A molecular description of cellulose biosynthesis.

Cellulose is the most abundant biopolymer on Earth, and certain organisms from bacteria to plants and animals synthesize cellulose as an extracellular polymer for various biological functions. Humans have used cellulose for millennia as a material and an energy source, and the advent of a lignocellulosic fuel industry will elevate it to the primary carbon source for the burgeoning renewable energy sector. Despite the biological and societal importance of cellulose, the molecular mechanism by which it is synthesized is now only beginning to emerge. On the basis of recent advances in structural and molecular biology on bacterial cellulose synthases, we review emerging concepts of how the enzymes polymerize glucose molecules, how the nascent polymer is transported across the plasma membrane, and how bacterial cellulose biosynthesis is regulated during biofilm formation. Additionally, we review evolutionary commonalities and differences between cellulose synthases that modulate the nature of the cellulose product formed.

A Mechanism for Sustained Cellulose Synthesis during Salt Stress.

Abiotic stress, such as salinity, drought, and cold, causes detrimental yield losses for all major plant crop species. Understanding mechanisms that improve plants' ability to produce biomass, which largely is constituted by the plant cell wall, is therefore of upmost importance for agricultural activities. Cellulose is a principal component of the cell wall and is synthesized by microtubule-guided cellulose synthase enzymes at the plasma membrane. Here, we identified two components of the cellulose synthase complex, which we call companion of cellulose synthase (CC) proteins. The cytoplasmic tails of these membrane proteins bind to microtubules and promote microtubule dynamics. This activity supports microtubule organization, cellulose synthase localization at the plasma membrane, and renders seedlings less sensitive to stress. Our findings offer a mechanistic model for how two molecular components, the CC proteins, sustain microtubule organization and cellulose synthase localization and thus aid plant biomass production during salt stress. VIDEO ABSTRACT.

Directed evolution of material-producing microorganisms.

Nature is home to a variety of microorganisms that create materials under environmentally friendly conditions. While this offers an attractive approach for sustainable manufacturing, the production of materials by native microorganisms is usually slow and synthetic biology tools to engineer faster microorganisms are only available when prior knowledge of genotype-phenotype links is available. Here, we utilize a high-throughput directed evolution platform to enhance the fitness of whole microorganisms under selection pressure and identify genetic pathways to enhance the material production capabilities of native species. Using Komagataeibacter sucrofermentans as a model cellulose-producing microorganism, we show that our droplet-based microfluidic platform enables the directed evolution of these bacteria toward a small number of cellulose overproducers from an initial pool of 40,000 random mutants. Sequencing of the evolved strains reveals an unexpected link between the cellulose-forming ability of the bacteria and a gene encoding a protease complex responsible for protein turnover in the cell. The ability to enhance the fitness of microorganisms toward a specific phenotype and to unravel genotype-phenotype links makes this high-throughput directed evolution platform a promising tool for the development of new strains for the sustainable manufacturing of materials.

MeGT2.6 increases cellulose synthesis and active gibberellin content to promote cell enlargement in cassava.

Cassava, a pivotal tropical crop, exhibits rapid growth and possesses a substantial biomass. Its stem is rich in cellulose and serves as a crucial carbohydrate storage organ. The height and strength of stems restrict the mechanised operation and propagation of cassava. In this study, the triple helix transcription factor MeGT2.6 was identified through yeast one-hybrid assay using MeCesA1pro as bait, which is critical for cellulose synthesis. Over-expression and loss-of-function lines were generated, and results revealed that MeGT2.6 could promote a significant increase in the plant height, stem diameter, cell size and thickness of SCW of cassava plant. Specifically, MeGT2.6 upregulated the transcription activity of MeGA20ox1 and downregulated the expression level of MeGA2ox1, thereby enhancing the content of active GA3, resulting in a large cell size, high plant height and long stem diameter in cassava. Moreover, MeGT2.6 upregulated the transcription activity of MeCesA1, which promoted the synthesis of cellulose and hemicellulose and produced a thick secondary cell wall. Finally, MeGT2.6 could help supply additional substrates for the synthesis of cellulose and hemicellulose by upregulating the invertase genes (MeNINV1/6). Thus, MeGT2.6 was found to be a multiple regulator; it was involved in GA metabolism and sucrose decomposition and the synthesis of cellulose and hemicellulose.

Biotropic liquid crystal phase transformations in cellulose-producing bacterial communities.

