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.

1Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides. 2The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject 3 of research interest among persistent clinical pathogens of global health importance. Our current 4 understanding of the underlying biochemical pathways responsible for biosynthesis of these 5 exopolysaccharides is largely limited to Gram-negative bacteria. Clostridia are a class of Gram-positive, 6 anaerobic and spore-forming bacteria, and include the important human pathogens Clostridium 7 perfringens, Clostridium botulinum, and Clostridioides difficile, among numerous others. Clostridia have 8 been reported to form biofilms composed of cellulose, although the specific loci which encode the 9 cellulose synthase have not been identified. Here, we report the discovery of a gene cluster, which we 10 named ccsABZCD, among selected bacteria within class Clostridia that appears to encode a synthase 11 complex responsible for polymerization, modification, and export of an O-acetylated cellulose 12 exopolysaccharide. To test this hypothesis, we subcloned the putative glycoside hydrolase CcsZ and 13 solved the X-ray crystal structure of both apo-and product-bound CcsZ. Our results demonstrate that 14 CcsZ is in fact an endo-acting cellulase belonging to glycoside hydrolase family 5 . This is in 15 contrast to the Gram-negative cellulose synthase, which instead encodes BcsZ, a structurally distinct GH-16 8. We further show CcsZ is capable of hydrolysis of the soluble mock substrate carboxymethylcellulose 17 (CMC) with a pH optimum of 4.5. The data we present here serves as an entry point to an understanding 18 of biofilm formation among class Clostridia and allowed us to predict a model for Clostridial cellulose 19 synthesis.20 Author summary 21 Biofilms are communities of microorganisms that enmesh themselves in a protective matrix of elf-22 produced polysaccharide materials. Biofilms have demonstrated roles in both virulence and persistence 23 among bacterial pathogens of global health importance. The class Clostridia are a polyphyletic grouping 24 of primarily Gram-positive, anaerobic and spore-forming bacteria which contain the important and well-25 studied human pathogens Clostridioides difficile, Clostridium botulinum, and Clostridium perfringens, 26among others. Bacteria belonging to class Clostridia have been anecdotally reported to form biofilms described. In this work, we identify a gene cluster, which we name ccsABZHI, for the Clostridial cellulose 29 synthase, which bears remarkable similarity to molecular machinery required for the production of 30 cellulose biofilms in other Gram-negative bacteria. We further biochemically characterize one of these 31 enzymes, CcsZ, a predicted endoglucanase which we predicted from our model should cleave cellulose 32 exopolysaccharides. We show that CcsZ is in fact capable of this activity and belongs to a broader family 33 of glycoside hydrolases with unexpected...

No abstract

No abstract