Discovery pipelines for marine resources: an ocean of opportunity for biotechnology?

Marine microbial diversity offers enormous potential for discovery of compounds of crucial importance in healthcare, food security and bioindustry. However, access to it has been hampered by the difficulty of accessing and growing the organisms for study. The discovery and exploitation of marine bioproducts for research and commercial development requires state-of-the-art technologies and innovative approaches. Technologies and approaches are advancing rapidly and keeping pace is expensive and time consuming. There is a pressing need for clear guidance that will allow researchers to operate in a way that enables the optimal return on their efforts whilst being fully compliant with the current regulatory framework. One major initiative launched to achieve this, has been the advent of European Research Infrastructures. Research Infrastructures (RI) and associated centres of excellence currently build harmonized multidisciplinary workflows that support academic and private sector users. The European Marine Biological Research Infrastructure Cluster (EMBRIC) has brought together six such RIs in a European project to promote the blue bio-economy. The overarching objective is to develop coherent chains of high-quality services for access to biological, analytical and data resources providing improvements in the throughput and efficiency of workflows for discovery of novel marine products. In order to test the efficiency of this prototype pipeline for discovery, 248 rarely-grown organisms were isolated and analysed, some extracts demonstrated interesting biochemical properties and are currently undergoing further analysis. EMBRIC has established an overarching and operational structure to facilitate the integration of the multidisciplinary value chains of services to access such resources whilst enabling critical mass to focus on problem resolution.

Cell wall is required for fixation of the embryonic axis in Fucus zygotes.

Establishment of a primary developmental axis generally is thought to involve rearrangements in the plasma membrane or cytoplasm of the egg. In this report the additional requirement for cell wall in polarization of Fucus zygotes was investigated. Protoplasts of fertilized eggs were tested for their ability to establish an axis in accordance with an orienting vector of unilateral light. The results demonstrate that cell wall is not required for axis formation. However, the orientation of the axis remains labile until new cell wall is synthesized. The presence of a cell wall is an absolute requirement for axis fixation.

Sulfated Oligosaccharides Mediate the Interaction between a Marine Red Alga and Its Green Algal Pathogenic Endophyte.

The endophytic green alga Acrochaete operculata completely colonizes the sporophytes of the red alga Chondrus crispus; however, it does not penetrate beyond the outer cell layers of the gametophytes. Given that the life cycle phases of C. crispus differ in the sulfation pattern of their extracellular matrix carrageenans, we investigated whether carra-geenan fragments could modulate parasite virulence. lambda-Carrageenan oligosaccharides induced release of H(2)O(2), stimulated protein synthesis, increased carrageenolytic activity, and induced specific polypeptides in the pathogen, resulting in a marked increase in pathogenicity. In contrast, kappa-carrageenan oligosaccharides did not induce a marked release of H(2)O(2) from A. operculata but hindered amino acid uptake and enhanced their recognition by the host, resulting in a reduced virulence. Moreover, C. crispus life cycle phases were shown to behave differently in their response to challenge with cell-free extracts of A. operculata. Gametophytes exhibited a large burst of H(2)O(2), whereas only low levels were released from the sporophytes.

Oligosaccharide recognition signals and defence reactions in marine plant-microbe interactions.

Recent findings on the involvement of oligosaccharide signals in pathogen recognition and defence reactions in marine algae shine a new light on the ecology of their interactions with associated microorganisms. Since the marine environment encompasses lineages that have diverged a long time ago from the terrestrial phyla, these results suggest that cell-cell recognition pathways typical of terrestrial plants appeared very early in the evolution of eukaryotes. Production of oligosaccharides from marine algae using microbial recombinant polysaccharidases is also of industrial interest as plants can be protected from infections by preincubation in the presence of appropriate signals that mimic the attacks by pathogens.

The kappa-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of Clan-B glycoside hydrolases.

