Isopeptide bonds of the major pilin protein BcpA influence pilus structure and bundle formation on the surface of Bacillus cereus

• Published in: Molecular microbiology Vol. 85; no. 1; pp. 152 - 163
• Main Authors: Hendrickx, Antoni P. A., Poor, Catherine B., Jureller, Justin E., Budzik, Jonathan M., He, Chuan, Schneewind, Olaf
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.07.2012; Blackwell
• Subjects: Bacillus cereus; Bacillus cereus - chemistry; Bacillus cereus - genetics; Bacteria; Bacteriology; Biological and medical sciences; Catalysts; Chemical bonds; Fimbriae Proteins - chemistry; Fimbriae Proteins - genetics; Fimbriae, Bacterial - chemistry; Fimbriae, Bacterial - ultrastructure; Fundamental and applied biological sciences. Psychology; Gram-positive bacteria; Microbiology; Microscopy; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Miscellaneous; Mutation; Peptides; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Tertiary; Bacteria; Pilus; Bacillus cereus; Bacillales; Bacillaceae; Protein
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/j.1365-2958.2012.08098.x

Summary Bacillus cereus strains elaborate pili on their surface using a mechanism of sortase‐mediated cross‐linking of major and minor pilus components. Here we used a combination of electron microscopy and atomic force microscopy to visualize these structures. Pili occur as single, double or higher order assemblies of filaments formed from monomers of the major pilin, BcpA, capped by the minor pilin, BcpB. Previous studies demonstrated that within assembled pili, four domains of BcpA – CNA1, CNA2, XNA and CNA3– each acquire intramolecular lysine‐asparagine isopeptide bonds formed via catalytic glutamic acid or aspartic acid residues. Here we showed that mutants unable to form the intramolecular isopeptide bonds in the CNA2 or CNA3 domains retain the ability to form pilus bundles. A mutant lacking the CNA1 isopeptide bond assembled deformed pilin subunits that failed to associate as bundles. X‐ray crystallography revealed that the BcpA variant Asp312Ala, lacking an aspartyl catalyst, did not generate the isopeptide bond within the jelly‐roll structure of XNA. The Asp312Ala mutant was also unable to form bundles and promoted the assembly of deformed pili. Thus, structural integrity of the CNA1 and XNA domains are determinants for the association of pili into higher order bundle structures and determine native pilus structure.