Biological functionality is often enabled by a fascinating variety of physical phenomena that emerge from orientational order of building blocks, a defining property of nematic liquid crystals that is also pervasive in nature. Out-of-equilibrium, "living" analogs of these technological materials are found in biological embodiments ranging from myelin sheath of neurons to extracellular matrices of bacterial biofilms and cuticles of beetles. However, physical underpinnings behind manifestations of orientational order in biological systems often remain unexplored. For example, while nematiclike birefringent domains of biofilms are found in many bacterial systems, the physics behind their formation is rarely known. Here, using cellulose-synthesizing Acetobacter xylinum bacteria, we reveal how biological activity leads to orientational ordering in fluid and gel analogs of these soft matter systems, both in water and on solid agar, with a topological defect found between the domains. Furthermore, the nutrient feeding direction plays a role like that of rubbing of confining surfaces in conventional liquid crystals, turning polydomain organization within the biofilms into a birefringent monocrystal-like order of both the extracellular matrix and the rod-like bacteria within it. We probe evolution of scalar orientational order parameters of cellulose nanofibers and bacteria associated with fluid-gel and isotropic-nematic transformations, showing how highly ordered active nematic fluids and gels evolve with time during biological-activity-driven, disorder-order transformation. With fluid and soft-gel nematics observed in a certain range of biological activity, this mesophase-exhibiting system is dubbed "biotropic," analogously to thermotropic nematics that exhibit solely orientational order within a temperature range, promising technological and fundamental-science applications.

Glucosylceramides impact cellulose deposition and cellulose synthase complex motility in Arabidopsis.

Cellulose is an abundant component of plant cell wall matrices, and this para-crystalline polysaccharide is synthesized at the plasma membrane by motile Cellulose Synthase Complexes (CSCs). However, the factors that control CSC activity and motility are not fully resolved. In a targeted chemical screen, we identified the alkylated nojirimycin analog N-Dodecyl Deoxynojirimycin (ND-DNJ) as a small molecule that severely impacts Arabidopsis seedling growth. Previous work suggests that ND-DNJ-related compounds inhibit the biosynthesis of glucosylceramides (GlcCers), a class of glycosphingolipid associated with plant membranes. Our work uncovered major changes in the sphingolipidome of plants treated with ND-DNJ, including reductions in GlcCer abundance and altered acyl chain length distributions. Crystalline cellulose content was also reduced in ND-DNJ-treated plants as well as plants treated with the known GlcCer biosynthesis inhibitor N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenyl ethyl]-decanamide (PDMP) or plants containing a genetic disruption in GLUCOSYLCERAMIDE SYNTHASE (GCS), the enzyme responsible for sphingolipid glucosylation that results in GlcCer synthesis. Live-cell imaging revealed that CSC speed distributions were reduced upon treatment with ND-DNJ or PDMP, further suggesting an important relationship between glycosylated sphingolipid composition and CSC motility across the plasma membrane. These results indicate that multiple interventions compromising GlcCer biosynthesis disrupt cellulose deposition and CSC motility, suggesting that GlcCers regulate cellulose biosynthesis in plants.

Cell wall damage attenuates root hair patterning and tissue morphogenesis mediated by the receptor kinase STRUBBELIG.

Cell wall remodeling is essential for the control of growth and development as well as the regulation of stress responses. However, the underlying cell wall monitoring mechanisms remain poorly understood. Regulation of root hair fate and flower development in Arabidopsis thaliana requires signaling mediated by the atypical receptor kinase STRUBBELIG (SUB). Furthermore, SUB is involved in cell wall integrity signaling and regulates the cellular response to reduced levels of cellulose, a central component of the cell wall. Here, we show that continuous exposure to sub-lethal doses of the cellulose biosynthesis inhibitor isoxaben results in altered root hair patterning and floral morphogenesis. Genetically impairing cellulose biosynthesis also results in root hair patterning defects. We further show that isoxaben exerts its developmental effects through the attenuation of SUB signaling. Our evidence indicates that downregulation of SUB is a multi-step process and involves changes in SUB complex architecture at the plasma membrane, enhanced removal of SUB from the cell surface, and downregulation of SUB transcript levels. The results provide molecular insight into how the cell wall regulates cell fate and tissue morphogenesis.

Bacterial nanocellulose: A versatile biopolymer production using a cost-effective wooden disc based rotary reactor.