BACKGROUND: kappa-carrageenans are gel-forming, sulfated 1,3-alpha-1,4-beta-galactans from the cell walls of marine red algae. The kappa-carrageenase from the marine, gram-negative bacterium Pseudoalteromonas carrageenovora degrades kappa-carrageenan both in solution and in solid state by an endoprocessive mechanism. This beta-galactanase belongs to the clan-B of glycoside hydrolases. RESULTS: The structure of P. carrageenovora kappa-carrageenase has been solved to 1.54 A resolution by the multiwavelength anomalous diffraction (MAD) method, using a seleno-methionine-substituted form of the enzyme. The enzyme folds into a curved beta sandwich, with a tunnel-like active site cavity. Another remarkable characteristic is the presence of an arginine residue at subsite -1. CONCLUSIONS: The crystal structure of P. carrageenovora kappa-carrageenase is the first three-dimensional structure of a carrageenase. Its tunnel-shaped active site, the first to be reported for enzymes other than cellulases, suggests that such tunnels are associated with the degradation of solid polysaccharides. Clan-B glycoside hydrolases fall into two subgroups, one with catalytic machinery held by an ancestral beta bulge, and the other in which it is held by a regular beta strand. At subsite -1, all of these hydrolases exhibit an aromatic amino acid that interacts with the hexopyranose ring of the monosaccharide undergoing catalysis. In addition, in kappa-carrageenases, an arginine residue recognizes the sulfate-ester substituents of the beta-linked kappa-carrageenan monomers. It also appears that, in addition to the nucleophile and acid/base catalysts, two other amino acids are involved with the catalytic cycle, accelerating the deglycosylation step.

mRNA expression in mitochondria of the red alga chondrus crispus requires a unique RNA-processing mechanism, internal cleavage of upstream tRNAs at pyrimidine 48.

The hypothesis that tRNAs are involved in the maturation of the large primary transcripts of Chondrus crispus mitochondrial DNA was addressed by primer extension mapping of the transcript 5' ends of the ten genes that are preceded by tRNA genes in C. crispus mitochondrial genome. Among the 12 tRNAs that were candidates as maturation signals, eight, namely tRNAArg, tRNALys, tRNAAsp, tRNAGln, tRNATrp, tRNAIle, tRNAPhe and tRNAGly, were cleaved internally upon maturation of C. crispus mitochondrial primary transcripts, all of them at the same base, invariant pyrimidine 48. Only four tRNAs departed from this pattern: tRNALeu and tRNACys, which are not maturation signals, tRNAMet, which appears to be excised as a whole from the orf94 primary trancript and tRNAAla, which is cleaved internally at positions other than Y48. Sequence comparisons between the cleaved and the uncleaved tRNAs suggest that their core tertiary structure is involved with their recognition and cleavage. However, the precursor transcripts are also processed at the 5' and 3' ends of the tRNAs to yield tRNA molecules that are stable and functional in translation. This indicates that two different RNA processing mechanisms coexist in C. crispus mitochondria, one required for the production of functional tRNAs and the other for the processing of mRNAs.

Transcription initiation and RNA processing in the mitochondria of the red alga Chondrus crispus: convergence in the evolution of transcription mechanisms in mitochondria.

The mitochondrial DNA (mt DNA) of the red alga Chondrus crispus is shown to be transcribed into two large RNA molecules. These primary transcripts are cleaved once, at the level of a tRNA, then the resulting products are processed via multiple maturation events into either mono- or poly-cistronic RNAs. Transcripts were detected for all genes and open reading frames, except for rps11 and orf172. For both transcription units the initiation of transcription was mapped by in vitro RNA capping and primer extension experiments within inverse repeated sequences at the north pole of the molecule. Consistent with primer extension mapping, putative promoter motifs sharing significant similarities with both chicken and Xenopus mitochondrial promoters were found in the C. crispus mitochondrial genome. Altogether C. crispus mitochondrial DNA appears to be transcribed as animal mtDNA is, suggesting that transcription mechanisms in mitochondria are dependent on the overall organization of the mitochondrial genome irrespective of the eukaryotic phylogeny.

Witnessing the evolution of transcription in mitochondria: the mitochondrial genome of the primitive brown alga Pylaiella littoralis (L.) Kjellm. Encodes a T7-like RNA polymerase.

A region of the mitochondrial genome of the primitive brown alga Pylaiella littoralis containing a plasmid-like insert which contains a transcribed T7-phage-type RNA polymerase gene is described. This is a first report of a phage-type RNA polymerase gene integrated in a mitochondrial genome. As the mitochondrial genome of this alga also contains sigma-70 proteobacterial promoter regions, i.e. traces of the ancestral alpha2betabeta'sigma-70 proteobacterial RNA polymerase, this genome witnesses two types of RNA polymerases. As such the mitochondrial genome of P. littoralis represents a unique stage in the evolution of transcription in mitochondria, which contrasts with that of the primitive protist Reclinomonas americana, which still retains the ancestral alpha2betabeta'sigma-70 proteobacterial RNA polymerase genes, and with animals, land plants and fungi, which use phage-type polymerases.

Complete sequence of the mitochondrial DNA of the rhodophyte Chondrus crispus (Gigartinales). Gene content and genome organization.

