WssI from the Gram-negative bacterial cellulose synthase is an O-acetyltransferase that acts on cello-oligomers with several acetyl donor substrates.

In microbial biofilms, bacterial cells are encased in a self-produced matrix of polymers (e.g., exopolysaccharides) that enable surface adherence and protect against environmental stressors. For example, the wrinkly spreader phenotype of Pseudomonas fluorescens colonizes food/water sources and human tissue to form robust biofilms that can spread across surfaces. This biofilm largely consists of bacterial cellulose produced by the cellulose synthase proteins encoded by the wss (WS structural) operon, which also occurs in other species, including pathogenic Achromobacter species. Although phenotypic mutant analysis of the wssFGHI genes has previously shown that they are responsible for acetylation of bacterial cellulose, their specific roles remain unknown and distinct from the recently identified cellulose phosphoethanolamine modification found in other species. Here, we have purified the C-terminal soluble form of WssI from P. fluorescens and Achromobacter insuavis and demonstrated acetylesterase activity with chromogenic substrates. The kinetic parameters (kcat/KM values of 13 and 8.0 M-1 s-1, respectively) indicate that these enzymes are up to four times more catalytically efficient than the closest characterized homolog, AlgJ from the alginate synthase. Unlike AlgJ and its cognate alginate polymer, WssI also demonstrated acetyltransferase activity onto cellulose oligomers (e.g., cellotetraose to cellohexaose) with multiple acetyl donor substrates (p-nitrophenyl acetate, 4-methylumbelliferyl acetate, and acetyl-CoA). Finally, a high-throughput screen identified three low micromolar WssI inhibitors that may be useful for chemically interrogating cellulose acetylation and biofilm formation.

Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ.

Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides. The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject of research interest among persistent clinical pathogens of global health importance. Our current understanding of the underlying biochemical pathways responsible for biosynthesis of these exopolysaccharides is largely limited to Gram-negative bacteria. Clostridia are a class of Gram-positive, anaerobic and spore-forming bacteria and include the important human pathogens Clostridium perfringens, Clostridium botulinum and Clostridioides difficile, among numerous others. Several species of Clostridia have been reported to produce a biofilm matrix that contains an acetylated glucan linked to a series of hypothetical genes. Here, we propose a model for the function of these hypothetical genes, which, using homology modelling, we show plausibly encode a synthase complex responsible for polymerization, modification and export of an O-acetylated cellulose exopolysaccharide. Specifically, the cellulose synthase is homologous to that of the known exopolysaccharide synthases in Gram-negative bacteria. The remaining proteins represent a mosaic of evolutionary lineages that differ from the described Gram-negative cellulose exopolysaccharide synthases, but their predicted functions satisfy all criteria required for a functional cellulose synthase operon. Accordingly, we named these hypothetical genes ccsZABHI, for the Clostridial cellulose synthase (Ccs), in keeping with naming conventions for exopolysaccharide synthase subunits and to distinguish it from the Gram-negative Bcs locus with which it shares only a single one-to-one ortholog. To test our model and assess the identity of the exopolysaccharide, we subcloned the putative glycoside hydrolase encoded by ccsZ and solved the X-ray crystal structure of both apo- and product-bound CcsZ, which belongs to glycoside hydrolase family 5 (GH-5). Although not homologous to the Gram-negative cellulose synthase, which instead encodes the structurally distinct BcsZ belonging to GH-8, we show CcsZ displays specificity for cellulosic materials. This specificity of the synthase-associated glycosyl hydrolase validates our proposal that these hypothetical genes are responsible for biosynthesis of a cellulose exopolysaccharide. The data we present here allowed us to propose a model for Clostridial cellulose synthesis and serves as an entry point to an understanding of cellulose biofilm formation among class Clostridia.