Structure of the Bacterial Cellulose Ribbon and Its Assembly-Guiding Cytoskeleton by Electron Cryotomography.

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength, and stiffness prevent dehydration and mechanical disruption of the biofilm. Bacteria in the genus Gluconacetobacter secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused-ion-beam milling of native bacterial biofilms to image cellulose-synthesizing Gluconacetobacter hansenii and Gluconacetobacter xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several micrometers in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, which we have named the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We found that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line to form higher-order cellulose structures, such as sheets and ribbons.IMPORTANCE This work's relevance for the microbiology community is twofold. It delivers for the first time high-resolution near-native snapshots of Gluconacetobacter spp. (previously Komagataeibacter spp.) in the process of cellulose ribbon synthesis, in their native biofilm environment. It puts forward a noncharacterized cytoskeleton element associated with the side of the cell where the cellulose synthesis occurs. This represents a step forward in the understanding of the cell-guided process of crystalline cellulose synthesis, studied specifically in the Gluconacetobacter genus and still not fully understood. Additionally, our successful attempt to use cryo-focused-ion-beam milling through biofilms to image the cells in their native environment will drive the community to use this tool for the morphological characterization of other studied biofilms.

Enhanced mechanical properties of bacterial cellulose nanocomposites produced by co-culturing Gluconacetobacter hansenii and Escherichia coli under static conditions.

Including additives in the culture media during bacterial cellulose (BC) biosynthesis is a traditional method to produce BC-based nanocomposites. This study examines a novel fermentation process, which is to co-culture Gluconacetobacter hansenii (G. hansenii) with Escherichia coli (E. coli) under static conditions, to produce BC pellicles with enhanced mechanical properties. The mannose-rich exopolysaccharides (EPS) synthesized by E. coli were incorporated into the BC network and affected the aggregation of co-crystallized microfibrils without significantly changing the crystal sizes of BC. When co-culturing G. hansenii ATCC 23769 with E. coli ATCC 700728, which produced a low concentration of EPS at 3.3 ± 0.7 mg/L, the BC pellicles exhibited a Young's modulus of 4,874 ± 1144 MPa and a stress at break of 80.7 ± 21.1 MPa, which are 81.9% and 79.3% higher than those of pure BC, respectively. The growth dynamics of the two co-cultured strains suggested that the production of BC and EPS were enhanced through co-culturing fermentation.