The complete nucleotide sequence of the circular mitochondrial (mt) DNA from the red alga Chondrus crispus was determined (25,836 nucleotides, A+T content 72.1%). Fifty one genes were identified. They include genes encoding three subunits of the cytochrome oxidase (cox1 to 3), apocytochrome b (cob), seven subunits of the NADH dehydrogenase complex (nad1 to 6, nad4L), two ATPase subunits (atp6 and atp9), three ribosomal RNAs (rrn5, srn and lrn), 23 tRNAs and four ribosomal proteins (rps3, rps11, rps12 and rpl16). Two subunits of the succinate dehydrogenase complex (sdhB and sdhC), usually found on nuclear genomes, are also located on the mtDNA of C. crispus. One group IIb intron is inserted in the tRNAIle gene. Six potentially functional open reading frames were identified, four of them having counterparts among green plant mtDNAs. The use of a modified genetic code and the absence of RNA editing, previously reported for the cox3 gene, appears as a general characteristic of this molecule. Mitochondrial genes are encoded on both DNA strands, in two opposite major transcriptional directions, suggesting the existence of two main transcriptional units. Two long and stable stem-loops were identified in intergenic regions, which are believed to be involved with transcription and replication. The main structural features of this genome are compared with the overall organization of mtDNAs and are discussed in view of the evolution of mitochondria.

The mitochondrial LSU rDNA of the brown alga Pylaiella littoralis reveals alpha-proteobacterial features and is split by four group IIB introns with an atypical phylogeny.

The mitochondrial DNA region coding for the large ribosomal RNA subunit from the brown alga Pylaiella littoralis (L.) Kjellm was sequenced. The LSU rRNA was folded into a secondary structure and aligned with homologous, mitochondrial and eubacterial sequences. Taking into account the primary and secondary structure levels, the mitochondrial LSU rRNA of P. littoralis shares more structural features with alpha-proteobacterial genes than do those of the green alga Prototheca wickerhamii and land plants. In phylogenetic trees, branches leading to brown algae, red algae, the protozoan Acanthamoeba castellanii and land plants, respectively, emerge approximately at the same time, as they do in nuclear-gene based phylogenies. This suggests that there is only one origin for the mitochondrial rRNA genes found in these lineages. The LSU rDNA is split by four group IIB introns. The first two introns each contain one open reading frame which encodes a reverse transcriptase-like protein. Comparison of their amino acid sequences with those of other reverse transcriptase-like genes contained in group II introns shows that these genes are more closely related to plastid and cyanobacterial homologous genes than to any known mitochondrial intronic reverse transcriptase.

The gene encoding the kappa-carrageenase of Alteromonas carrageenovora is related to beta-1,3-1,4-glucanases.

The nucleotide (nt) and deduced amino acid (aa) sequences are reported for the structural gene cgkA encoding kappa-carrageenase (M(r) 44,412) from the marine bacterium Alteromonas carrageenovora (ATCC 43555), a hydrolase involved in the degradation of kappa-carrageenan. The cgkA gene codes for a protein of 397 aa, with a signal peptide of 25 aa. The enzyme is a new member of family 16 of glycosyl hydrolases, which comprises beta-1,3-1,4-glucanases from various sources and the beta-agarase of Streptomyces coelicolor. It is proposed that residue Glu163 in the kappa-carrageenase from A. carrageenovora and Glu155 in the beta-agarase from S. coelicolor are important for catalysis.

The mitochondrial Pylaiella littoralis nad11 gene contains only the N-terminal FeS-binding domain.

We describe a nad11 gene located on the mitochondrial genome of the brown alga Pylaiella littoralis. This gene is cotranscribed with other neighbouring nad genes. It encodes the first domain only of the Nad11 polypeptide, i.e. a 23-kDa, FeS-binding domain instead of the usual 75/80-kDa protein found in the mitochondrial or alpha-proteobacterial complex I enzymes. The second domain of the protein, of unknown function, seems to be entirely missing in this alga. Cyanobacteria, beta-proteobacteria and actinomycetes also feature small homologous genes, known as hoxU, and it has been suggested that these could function in complex I of cyanobacteria. These observations indicate that complex I can probably function with the first domain only of the 75-kDa protein. P. littoralis represents the first such example within the alpha-proteobacterial/mitochondrial lineage.

New natural polysaccharides with potent antithrombic activity: fucans from brown algae.

Fucans extracted from brown algae exhibit anticoagulant properties, as demonstrated by coagulation assays and kinetic study of the inactivation of thrombin in the presence of antithrombin III. These derivatives exert a direct antithrombic activity by an antithrombin III-independent pathway. Besides, their anticoagulant activity is not mediated by a potency to inhibit factor Xa. Relatively low-molecular-mass fractions (50,000 Daltons) prepared by chromatographic fractionation of the crude fucans also exhibit anticoagulant activity. They may prove useful as anticoagulant drugs.

