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.

Processing of cellulose synthase (AcsAB) from Gluconacetobacter hansenii 23769.

The cellulose synthase protein (AcsAB) is encoded by a single gene in Gluconacetobacter hansenii ATCC 23769. We have examined the processing pattern of this enzyme and the localization of the cleavage products by heterologously expressing the truncated portions of the AcsAB protein and using specific antibodies generated against these regions. We found that the AcsAB protein is processed into three polypeptide subunits of molecular masses 46kDa, 34kDa and 95kDa. The 46kDa polypeptide (AcsA(cat)) harbors the conserved glycosyltransferase domain and hence contains the catalytic subunit of the enzyme. This polypeptide is localized in the cytoplasmic membrane. The 34kDa polypeptide (AcsA(reg)) is the regulatory subunit with the cyclic diGMP-binding PilZ domain. This polypeptide is largely cytoplasmic. The 95kDa subunit (AcsB) is of unknown function and contains a predicted signal peptide at its N-terminus. This subunit is localized in the outer membrane. In addition to this, we have also localized the AcsC protein in the outer membrane, confirming its predicted localization based on the OM-signal sequence at its N-terminus.