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β-(1,3)-Glucan, the major fungal cell wall component, ramifies through β-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(14C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2Δ and gas1Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced β-(1,6)-branching on the β-(1,3)-oligomers following its β-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear β-(1,3)-oligomers as well as Bgl2p-catalyzed products [short β-(1,3)-oligomers linked by a linear β-(1,6)-linkage]. The double S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual β-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual β-(1,3)-glucan elongating and branching activity. Our report unravels the β-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life., IMPORTANCE The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core β-(1,3)-glucan ramification, is poorly understood; the ramified β-(1,3)-glucan interconnects other cell wall components. Once linear β-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using Saccharomyces cerevisiae as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the β-(1,3)-glucan branching. The sick phenotype of the double Scgas1Δ bgl2Δ mutant suggested that β-(1,3)-glucan branching is essential. In addition to ScGas1p, GH72 family ScGas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual β-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall β-(1,3)-glucan branching mechanism. The essentiality of β-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.

International audience; Despite its essential role in the yeast cell wall, the exact composition of the beta-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant beta-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of approximately 38 kDa and could be hydrolyzed only by beta-(1,6)-glucanase. Gas chromatography mass spectrometry and NMR ((1)H and (13)C) analyses confirmed it to be a beta-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two beta-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the beta-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[(14)C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched beta-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for beta-(1,6)-glucan synthetic activity were defined.

International audience

International audience; A 1,3-beta-glucanosyltransferase which introduces intrachain 1,6-beta linkages into 1,3-beta-glucan was isolated from the cell wall of Aspergillus fumigatus. The biochemical and molecular characterization of the A. fumigatus transferase showed it was homologous to the Saccharomyces cerevisiae and Candida albicans transferase Bgl2p. A null mutant constructed in A. fumigatus by gene replacement did not show a distinct phenotype from the parental strain. The putative function of this major cell-wall-associated protein is discussed.

International audience; A 1,3-beta-glucanosyltransferase which introduces intrachain 1,6-beta linkages into 1,3-beta-glucan was isolated from the cell wall of Aspergillus fumigatus. The biochemical and molecular characterization of the A. fumigatus transferase showed it was homologous to the Saccharomyces cerevisiae and Candida albicans transferase Bgl2p. A null mutant constructed in A. fumigatus by gene replacement did not show a distinct phenotype from the parental strain. The putative function of this major cell-wall-associated protein is discussed.

International audience; Cell wall transferases utilizing beta-(1-3)-glucan chains as substrates may play important roles in cell wall assembly and rearrangement, as beta-(1-3)-glucan is a major structural component of the cell wall of many fungi. A novel beta-(1-3)-glucanosyltransferase was purified to apparent homogenei ty from an autolysate of the cell wall of Aspergillus fumigatus. The enzyme had a molecular mass of 49 kDa and contained approximately 5 kDa of N-linked carbohydrate. The enzyme catalyzed an initial endo-type splitting of a beta-(1-3)-glucan molecule, followed by linkage of the newly generated reducing end to the nonreducing end of another beta-(1-3)-glucan molecule. Laminarioligosaccharides of size G10 and greater were donor substrates for the transferase. Laminarioligosaccharides of size G5 and greater formed acceptors. The enzyme was able to reuse initial transferase products as donors and acceptors in extended incubations, resulting in the formation of increasingly larger transferase products until they became insoluble. The major initial products from an incubation of the transferase with borohydride-reduced G11 (rG11) were rG6 and rG16. 1H NMR analysis of the rG16 transferase product showed it was a laminarioligosaccharide, indicating that the enzyme forms a beta-(1-3)-linkage during transfer. The enzyme may have a key function in vivo by allowing the integration of newly synthesized glucan into the wall and promoting cell wall expansion during cell growth.

International audience; The oxidized and reduced forms of the [Fe4-S4] ferredoxin I from Desulfovibrio desulfuricans Norway were investigated by 1H NMR spectroscopy with the aim of obtaining the complete assignment of the cysteines ligating the cluster. A combination of TOCSY and NOESY measurements together with information from the x-ray crystallographic structure of related ferredoxins provided the sequence-specific assignment of the four cysteines coordinated to the cluster. Through EXSY experiments, the hyperfine shifted resonance signals in the reduced ferredoxin were also assigned. The temperature dependence of the contact-shifted cysteinyl residues of the reduced ferredoxin reveals that two cysteines exhibit anti-Curie behavior whereas the other two cysteines display Curie behavior; that identifies Cys 9 (I) and Cys 15 (III) as ligated to the mixed-valence iron ions.