Site-specific processing of phosphoethanolamine cellulose by the BcsZ cellulase reveals stochastic biofilm cellulose modification

Abstract

Cellulose is a common component of bacterial biofilms where it interacts with other biopolymers to form a 3-dimensional matrix enclosing the bacteria. Synthesized and secreted by the synthase-dependent biosynthesis pathway common to many bacterial exopolysaccharides, its surface exposure depends on the presence of the periplasmic cellulase BcsZ. During export across the periplasm, E. coli and other Enterobacteriaceae modify cellulose with lipid-derived phosphoethanolamine (pEtN). How BcsZ hydrolyzes pEtN-cellulose in the periplasm is unknown and so is the native distribution pattern of pEtN on cellulose. Here, we used carbohydrate synthesis, X-ray crystallography, native mass spectrometry, and super-resolution MINFLUX nanoscopy to delineate BcsZ’s role during cellulose biosynthesis. Crystal structures of BcsZ bound to chemically synthesized pEtN cello-oligosaccharides identify how the enzyme recognizes pEtN-modified glucosyl units. Comparing mono and double substituted cellohexaoses, we identify varying binding poses that are determined by two pEtN coordination sites within BcsZ’s catalytic pocket. Combined, our structural analyses reveal an ideal BcsZ cellohexaose ligand containing two pEtN modified units separated by an unmodified cellotriosyl unit. The enzyme binds and hydrolyzes this compound with substantially increased affinity and efficiency. Further, BcsZ digestion of native pEtN cellulose combined with native mass spectrometry analyses reveals the stochastic distribution of pEtN on biofilm cellulose. Additionally, MINFLUX co-localization of BcsZ with other components of the biosynthetic complex demonstrates BcsZ’s random distribution across the periplasm. Our data suggest BcsZ functions independently of the biosynthetic complex to clear mislocalized pEtN cellulose from the periplasm. Significance Statement Biofilms are an abundant form of bacterial growth and responsible for the majority of hospital-derived infections. Uropathogenic E. coli produces phosphoethanolamine cellulose as a stabilizing extracellular polysaccharide. Surprisingly, the periplasmic cellulase BcsZ, encoded in the cellulose biosynthesis operon, is necessary for efficient bacterial cellulose production. Crystal structures of BcsZ bound to chemically synthesized phosphoethanolamine cellulose fragments reveal how the enzyme recognizes and cleaves its unique substrate. Further, super-resolution fluorescence microscopy shows that BcsZ does not form a stable complex with other cellulose synthase components and likely diffuses in the E. coli periplasm. Finally, mass spectrometry of oligosaccharides released by BcsZ from biofilm E. coli indicates the stochastic modification of cellulose with phosphoethanolamine groups in vivo.