β-(1,3)-glucans are important for the integrity of the fungal cell wall and the interaction of fungi with the (a)biotic environment. For instance, the β-(1,3)(1,6)-glucan schizophyllan protects against thermal stress, starvation, and competition. Agaricomycetes like Schizophyllum commune have two β-(1,3)-glucan synthase (bgs) genes. Here, deletion constructs for the bgs1 and bgs2 genes were introduced in S. commune. A Δbgs2 strain was not obtained. Inactivation of bgs1 resulted in an approximately twofold lower production of water-soluble polysaccharide with FTIR characteristics consistent with schizophyllan. In addition, solid state NMR showed a relative 5-fold decrease in β-(1,3)-glucan / β-(1,3)(1,6)-glucan (schizophyllan) levels in the rigid part of the cell wall of the deletion strain, which was accompanied by a relative 3.3-fold and 2.7-fold increase in chitin and α-(1–3)-glucan, respectively. These changes in cell wall composition were accompanied by reduced growth, impaired fruiting body formation, and absence of spore production. Together, these data show for the first time the impact of a deletion of a β-(1,3)-glucan synthase gene in a mushroom-forming fungus on cell wall composition, growth, and fruiting body development.
The fungal cell wall is a dynamic structure whose composition and integrity are closely coupled to metabolism, morphogenesis, and environmental fitness. Glutamine fructose-6-phosphate amidotransferase (Gfa1), the committed enzyme of the hexosamine biosynthetic pathway, supplies precursors for UDP-N-acetylglucosamine production and cell-wall chitin synthesis. Although Gfa1 is essential for growth and virulence in Aspergillus fumigatus, its role in the cell-wall biology and developmental program of the aflatoxigenic fungus Aspergillus flavus remains unclear. Here, we characterized a gfa1 deletion mutant and a complemented strain of A. flavus. Loss of gfa1 caused glucosamine/N-acetylglucosamine auxotrophy and markedly impaired vegetative growth and conidiation. Despite amino-sugar supplementation, the mutant exhibited increased sensitivity to cell-wall perturbing agents and significant reductions in cell-wall chitin and glucan, indicating disrupted cell-wall homeostasis. Gfa1 deficiency also severely impaired sclerotial formation and increased sensitivity to several environmental stresses. Importantly, the mutant showed markedly reduced colonization of peanut and maize seeds and no detectable aflatoxin B1 accumulation under the conditions tested. Consistent with its broad fitness defects, Δgfa1 displayed strongly attenuated virulence in Galleria mellonella. These findings establish Gfa1-dependent hexosamine metabolism as an important determinant of cell-wall homeostasis and developmental competence in A. flavus and reveal a link between amino-sugar metabolism, fungal surface integrity, sclerotial differentiation, and crop-associated fitness.
Plant cell walls contain multiple matrix polysaccharides whose interactions help determine wall mechanics, growth, and integrity, but it remains unclear how disruption of one biosynthetic or modification pathway reshapes other, non-target polymers. We used gel permeation chromatography coupled with enzyme-linked immunosorbent assay (GPC-ELISA) to compare mass distributions of major matrix polysaccharides in Arabidopsis thaliana mutants affecting xylan biosynthesis and decoration (irx14L, irx15, gux1-1, and gux2-2), glucomannan synthesis (csla2-1), pectin homogalacturonan (HG) biosynthesis (gaut10-2 and gaut11-2), and wall esterification (axy4-4, tbl31, tbl2, tbl3, and tbr). Three biological replicates per genotype, each from independent cell wall extractions, were profiled. Disruption of a single pathway frequently altered the distribution of non-target polymers. Xylan-related mutants altered not only glucuronoxylan but also HG and, in some cases, xyloglucan, with gux1-1 and gux2-2 producing particularly contrasting GX and HG profiles. By contrast, csla2-1 showed relatively limited secondary effects, whereas gaut10-2 and gaut11-2 showed increased low-molecular-size unesterified HG together with localised redistribution of several hemicellulose epitopes. Esterification mutants separated into distinct redistribution patterns rather than a single shared phenotype. Together, these findings support a directional and asymmetric model of cross-polymer coupling within extractable wall fractions, in which perturbation of HG or GX pathways has broader effects on the wall matrix than disruption of mannan biosynthesis.
Fucoidans, also known as fucose-containing sulfated polysaccharides (FCSPs), are abundant and structurally complex glycans found in the cell wall of brown algae. These compounds have been shown to have fundamental roles in the development and physiology of these organisms, to provide a major contribution to marine carbon sequestration, and to exhibit numerous bioactivities. Despite this, we currently have a limited understanding of the metabolic pathways and enzymes involved in synthesis and remodelling of fucoidans, their spatial and temporal dynamics in brown algae and in the marine environment, and how their structure and composition influence their bioactivities. Here, we highlight some of the challenges that have hindered progress on fucoidan research. We then moved on to underline some of the recent technical progress and new resources that would contribute to alleviate these challenges when combined together. This includes new technical approaches in analytical chemistry and glycobiology, as well the extension of genomic resources and genetics tools for brown algae. Finally, we suggest how progress in fucoidan research should contribute to advance understanding on brown algae biology, while supporting current and developing new aspects in fucoidan applications and seaweed biotechnology as part of the growing blue bioeconomy.
Mycolic acids are key components of the mycobacterial cell envelope, contributing to its structural integrity and intrinsic resistance to environmental stress. The polyketide synthase Pks13 catalyzes the final Claisen condensation step in mycolic acid biosynthesis and is conserved across Corynebacteriales. While the catalytic cysteine within the ketosynthase (KS) domain is established as essential for activity, the role of surrounding residues in supporting catalysis remains less well understood. Herein, we utilized a Mycobacterium smegmatis conditional pks13 mutant, and subsequent complemented strains harbouring a plasmid-borne copy of pks13 to probe the functional importance of selected residues within the KS domain from previous structural studies. These site-directed mutagenesis studies identified Asp264 as critical for Pks13 function. The D264A strain showed severely impaired growth along with a pronounced loss of mycolate synthesis and mycolate-containing lipids: trehalose dimycolate (TDM) and trehalose monomycolate (TMM). In contrast, other mutations had either little or no significant effect on cell viability or mycolate synthesis or mycolate-containing lipids. Structural modelling of the KS domain suggests that loss of Asp264 disrupts the local active-site environment, resulting in compaction of the binding pocket and altered conformation of a loop proximal to the catalytic residue Cys267. Together, these findings demonstrate that Asp264 is essential for Pks13 function and likely contributes to maintaining the structural environment of the KS domain.
