Bacterial Cellulose Synthase Modification and Export

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Many bacteria form highly resistant biofilms composed of extracellular polysaccharides, like cellulose, that promote the adherence to various biotic and abiotic surfaces. The biofilm matrix also affords bacteria protection from immune system components, antimicrobial agents, detergents and harsh environmental factors. Bacterial cellulose is also gaining industrial significance, as it has unique physical/chemical properties that provide significant advantages when compared to algal- or plant-derived cellulose (eg., nano-scale, pliable and durable). Even though the biological and industrial implications of bacterial cellulose are expanding, the synthesis, modification and export of this polymer by bacteria is still being elucidated. The bcsABZC genes comprise the core bacterial cellulose synthase genes, however, important neighboring loci have also been noted for proper polymer production, modification and release. For example, bcsEFG and wssFGHI have been implicated in the installation of phosphoethanolamine (pEtN) or acetyl (Ac) groups onto newly synthesized cellulose chains, respectively. Despite the growing number of bacteria identified with these genes for distinct cellulose synthases, biochemical verification of the precise activities of the inherent enzymes that produce these uniquely modified polymers is lacking. Adding to this ambiguity, the associated cellulases that are predicted to release cellulose chains from the cell into the biofilm matrix are from diverse glycosyl hydrolase (GH) families. BcsZ enzymes in Gram negatives belong to GH-8 and BglX is a GH-3, but the CcsZ Gram positive homologs in Clostridia are GH-5 enzymes. The biological reason for this deviation and how these enzymes specifically recognize their respectively decorated cellulose polymers is unknown. This research proposal aims to address these deficiencies by a gene-to-molecule approach. We will phenotypically analyse environmental conditions that lead to cellulose biofilm formation and link this to expression of specific genes for our panel of organisms containing novel synthase complexes. The distinct cellulose-active enzymes from these synthases (Ac-/pEtN-transferases and GH enzymes) will be biochemically characterized in parallel to uncover molecular determinants responsible for polymer recognition, modification and turnover. These combined results will solve the ambiguity regarding fundamental requirements/synergies of enzymes in the distinct synthases of Gram negatives and positives and relate this to the importance of specific polymers to bacterial colonization and persistence. Exploitation of our toolkit (strains, enzymes, substrates and assays) will also be used to develop biocatalytic alternatives for the installment of desirable functionalities and physicochemical properties to cellulose polymers for various bioapplications. Thus, this combined research has immense potential for basic discoveries, as well as avenues that enhance further applied research.