CELL-SURFACE DISPLAY OF A PSEUDOMONAS-AERUGINOSA STRAIN-K PILIN PEPTIDE WITHIN THE PARACRYSTALLINE S-LAYER OF CAULOBACTER-CRESCENTUS

The paracrystalline surface (S)-layer of Caulobacter crescentus is com posed of a single secreted protein (RsaA) that interlocks in a hexagon al pattern to completely envelop the bacterium. Using a genetic approa ch, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa st rain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inser ted pilin peptide on the C. crescentus cell surface as a part of the S -layer. Eleven such sites (widely separated in the primary sequence) w ere identified, demonstrating for the first time that S-layers can be readily exploited as carrier proteins to display 'epitope-size' hetero logous peptides on bacterial cell surfaces. Whereas intact RsaA molecu les carrying a pilin peptide could always be found on the surface of C . crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolyti c cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin pept ide insert with both fragments of the S-layer protein remaining anchor ed to the outer membrane. The other proteolytic phenomenon was charact erized by cleavage of the S-layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface-anchored 26 kDa proteolytic fragment bearing the R saA N-terminus; the C-terminal cleavage product carrying the pilin pep tide was released into the growth medium. When the results of this wor k were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the loca tion of a peptide insertion and its effect on S-layer biogenesis: (i) insertions in the extreme N-terminus of RsaA either produce no apparen t effect on S-layer biogenesis or disrupt surface-anchoring of the pro tein; (ii) insertions in the extreme C-terminus either produce no appa rent effect on S-layer biogenesis or disrupt protein secretion; and (i ii) insertions more centrally located in the protein either have no ap parent effect on S-layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignme nt of the RsaA N- and C-terminus as regions that are important for sur face anchoring and secretion respectively.