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The Neurospora crassa genome has a gene cluster for the synthesis of galactosaminogalactan (GAG). The gene cluster includes the following: (1) UDP-glucose-4-epimerase to convert UDP-glucose and UDP-N-acetylglucosamine to UDP-galactose and UDP-N-acetylgalactosamine (NCU05133), (2) GAG synthase for the synthesis of an acetylated GAG (NCU05132), (3) GAG deacetylase (/NCW-1/NCU05137), (4) GH135-1, a GAG hydrolase with specificity for N-acetylgalactosamine-containing GAG (NCU05135), and (5) GH114-1, a galactosaminidase with specificity for galactosamine-containing GAG (NCU05136). The deacetylase was previously shown to be a major cell wall glycoprotein and given the name of NCW-1 (non-GPI anchored cell wall protein-1). Characterization of the polysaccharides found in the growthmore » medium from the wild type and the GAG synthase mutant demonstrates that there is a major reduction in the levels of polysaccharides containing galactosamine and N-acetylgalactosamine in the mutant growth medium, providing evidence that the synthase is responsible for the production of a GAG. The analysis also indicates that there are other galactose-containing polysaccharides produced by the fungus. Phenotypic characterization of wild-type and mutant isolates showed that deacetylated GAG from the wild type can function as an adhesin to a glass surface and provides the fungal mat with tensile strength, demonstrating that the deacetylated GAG functions as an intercellular adhesive. The acetylated GAG produced by the deacetylase mutant was found to function as an adhesive for chitin, alumina, celite (diatomaceous earth), activated charcoal, and wheat leaf particulates.« less

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“t$$\bar{a}$$pirins,” origin from M$$\bar{a}$$ori: to join), not directly associated with cellulases, thatmore » mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two t$$\bar{a}$$pirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, t$$\bar{a}$$pirins are specific to these extreme thermophiles. T$$\bar{a}$$pirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the t$$\bar{a}$$pirins for cellulose. Crystallization of a cellulose-binding truncation from one t$$\bar{a}$$pirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. In addition, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ t$$\bar{a}$$pirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.« less