NMR spectroscopic investigation of agarose oligomers produced by an alpha-agarase.

The 13C NMR signals of various even and odd agarose oligosaccharides with either D-galactose or 3,6-anhydro-alpha-L-galactose at the reducing end have been assigned. The chemical shifts in water of the agaro- and the neoagaro-oligosaccharides are compared and the influence of dimethyl sulfoxide on the chemical structure of the agaro-oligosaccharides is reported. The 3,6-anhydro-L-galactose residue at the reducing end of agaro-oligosaccharides is in the hydrated form.

HPLC analysis of saturated or unsaturated oligoguluronates and oligomannuronates. Application to the determination of the action pattern of Haliotis tuberculata alginate lyase.

The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-beta-D-mannuronate blocks, leading to the release of O-(4-deoxy-alpha-L-erythro-hex-4-enopyranosyluronic acid)-(1-->4)-O-(beta-D-mannopyranosyluronic acid)-(1-->4)-O-beta-D-mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligomannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligomannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G-M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G-G nor the M-G diads. This lyase should therefore be referred to as a mannuronate beta-eliminase, indicating that the enzyme performs beta-elimination on mannuronate residues only, from both the M-M and G-M diads of alginates.

NMR spectroscopy analysis of oligoguluronates and oligomannuronates prepared by acid or enzymatic hydrolysis of homopolymeric blocks of alginic acid. Application to the determination of the substrate specificity of Haliotis tuberculata alginate lyase.

The 1H and 13C NMR chemicals shifts of the various saturated and unsaturated timers obtained by acid or enzymatic depolymerisation of homopolymeric blocks of alginates are reported. In addition, 13C NMR chemical shifts are assigned for several saturated oligomers of higher polymerisation degrees. Breakdown of alginate and of homopolymeric alginate blocks by Haliotis tuberculata alginate lyase was monitored with 1H NMR spectroscopy and the signals relevant to the identification of the lyase products are pointed out. The enzymes performs beta-elimination on the mannuronic acid residues, independently of their immediately surrounding neighbours. Application of this approach to the analysis of the substrate specificity of alginate lyases is discussed.

Purification and determination of the action pattern of Haliotis tuberculata laminarinase.

The major laminarinase activity (EC 3.2.1.39) from the gastropodean marine mollusc Haliotis tuberculata was purified to homogeneity by cation exchange chromatography and its action pattern was investigated by HPAEC-PAD analysis of the degradation of various laminarin samples. It consists of a 60 kDa protein capable of depolymerizing the unbranched portions of the beta-(1-->3), beta-(1-->6)-glucan, down to laminaritriose. The enzyme operates via a molecular mechanism retaining the anomeric configuration. As the purified protein does not cleave the beta-(1-->6) linkages, it can be used for the structural analysis of laminarins.

A rapid method for the separation and analysis of carrageenan oligosaccharides released by iota- and kappa -carrageenase.

Based on the improved performances in speed of chromatographic separation on Superdex-type materials (Pharmacia) compared to conventional media such as Sephadex and Bio Gel-type, a rapid size-exclusion chromatography (SEC) method was developed for the separation and analysis of carrageenan oligosaccharides. It was used to evaluate the elution profiles of hydrolysates produced by carrageenases specific for kappa- and iota-carrageenans. Oligosaccharide peaks ranging from di- to dodeca-saccharides were obtained in about 20 min on an analytical scale, whereas preparative runs were completed in a few hours. The method may also be used to monitor polysaccharide degradation.

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae).

Protoplast were isolated from a filamentous brown alga, Sphacelaria sp. (Sphacelariales, Phaeophyta), using alginate-lyases extracted from marine molluscs, and commercial pectinase and cellulase. Yields were about 4000 protoplasts per gram of fresh tissue. Different types of protoplasts, originating from apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells produced a higher percentage of protoplasts (approx. 2%), compared with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types of protoplasts divided at least once in culture, but did not develop further.

Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B.

The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar [Young, K. S. Bhattacharjee, S. S. & Yaphe, W. (1978) Carbohydr. Res. 66, 207-212], were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonas and the new combination A. agarlyticus (Cataldi) is proposed. An alpha-agarase, i.e. specific for the alpha(1-->3) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CL-6B) and by anion-exchange chromatography (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDS/PAGE, the purified alpha-agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0-9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45 degrees C or by removing calcium. In addition, a beta-galactosidase specific for the end products of the alpha-agarase was present in the alpha-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues.