Characterization of Redox Sensitive Brown Algal Mannitol-1-Phosphatases

Macroalgae (seaweeds) are key primary producers in marine coastal habitats and largely contribute to global ocean carbon fluxes. They also represent attractive renewable feedstock for the production of biofuels, food, feed, and bioactive. Brown algae are seaweeds that produce alginates and fucose containing sulfated polysaccharides in their cell wall and laminarin and mannitol for carbon storage. The availability of genomes of the kelp Saccharina japonica and of the filamentous Ectocarpus sp. paved the way for the biochemical characterization of recombinant enzymes involved in their polysaccharide and carbohydrates synthesis, including, notably, mannitol. Brown algal mannitol biosynthesis starts with the conversion of fructose-6-phospate into mannitol-1-phosphate (mannitol-1P), and this intermediate is hydrolysed by a haloacid dehalogenase phosphatase (M1Pase) to produce mannitol. We report here the biochemical characterization of a second M1Pase in Ectocarpus sp. (EsM1Pase1). Both Ectocarpus M1Pases were redox-sensitive enzymes, with EsM1Pase1 active only in presence of the reducing agent. Such catalytic properties have not been observed for any M1Pases yet. EsM1Pases were specific to mannitol-1-P, in contrast to S. japonica M1Pases that could act on other phosphorylated sugars. Finally, brown algal M1Pases formed two well-supported clades, with possible distinct subcellular localization and physiological role(s) under diverse environmental conditions and/or life cycle stages

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

Background: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. Results: We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. Conclusions: The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation

Low mannitol concentrations in Arabidopsis thaliana expressing Ectocarpus genes improve salt tolerance

Mannitol is abundant in a wide range of organisms, playing important roles in biotic and abiotic stress responses. Nonetheless, mannitol is not produced by a vast majority of plants, including many important crop plants. Mannitol-producing transgenic plants displayed improved tolerance to salt stresses though mannitol production was rather low, in the µM range, compared to mM range found in plants that innately produce mannitol. Little is known about the molecular mechanisms underlying salt tolerance triggered by low concentrations of mannitol. Reported here is the production of mannitol in Arabidopsis thaliana, by expressing two mannitol biosynthesis genes from the brown alga Ectocarpus sp. strain Ec32. To date, no brown algal genes have been successfully expressed in land plants. Expression of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase genes was associated with the production of 42.3–52.7 nmol g−1 fresh weight of mannitol, which was sufficient to impart salinity and temperature stress tolerance. Transcriptomics revealed significant differences in the expression of numerous genes, in standard and salinity stress conditions, including genes involved in K+ homeostasis, ROS signaling, plant development, photosynthesis, ABA signaling and secondary metabolism. These results suggest that the improved tolerance to salinity stress observed in transgenic plants producing mannitol in µM range is achieved by the activation of a significant number of genes, many of which are involved in priming and modulating the expression of genes involved in a variety of functions including hormone signaling, osmotic and oxidative stress, and ion homeostasis

Stanniocalcin 1 effects on the renal gluconeogenesis pathway in rat and fish

The mammalian kidney contributes significantly to glucose homeostasis through gluconeogenesis. Considering that stanniocalcin 1 (STC1) regulates ATP production, is synthesized and acts in different cell types of the nephron, the present study hypothesized that STC1 may be implicated in the regulation of gluconeogenesis in the vertebrate kidney. Human STC1 strongly reduced gluconeogenesis from C-14-glutamine in rat renal medulla (MD) slices but not in renal cortex (CX), nor from C-14-lactic acid. Total PEPCK activity was markedly reduced by hSTC1 in MD but not in CX. Pck2 (mitochondrial PEPCK isoform) was down-regulated by hSTC1 in MD but not in CX. In fish (Dicentrarchus labrax) kidney slices, both STC1-A and -B isoforms decreased gluconeogenesis from C-14-acid lactic, while STC1-A increased gluconeogenesis from C-14-glutamine. Overall, our results demonstrate a role for STC1 in the control of glucose synthesis via renal gluconeogenesis in mammals and suggest that it may have a similar role in teleost fishes. (C) 2015 Elsevier Ireland Ltd. All rights reserved.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) Brazil; Foundation for Science and Technology of Portugal [PTDC/MAR/121279/2010]; bilateral programme CAPES (Brazil)/GRICES (Portugal) CAPES/GRICES [215/08]info:eu-repo/semantics/publishedVersio

