A Forward Genetic Approach in Chlamydomonas reinhardtii as a Strategy for Exploring Starch Catabolism

International audienceA screen was recently developed to study the mobilization of starch in the unicellular green alga Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding β-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae

Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1

13siopenBackground: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. Methods: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. Results: A short cis-regulatory region restricted to the −72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. Conclusions: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.openWavelet-Vermuse C.; Groux-Degroote S.; Vicogne D.; Cogez V.; Venturi G.; Trinchera M.; Brysbaert G.; Krzewinski-Recchi M.-A.; Bachir E.H.; Schulz C.; Vincent A.; Van Seuningen I.; Harduin-Lepers A.Wavelet-Vermuse, C.; Groux-Degroote, S.; Vicogne, D.; Cogez, V.; Venturi, G.; Trinchera, M.; Brysbaert, G.; Krzewinski-Recchi, M. -A.; Bachir, E. H.; Schulz, C.; Vincent, A.; Van Seuningen, I.; Harduin-Lepers, A

Relation entre structure et fonctions des glucanes périplasmiques osmorégulés chez les Protéobactéries

Les glucanes périplasmiques osmorégulés (OPG) sont des composants intrinsèques de l'enveloppe des Protéobactéries. Rhodobacter sphaeroides produit des OPG cycliques [bêta]-1,2 avec une liaison [alpha]-1,6 qui peuvent être substitués par des résidus de succinyle et d'acétyle. L'inactivation du gène opgC par insertion de transposon conduit à la production d'OPG neutres dépourvus de la substitution par les résidus de succinyle. En amont d'opgC et organisés en opéron avec celui-ci, trois gènes, opgGIH, ont été identifiés ; ces gènes sont nécessaires à la synthèse du squelette glucanique. Les protéines OpgG et OpgH présentent de fortes similitudes avec les produits des gènes qui gouvernent la synthèse des OPG linéaires branchés [bêta]-1,2, [bêta]-1,6 chez les Entérobacteries. L'expression hétérologue des gènes opgIHC chez Escherichia coli induit la synthèse de glucanes linéaires de plus grande taille et partiellement substitués par des résidus de succinyle. L'entérobacterie phytopathogène, Erwinia chrysanthemi, synthétise des OPG substitués par des résidus de succinyle et d'acétyle. Cette substitution dépend des conditions de culture. Les mutants déficients dans la synthèse des OPG présentent un phénotype pléiotrope (sécrétion réduite d'exo-enzymes, sensibilité aux sels biliaires, motilité réduite) et ne sont plus virulents. Ces phénotypes ont permis d'isoler des pseudo-révertants. Les mutations additionnelles restaurent à divers degrés le comportement de la souche sauvage.LILLE1-BU (590092102) / SudocSudocFranceF

Phylogenetic-Derived Insights into the Evolution of Sialylation in Eukaryotes: Comprehensive Analysis of Vertebrate β-Galactoside α2,3/6-Sialyltransferases (ST3Gal and ST6Gal).

International audienceCell surface of eukaryotic cells is covered with a wide variety of sialylated molecules involved in diverse biological processes and taking part in cell-cell interactions. Although the physiological relevance of these sialylated glycoconjugates in vertebrates begins to be deciphered, the origin and evolution of the genetic machinery implicated in their biosynthetic pathway are poorly understood. Among the variety of actors involved in the sialylation machinery, sialyltransferases are key enzymes for the biosynthesis of sialylated molecules. This review focus on β-galactoside α2,3/6-sialyltransferases belonging to the ST3Gal and ST6Gal families. We propose here an outline of the evolutionary history of these two major ST families. Comparative genomics, molecular phylogeny and structural bioinformatics provided insights into the functional innovations in sialic acid metabolism and enabled to explore how ST-gene function evolved in vertebrates

Characterization of the Erwinia chrysanthemi gan Locus, Involved in Galactan Catabolism▿ †

β-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK2. Degradation of galactans would be catalyzed by the periplasmic 1,4-β-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-β-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny

Biosynthesis of Osmoregulated Periplasmic Glucans in Escherichia coli : The Phosphoethanolamine Transferase Is Encoded by opgE

International audienceOsmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity

Probing the CMP-Sialic Acid Donor Specificity of Two Human β- d -Galactoside Sialyltransferases (ST3Gal I and ST6Gal I) Selectively Acting on O- and N-Glycosylproteins

International audienc

Vertebrate Alpha2,8-Sialyltransferases (ST8Sia): A Teleost Perspective

International audienc