Cellulose is the most abundant biopolymer on Earth and while it is the predominant building constituent of plants, it is also a key extracellular matrix component in the biofilms of many bacteria.
In our seminal 2017 study (Krasteva et al. 2017), we showed that in Escherichia coli, the BcsAB cellulose synthase tandem assembles into a stable megadalton-sized macrocomplex with multiple accessory subunits. In recent works (Zouhir et al. 2020, Abidi et al. 2021 and Anso et al. 2024), we presented high-resolution cryo-EM structures of the assembled Bcs secretion macrocomplex and sub-assemblies, as well as multiple crystallographic structures of individual Bcs subunits and/or multi-component complexes. We demonstrated that the E. coli Bcs macrocomplex features an unexpected subunit stoichiometry, which begs a rethinking of the generally accepted model for equimolar BcsAB synthase assemblies throughout the bacterial domain of life. In it, the essential for secretion BcsRQ appears to play essential roles in both ATP-dependent membrane targeting of the synthase and in its post-translational catalytic activation. We also showed an unexpected mechanism for asymmetric secretion system assembly via superhelical periplasmic BcsB oligomerization, which is likely conserved among enterobacteria. We further revealed that enterobacterial BcsA has evolved an additional N-terminal domain that recruits three copies of the phosphoethanolamine-transferase BcsG for efficient polymer modification. We also demonstrated that enterobacterial BcsE packs a subtle but diverse toolkit to fine-tune cellulose production and provided structural and functional data that support multidomain evolution, fold conservation and synthase-activating, intercalated c-di-GMP complexation on one hand, together with BcsRQ recruitment and membrane targeting through BcsF interactions on the other. We further showed that through discrete conformations and dimerization interfaces BcsE then acts as a high-affinity c-di-GMP sensor that recruits the synthase-activating BcsRQ tandem even in the absence of direct dinucleotide-synthase interactions. Together, these results can explain cellulose secretion initiation in diverse layers of the mature biofilm architecture.
Despite strong conservation of the BcsAB tandem, in the economically relevant cellulose superproducer G. hansenii cellulose is secreted in a drastically different manner: a longitudinal nanoarray of synthase terminal complexes (TCs) assembles the EPS into a crystalline cellulose ribbon with implications in cell motility, flotation and substrate colonization. Crystalline BC secretion is dependent on two accessory subunits earlier proposed to interact in the periplasm, BcsD and BcsH. We recently provided the first atomic-resolution insights into BcsHD mediated BC crystallinity: proline-rich BcsH drives BcsD oligomerization into a three-dimensional supramolecular scaffold. We showed that, in situ, the BcsHD assemblies share remarkable morphological similarities with the recently discovered cortical belt, namely an intracellular cytoskeleton that spatially correlates with the cellulose exit sites and the assembled crystalline cellulose ribbon. Finally, we detected specific protein-protein interactions between the BcsHD components and the regulatory BcsAPilZ module, further supporting that BcsHD features an unexpected intracellular localization for inside-out control of TC array formation and crystalline cellulose secretion (Abidi et al. 2022).
Interestingly, while BcsH has been postulated as specific for the alpha-proteobacterial Gluconacetobacter lineage, BcsD homologs are widespread in plant-colonizing bacteria, including symbionts and pathogens secreting semi-crystalline or chemically modified cellulose. We demonstrated that in beta and gamma-Proteobacteria, BcsD can adopt a different quaternary structure via an additional N-terminal alpha-helix and together with alternative proline-rich partners (BcsP and BcsO) can form intracellular, tiled, synthase-interaction cytoskeletal scaffolds likely serving to drive synthase array formation and recruit regulatory complexes that determine cellulose secretion and/or biofilm architecture (Sana et al. 2024).
