Conservation and Specificity in Bacillus Biofilm Dynamics: On Structure and Function of B. cereus Camelysins

• The mechanism of biofilm filament genesis by the B. cereus TasA-like protein CalY2 via donor strand complementation is unraveled and found to be different from B. subtilis where initiation by TapA is important. • Surprisingly, its second TasA-like protein CalY1 does not form filaments in vitro, despite 60% sequence identify, since it occurs as polydisperse mixture of oligomers as known from small heat shock proteins. • The C-terminal sequence segments of TasA-like proteins are very different even between related bacteria and shown here to contribute substantially to specificity in filament formation by a combination of solid-state NMR, Alphafold and the generation of a CalY2 deletion variant lacking the four C-terminal residues. • CalY1 and CalY2 appear both as folded monomers in solution of which the structure of the functionally important β1-β2-β3 unit is incorrectly predicted by AlphaFold 3 in a consistent manner. The B. cereus family comprises members highly pathogenic for mammals or insects, with B. anthracis and B. thuringiensis respectively as notable examples. The biofilm operon of these bacteria encodes two TasA-like proteins, the 60% identical Camelysins CalY1 and CalY2. In this study, we observed that at neutral pH CalY2 alone polymerizes readily into filaments, whereas CalY1 forms a polydispersed mixture of oligomers without filament formation. However, at basic or acidic pH CalY1 also modestly polymerizes. CalY2 polymerization into filaments involves ß-sheet remodeling via donor strand complementation, as demonstrated here by a combination of NMR and AlphaFold studies. In contrast to TasA of B. subtilis , this process is spontaneous and does not require initiation by a TapA homolog. NMR studies show that the functionally relevant region (β1-β2-β3) of the CalY2 monomer structure closely resembles that of B. subtilis TasA, and differs from AlphaFold models. A survey of AlphaFold 2 predictions on 12 homologous B. cereus group Camelysins yielded only four correctly predicted β1-β2-β3 segments, which decreased to one when using AlphaFold 3. Since crucial residues in the protomer contact region are conserved among TasA-like proteins, we investigated whether family members of different species could form mixed filaments. NMR revealed features in CalY2 filaments that are structurally conserved with TasA filaments but sequentially different, promoting specificity. These interactions and differences, respectively, involve the C-terminus and the beginning of β3, which most likely hinder joint TasA and CalY1 copolymerization. A protease activity could not be observed for the heterologously expressed B. cereus Camelysins. The B. cereus group includes extremely harmful and surprisingly benign bacterial strains. The Anthrax-toxin-producing B. anthracis is one of the most toxic bacterial threats to man, whereas B. thuringiensis toxin is used as a biological insecticide. Other B. cereus strains pose problems in food production and medical implant usage. These bacteria can exist as biofilms allowing them to survive and proliferate, an essential feature of which are protein filaments. Here we characterize the B. cereus Camelysins CalY1 and CalY2 and compare their structure and filament formation with B. subtilis filaments to understand principles determining patterns of conservation and specificity. This investigation provides the basis for developing novel means to suppress or enhance biofilms with potential benefits for plant protection.