The mannitol utilization system of the marine bacterium <em>Zobellia galactanivorans</em>

International audienceMannitol is a polyol which occurs in a wide range of living organisms where it fulfills different physiological roles. Particularly, mannitol can account up to 20-30% of the dry weight of brown algae, and is likely to be an important source of carbon for marine heterotrophic bacteria. Zobellia galactanivorans (Flavobacteria) is a model to study pathways involved in degradation of seaweed carbohydrates. Annotation of its genome revealed the presence of genes potentially involved in mannitol catabolism, and we describe here the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) and of a fructokinase (FK). Among the observations, the M2DH of Z. galactanivorans was active as a monomer, did not require metal ions for catalysis, and features narrow substrate specificity. The characterized FK was active on fructose and mannose in presence of a monocation, preferentially K+. Furthermore, genes coding for both proteins were adjacent in the genome, and located directly downstream three loci likely to encode an ATP binding cassette (ABC) transporter complex, suggesting organization into an operon. Gene expression analysis supported this hypothesis, and showed the induction of these five genes after culturing Z. galactanivorans in presence of mannitol as sole source of carbon. This operon for mannitol catabolism was identified in only six genomes of Flavobacteriaceae among the 76 publicly available at the time of the analysis. It is not conserved in all Bacteroidetes because some species contained a predicted mannitol permease instead of a putative ABC transporter complex upstream M2DH and FK ortholog genes

Genetic diversity and habitats of two enigmatic marine alveolate lineages

Systematic sequencing of environmental SSU rDNA genes amplified from different marine ecosystems has uncovered novel eukaryotic lineages, in particular within the alveolate and stramenopile radiations. The ecological and geographic distribution of 2 novel alveolate lineages (called Group I and II in previous papers) is inferred from the analysis of 62 different environmental clone libraries from freshwater and marine habitats. These 2 lineages have been, up to now, retrieved exclusively from marine ecosystems, including oceanic and coastal waters, sediments, hydrothermal vents, and permanent anoxic deep waters and usually represent the most abundant eukaryotic lineages in environmental genetic libraries. While Group I is only composed of environmental sequences (118 clones), Group II contains, besides environmental sequences (158 clones), sequences from described genera (8) (Hematodinium and Amoebophrya) that belong to the Syndiniales, an atypical order of dinoflagellates exclusively composed of marine parasites. This suggests that Group II could correspond to Syndiniales, although this should be confirmed in the future by examining the morphology of cells from Group II. Group II appears to be abundant in coastal and oceanic ecosystems, whereas permanent anoxic waters and hydrothermal ecosystems are usually dominated by Group I. Based upon the similarity of partial sequences, we organized these 2 groups into clusters, The diversity of Group II (16 clusters) is wider than that of Group 1 (5 clusters). Two clusters from Group I have a widespread distribution and are found in all explored marine habitats. In contrast, all other clusters seem to be limited to specific marine habitats. For example, some clusters belonging to Group I and Group II are only detected in extreme environments (anoxic and hydrothermal vents), whereas many clusters from Group II have only been retrieved from coastal waters. We determined near-complete SSU rRNA gene sequences for 26 environmental clones, selected in order to obtain at least one complete sequence per cluster. Phylogenetic analyses (maximum likelihood, neighbor joining, maximum parsimony, and Bayesian reconstruction) based upon complete sequences all concurred to place both Group I and II as sister lineages of dinoflagellates. This result contradicts several published studies, which placed both groups within dinoflagellates