carriers, or Persistently Infected (PI) animals, eradication are important. If the virus presence was previously suspected in Bolivia, since 2009 it is certain that there is BVDV infection and the prevalence is high in comparison with others areas. Furthermore, this virus can also infect « New Worlds camelids » which are the alpacas, lamas, vicunas and guanacos species living in South-America. The aim of this study was to implement a BVDV detection method for the Foundation of Andeans Productions Promotion PROINPA in Bolivia. This objective was first met by a prevalence evaluation of the virus using ELISA anti-antibodies analysis. Then, a screening of the tested herds containing animals with a positive serology was done by real-time PCR and finally the PI animals were detected by individual real-time PCR analysis or by two ELISA anti-antigens analysis’s in a 3 week's interval. There was no problem from an infrastructure viewpoint; the laboratories have all the necessary material and equipment and the labor was qualified for both analyses. But the logistic part was more difficult, because the molecular kit has to be preserved at minus twenty degrees. And this kit took 10 days to arrive from Belgium. Because none of the molecular analysis’s succeeded, we concluded that the kit had been damaged during the travel and had been degraded. The kit conservation conditions seemed to be a problem and that is why the elaboration of a new lyophilized kit was tried in Belgium. The lyophilisation of the BVDV sequences, the IPC and EPC was easy but wasn’t possible for the Master MIX (containing the Taq Polymerase (Taq)) and the Reverse Transcriptase (RT). The « Illustra Ready-To-GoTM RT-PCR Beads », from GE Healthcare, are beads made of Taq and RT that can be stored at room temperature. The combination of a lyophilized part of the LSI kit (BVDV sequences, EPC and IPC) with those beads will lead to a molecular BVDV detection kit that can be stored at room temperature. However, this combination doesn’t seem to work. This can be due to a lot of different things like the incompatibility between the primers size and design and the Taq, the temperature activities of the Taq and the RT, the salt concentration, etc. In conclusion, different experiments are required to finalize a qPCR kit resistant to long trip conditions. [less ▲]

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