Bacterial nanocellulose (BNC) has various unique qualities, including high mechanical strength, crystallinity, and high water-holding capacity, which makes it appropriate for a wide range of industrial applications. But its lower yield coupled with its high production cost creates a barrier to its usage. In this study, we have demonstrated the better yield of BNC from an indigenous strain Komagataeibacter rhaeticus MCC-0157 using a rotary disc bioreactor (RDB) having a wooden disc. The RDB was optimized based on the type of disc material, distance between the disc, and rotation speed to get the highest yield of 13.0 g/L dry material using Hestrin-Schramm (H-S) medium. Further, the bioreactor was compared for the BNC production using reported medium, which is used for static condition; the RDB showed up to fivefold increase in comparison with the static condition reported. Komagataeibacter rhaeticus MCC-0157 was previously reported to be one of the highest BNC producing stains, with 8.37 g/L of dry yield in static condition in 15 days incubation. The designed RDB demonstrated 13.0 g/L dry yield of BNC in just 5 days. Other characteristics of BNC remain same as compared with static BNC production, although the difference in the crystallinity index was observed in RDB (84.44%) in comparison with static (89.74%). For the first time, wooden disc was used for rotary bioreactor approach, which demonstrated higher yield of BNC in lesser time and can be further used for sustainable production of BNC at the industrial level.

Overproduction of bacterial cellulose from Acetobacter xylinum BPR2001 using food industries wastes.

In this study, a cost-effective complex culture media containing molasses and corn steep liquor (CSL) was developed for the high production of bacterial cellulose (BC) by investigating the effect of four effective factors on BC production at three levels using Taguchi and combined methods. The predicted and actual values of BC production in optimal conditions by Taguchi and combined methods were 8.41 and 14.52 g/L, respectively. These results showed that the combined method was more suitable for predicting the optimal conditions in the optimization of BC production, the cost of developed culture medium was around 94% cost of HS medium preparation, molasses was the most effective factor in both experimental design methods, and initial pH adjustment had little impact on BC production. Then, the effect of inoculation conditions containing three factors of inoculation age, ethanol addition time, and agitation rate on the increase of BC production at three levels was investigated using the response surface methodology with the Box-Behnken design algorithm. Under the optimal conditions including inoculum age of 3 days, ethanol addition time of 10 days, and stirring speed of 100 rpm, the predicted and experimental results of BC production were 21.61 and 20.21 g/L, respectively. This is among the highest ever reported for BC production, which was achieved with a more cost-effective culture medium containing molasses and CSL.

Photosynthesis-assisted remodeling of three-dimensional printed structures.

The mechanical properties of engineering structures continuously weaken during service life because of material fatigue or degradation. By contrast, living organisms are able to strengthen their mechanical properties by regenerating parts of their structures. For example, plants strengthen their cell structures by transforming photosynthesis-produced glucose into stiff polysaccharides. In this work, we realize hybrid materials that use photosynthesis of embedded chloroplasts to remodel their microstructures. These materials can be used to three-dimensionally (3D)-print functional structures, which are endowed with matrix-strengthening and crack healing when exposed to white light. The mechanism relies on a 3D-printable polymer that allows for an additional cross-linking reaction with photosynthesis-produced glucose in the material bulk or on the interface. The remodeling behavior can be suspended by freezing chloroplasts, regulated by mechanical preloads, and reversed by environmental cues. This work opens the door for the design of hybrid synthetic-living materials, for applications such as smart composites, lightweight structures, and soft robotics.

GhMYB52 Like: A Key Factor That Enhances Lint Yield by Negatively Regulating the Lignin Biosynthesis Pathway in Fibers of Upland Cotton (Gossypium hirsutum L.).

In the context of sustainable agriculture and biomaterial development, understanding and enhancing plant secondary cell wall formation are crucial for improving crop fiber quality and biomass conversion efficiency. This is especially critical for economically important crops like upland cotton (Gossypium hirsutum L.), for which fiber quality and its processing properties are essential. Through comprehensive genome-wide screening and analysis of expression patterns, we identified a particularly high expression of an R2R3 MYB transcription factor, GhMYB52 Like, in the development of the secondary cell wall in cotton fiber cells. Utilizing gene-editing technology to generate a loss-of-function mutant to clarify the role of GhMYB52 Like, we revealed that GhMYB52 Like does not directly contribute to cellulose synthesis in cotton fibers but instead represses a subset of lignin biosynthesis genes, establishing it as a lignin biosynthesis inhibitor. Concurrently, a substantial decrease in the lint index, a critical measure of cotton yield, was noted in parallel with an elevation in lignin levels. This study not only deepens our understanding of the molecular mechanisms underlying cotton fiber development but also offers new perspectives for the molecular improvement of other economically important crops and the enhancement of biomass energy utilization.

PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar.

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. This network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription - quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector-reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis.

