Functional analysis of the accessory protein TapA in Bacillus subtilis amyloid fiber assembly.
Bacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules.
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
Diego
D
Romero
Hera
H
Vlamakis
Richard
R
Losick
Roberto
R
Kolter
D019943:Amino Acid Substitution; D000682:Amyloid / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development; D004252:DNA Mutational Analysis; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization
D000682:Amyloid; D001426:Bacterial Proteins; C119938:TasA protein, Bacillus subtilis