Type IV pili-associated secretion of a biofilm matrix protein from that forms intermolecular isopeptide bonds.

is a Gram-positive, anaerobic, spore-forming, bacterial pathogen of humans and animals. also produces type IV pili (T4P) and has two complete sets of T4P-associated genes, one of which has been shown to produce surface pili needed for cell adherence. One hypothesis about the second set of T4P genes is that they comprise a system analogous to the type II secretion systems (TTSS) found in Gram-negative bacteria, which is used to export folded proteins from the periplasm through the outer membrane to the extracellular environment. Gram-positive bacteria have a similar secretion barrier in the thick peptidoglycan (PG) layer, which blocks secretion of folded proteins >25 kD. To determine if the T4P-associated genes comprise a Gram-positive TTSS, the secretome of mutants lacking type IV pilins were examined and a single protein, a von Willebrand A domain containing protein, BsaC (CPE0517), was identified as being dependent on pilin PilA3 for secretion. The gene is in an operon with genes encoding a SipW signal peptidase and two putative biofilm matrix proteins BsaA and BsaB, both of which have remote homology to biofilm protein TasA. Since BsaA forms long oligomers that are secreted, we analyzed BsaA monomer interactions with modeling. These models projected that the monomers formed isopeptide bonds as part of a donor strand exchange process, in which an N-terminal disordered loop of one monomer intercalates into a beta sheet structure of an adjacent monomer and reforms into a beta sheet with subsequent isopeptide bond formation. Mutations in residues predicted to form the isopeptide bonds led to loss of oligomerization, supporting an exchange and lock mechanism. Phylogenetic analysis showed the BsaA family of proteins are widespread among bacteria and archaea but only a subset is predicted to form isopeptide bonds.