Production and analysis of synthesized bacterial cellulose by Enterococcus faecalis strain AEF using Phoenix dactylifera and Musa acuminata fruit extracts.

Bacterial cellulose (BC) is a highly versatile biopolymer renowned for its exceptional mechanical strength, water retention, and biocompatibility. These properties make it a valuable material for various industrial and biomedical applications. In this study, Enterococcus faecalis synthesized extracellular BC, utilizing Phoenix dactylifera and Musa acuminata fruit extracts as sustainable carbon sources. LC-MS analysis identified glucose as the primary carbohydrate in these extracts, providing a suitable substrate for BC production. Scanning Electron Microscopy (SEM) revealed a network of BC nanofibers on Congo red agar plates. ATR-FTIR spectroscopy confirmed the presence of characteristic cellulose functional groups, further supporting BC synthesis. X-ray diffraction (XRD) analysis indicated a high crystallinity index of 71%, consistent with the cellulose I structure, as evidenced by peaks at 16.22°, 21.46°, 22.52°, and 34.70°. Whole-genome sequencing of E. faecalis identified vital genes involved in BC biosynthesis, including bcsA, bcsB, diguanylate cyclase (DGC), and 6-phosphofructokinase (pfkA). Antibiotic susceptibility tests revealed resistance to cefotaxime, ceftazidime, and ceftriaxone, while susceptibility to imipenem was observed. Quantitative assessment demonstrated that higher concentrations of fruit extracts (5.0-20 mg/mL) significantly enhanced BC production. Cytotoxicity testing via the MTT assay confirmed excellent biocompatibility with NIH/3T3 fibroblast cells, showing high cell viability (97-105%). Unlike commonly studied Gram-negative bacteria like Acetobacter xylinum for BC production, this research focuses on Gram-positive Enterococcus faecalis and utilizes Phoenix dactylifera and Musa acuminata fruit extracts as carbon sources. This approach offers a sustainable and promising avenue for BC production.

Expression Atlas of Selaginella moellendorffii Provides Insights into the Evolution of Vasculature, Secondary Metabolism, and Roots.

Selaginella moellendorffii is a representative of the lycophyte lineage that is studied to understand the evolution of land plant traits such as the vasculature, leaves, stems, roots, and secondary metabolism. However, only a few studies have investigated the expression and transcriptional coordination of Selaginella genes, precluding us from understanding the evolution of the transcriptional programs behind these traits. We present a gene expression atlas comprising all major organs, tissue types, and the diurnal gene expression profiles for S. moellendorffii We show that the transcriptional gene module responsible for the biosynthesis of lignocellulose evolved in the ancestor of vascular plants and pinpoint the duplication and subfunctionalization events that generated multiple gene modules involved in the biosynthesis of various cell wall types. We demonstrate how secondary metabolism is transcriptionally coordinated and integrated with other cellular pathways. Finally, we identify root-specific genes and show that the evolution of roots did not coincide with an increased appearance of gene families, suggesting that the development of new organs does not coincide with increased fixation of new gene functions. Our updated database at conekt.plant.tools represents a valuable resource for studying the evolution of genes, gene families, transcriptomes, and functional gene modules in the Archaeplastida kingdom.

Endosidin20 Targets the Cellulose Synthase Catalytic Domain to Inhibit Cellulose Biosynthesis.

Plant cellulose is synthesized by rosette-structured cellulose synthase (CESA) complexes (CSCs). Each CSC is composed of multiple subunits of CESAs representing three different isoforms. Individual CESA proteins contain conserved catalytic domains for catalyzing cellulose synthesis, other domains such as plant-conserved sequences, and class-specific regions that are thought to facilitate complex assembly and CSC trafficking. Because of the current lack of atomic-resolution structures for plant CSCs or CESAs, the molecular mechanism through which CESA catalyzes cellulose synthesis and whether its catalytic activity influences efficient CSC transport at the subcellular level remain unknown. Here, by performing chemical genetic analyses, biochemical assays, structural modeling, and molecular docking, we demonstrate that Endosidin20 (ES20) targets the catalytic site of CESA6 in Arabidopsis (Arabidopsis thaliana). Chemical genetic analysis revealed important amino acids that potentially participate in the catalytic activity of plant CESA6, in addition to previously identified conserved motifs across kingdoms. Using high spatiotemporal resolution live cell imaging, we found that inhibiting the catalytic activity of CESA6 by ES20 treatment reduced the efficiency of CSC transport to the plasma membrane. Our results demonstrate that ES20 is a chemical inhibitor of CESA activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants.

