Surprisingly, the periplasmic cellulase BcsZ, encoded in the cellulose biosynthesis operon, is necessary for efficient bacterial cellulose production and functions independently of the biosynthetic complex to clear mislocalized pEtN cellulose from the periplasm.
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.
It is shown that cellulosomal principles can operate in highly reduced cohesin‐dockerin systems, and two additional candidate interacting partners for ClcC appear to bind via a non‐canonical interface.
John Allan, Amias Alstrom-Moore, Gary W. Black· FEBS Open Bio· 0 citations
Synthetic biology has advanced microorganisms to be programmed as production hosts, but its application to bacteria that inherently assemble extracellular materials remains limited. Komagataeibacter spp., natively synthesizes cellulose at the bacterial cell surface, creating a material-forming interface that has not been used as a programmable recruitment platform. Here we establish cell-surface display in Komagataeibacter intermedius and show that this interface can recruit defined proteins, making functionalization part of cellulose formation. By engineering Lpp’OmpA, we displayed a fluorescent protein and genetically encoded capture modules (SpyTag and SilkTag) to selectively capture catcher-fused protein cargos onto K. intermedius cell surface. Recruitment of silk-derived structural protein before cellulose production generated silk-associated fibrous structures within the pellicles, with retained cargo signal after washing. The resulting biocomposite showed reorganized fibre-network morphology, increased surface hydrophobicity, mesoscale ordering, and improved wet-state compressive strength. Wild-type cells exposed to same conditions did not reproduce these changes, demonstrating that material properties arise from surface-directed recruitment rather than protein exposure alone. This work demonstrates the material-forming bacterial surface as a programmable engineering interface for organizing extracellular proteins, providing a general strategy for engineering living materials.
Shuang Zhang, Chengji Yang, Ruxia Fan et al.· bioRxiv· 0 citations
Bacterial species that inhabit diverse nutritional niches, such as plants, the human gut, and aquatic environments, exhibit common and unique molecular mechanisms for acquiring nutrients from plant polysaccharides like xylan. The oligotrophic bacterium Caulobacter vibrioides (Syn. Caulobacter crescentus) shows unique adaptations for growth in low-nutrient conditions and codes for a repertoire of genes that facilitate xylan utilisation as a carbon source. Investigation of xylan and xylan-derivative utilisation by C. vibrioides shows membrane-bound xylanase-mediated xylan deconstruction on the cell surface and further deconstruction of the xylo-oligosaccharides in the periplasm by xylosidases and other accessory enzymes. Proton motive force disruption studies suggest involvement of TonB-dependent transporters, Major Facilitator Superfamily transporters, or ATP-Binding Cassette transporters in the transport of xylo-oligosaccharides and other xylan-derivatives across the membranes of C. vibrioides. Results from our studies using enzyme assays and chromatographic analyses suggest that bacteria deconstruct xylan in a surface-bound manner leading to efficient uptake of xylo-oligosaccharides across the outer membrane, without loss of nutrients to the environment. This proposed model for C. vibrioides xylan utilisation highlights both unique and similar mechanistic features found in gut and plant pathogenic bacteria, advancing the molecular understanding of nutrient acquisition in oligotrophic Gram-negative bacteria.
V. Bùi, Rhoda E Inkoom, Hansini K Gamage Don et al.· Environmental Microbiology· 0 citations
Host-pathogen interactions frequently depend on key components of the bacterial cell surface, such as capsules and lipopolysaccharides in Gram-negative bacteria. The first step in the synthesis of lipid-linked polysaccharides is the substitution of a uridine diphosphate (UDP)-sugar by a lipid monophosphate catalysed by a phosphoglycosyl transferase (PGT). We report the 3.0 Å cryo-electron microscopy apo-structure of the PGT enzyme WbaP from Escherichia coli, a UDP-galactose:undecaprenolphosphate galactose-1-phosphoryl transferase. The structure is a dimer with each monomer formed of four N-terminal transmembrane helices, a small α/β domain with a distinctive β-hairpin that inserts into the other monomer, and a catalytic domain, which sits perpendicular to the transmembrane domain. A complex of WbaP with the UDP-galactose substrate shows binding of the UDP moiety by R319 and R377. Mutations of R319 and R377, along with K331 and R401, highlighted the essential nature of these residues for the catalytic activity of the protein, as confirmed by an in vivo functional assay. Our results provide new insights into the PGT family of enzymes.
M. Weckener, Audrey Le Bas, Philip N. Ward et al.· Open Biology· 0 citations
Lignocellulosic biomass is a heterogeneous solid matrix whose biological deconstruction is limited by cellulose accessibility, lignin exposure, pore-scale transport, and enzyme stability at solid-liquid interfaces. Although bacterial cellulosomes provide a natural strategy for organizing multiple enzymes through scaffoldin-mediated cohesin-dockerin interactions, their engineering value depends on more than enzyme colocalization. This review frames cellulosome engineering as a process-aware macromolecular assembly problem, in which catalytic balance, substrate targeting, assembly size, inter-domain spacing, and environmental robustness must be optimized together. Natural cellulosome architecture is discussed in terms of its roles in enzyme recruitment and catalytic synergy. In addition, cellulosomal systems are compared with industrial fungal enzyme cocktails regarding production, scalability, and substrate accessibility. Rather than viewing engineered cellulosomes as universally superior multienzyme complexes, we evaluate how their performance is constrained by lignin adsorption, steric exclusion, diffusion limitations, high-solids conditions, host burden, catalyst recovery, AI-guided design validation, and the mismatch between model substrates and industrial feedstocks. Future progress will require experimentally validated, substrate-specific, and process-compatible cellulosome designs that balance catalytic diversity with assembly stability, production feasibility, and techno-economic performance.
Menglan Liao, Zi-Xuan Wan, Long-Qing Wang et al.· International Journal of Bio...· 0 citations