Amide bonds assemble pili on the surface of bacilli

Abstract

Pilin precursors are the building blocks of pili on the surface of Gram-positive bacteria; however, the assembly mechanisms of these adhesive fibers are unknown. Here, we describe the chemical bonds that assemble BcpA pilin subunits on the surface of Bacillus cereus. Sortase D cleaves BcpA precursor between the threonine (T) and the glycine (G) residues of its LPXTG sorting signal and catalyzes formation of an amide bond between threonine (T) of the sorting signal and lysine (K) in the YPKN motif of another BcpA subunit. Three CNA B domains of BcpA generate intramolecular amide bonds, and one of these contributes also to pilus formation. Conservation of catalysts and structural elements in pilin precursors in Gram-positive bacteria suggests a universal mechanism of fiber assembly.CNA B domain ͉ LPXTG sorting signal ͉ sortase ͉ YPKN motif

Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili

“…5A). Asparagine 163 (YPKN 163 ) of BcpA forms an intramolecular isopeptide bond that is dispensable for pilus assembly, as substitution of asparagine 163 with alanine abolished isopeptide bond formation without affecting pilus assembly (16). As expected, introduction of the bcpA N163A mutation in pJB213 (bcpA N163A, LAVAA , srtD, bcpB MH6 ) had no effect on the ability of sortase D to generate the transpeptidation product BcpB MH6 -BcpA LAVAA (Fig.…”

“…BcpB H6 -BcpA was purified by a second round of Ni-NTA affinity chromatography under denaturing conditions and separated by reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection using a C18 column with a linear gradient from 1 to 99% acetonitrile (CH 3 CN) in 0.1% formic acid in 100 min, as previously reported for BcpA pilin peptides (16) and the BcpA anchor structure (19).…”

“…Finally, sortase D catalyzes transpeptidation reactions in the assembly of pili (14,15). Sortase D recognizes the amino group of lysine residues within the YPKN motif of pilin subunits as nucleophiles (16). The resultant sortase D-catalyzed amide bond links adjacent pilin subunits to grow the pilus fiber (16,17).…”

“…After translocation across the plasma membrane, P2 precursor species arise from removal of the signal peptide from P1 precursors by a signal peptidase (16). Bacillus cereus pili are composed of two subunits; that is, the major pilin, BcpA, and the minor pilin, BcpB (15).…”

“…In contrast to BcpA, which is deposited throughout the pilus, BcpB is found at fiber tip (15). Sortase D cleaves the BcpA LPXTG motif sorting signal between the threonine and glycine residues to form an amide bond to the ⑀-amino group of the lysine within the YPKN motif of adjacent BcpA subunits (16). However, sortase A also cleaves BcpA precursors, which are subsequently linked to the side chain amino group of meso-diaminopimelic acid within lipid II (19).…”