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Endosidin20-1 is more potent than endosidin20 in inhibiting plant cellulose biosynthesis and molecular docking analysis of cellulose biosynthesis inhibitors on modeled cellulose synthase structure.

Endosidin20 (ES20) is a recently identified cellulose biosynthesis inhibitor (CBI) that targets the catalytic site of plant cellulose synthase (CESA). Here, we screened over 600 ES20 analogs and identified nine active analogs named ES20-1 to ES20-9. Among these, endosidin20-1 (ES20-1) had stronger inhibitory effects on plant growth and cellulose biosynthesis than ES20. At the biochemical level, we demonstrated that ES20-1, like ES20, directly interacts with CESA6. At the cellular level, this molecule, like ES20, induced the accumulation of cellulose synthase complexes at the Golgi apparatus and inhibited their secretion to the plasma membrane. Like ES20, ES20-1 likely targets the catalytic site of CESA. However, through molecular docking analysis using a modeled structure of full-length CESA6, we found that both ES20 and ES20-1 might have another target site at the transmembrane regions of CESA6. Besides ES20, other CBIs such as isoxaben, C17, and flupoxam are widely used tools to dissect the mechanism of cellulose biosynthesis and are also valuable resources for the development of herbicides. Here, based on mutant genetic analysis and molecular docking analysis, we have identified the potential target sites of these CBIs on a modeled CESA structure. Some bacteria also produce cellulose, and both ES20 and ES20-1 inhibited bacterial cellulose biosynthesis. Therefore, we conclude that ES20-1 is a more potent analog of ES20 that inhibits intrinsic cellulose biosynthesis in plants, and both ES20 and ES20-1 show an inhibitory effect on bacterial growth and cellulose synthesis, making them excellent tools for exploring the mechanisms of cellulose biosynthesis across kingdoms.

HOMEODOMAIN GLABROUS2 regulates cellulose biosynthesis in seed coat mucilage by activating CELLULOSE SYNTHASE5.

Numerous proteins involved in cellulose biosynthesis and assembly have been functionally characterized. Nevertheless, we have a limited understanding of the mechanisms underlying the transcriptional regulation of the genes that encode these proteins. Here, we report that HOMEODOMAIN GLABROUS2 (HDG2), a Homeobox-Leucine Zipper IV transcription factor, regulates cellulose biosynthesis in Arabidopsis (Arabidopsis thaliana) seed coat mucilage. HDG2 is a transcriptional activator with the transactivation domain located within its Leucine-Zipper domain. Transcripts of HDG2 were detected specifically in seed coat epidermal cells with peak expression at 10 d postanthesis. Disruptions of HDG2 led to seed coat mucilage with aberrant morphology due to a reduction in its crystalline cellulose content. Electrophoretic mobility shift and yeast one-hybrid assays, together with chromatin immunoprecipitation and quantitative PCR, provided evidence that HDG2 directly activates CELLULOSE SYNTHASE5 (CESA5) expression by binding to the L1-box cis-acting element in its promoter. Overexpression of CESA5 partially rescued the mucilage defects of hdg2-3. Together, our data suggest that HDG2 directly activates CESA5 expression and thus is a positive regulator of cellulose biosynthesis in seed coat mucilage.

The Regulation of Cellulose Biosynthesis in Plants.

Cell walls define the shape of plant cells, controlling the extent and orientation of cell elongation, and hence organ growth. The main load-bearing component of plant cell walls is cellulose, and how plants regulate its biosynthesis during development and in response to various environmental perturbations is a central question in plant biology. Cellulose is synthesized by cellulose synthase (CESA) complexes (CSCs) that are assembled in the Golgi apparatus and then delivered to the plasma membrane (PM), where they actively synthesize cellulose. CSCs travel along cortical microtubule paths that define the orientation of synthesis of the cellulose microfibrils. CSCs recycle between the PM and various intracellular compartments, and this trafficking plays an important role in determining the level of cellulose synthesized. In this review, we summarize recent findings in CESA complex organization, CESA posttranslational modifications and trafficking, and other components that interact with CESAs. We also discuss cell wall integrity maintenance, with a focus on how this impacts cellulose biosynthesis.

Observing cellulose biosynthesis and membrane translocation in crystallo.

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. Here, in crystallo enzymology with the catalytically active bacterial cellulose synthase BcsA-BcsB complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate- and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a 'finger helix' that contacts the polymer's terminal glucose. Cooperating with BcsA's gating loop, the finger helix moves 'up' and 'down' in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA's transmembrane channel. This mechanism is validated experimentally by tethering BcsA's finger helix, which inhibits polymer translocation but not elongation.