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Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus
Author(s) -
Bingle Wade H.,
Nomellini John F.,
Smit John
Publication year - 1997
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1997.5711932.x
Subject(s) - caulobacter crescentus , pilin , pilus , s layer , cleavage (geology) , biology , signal peptide , peptide , peptide sequence , signal peptidase , biochemistry , microbiology and biotechnology , biophysics , gene , bacterial protein , escherichia coli , paleontology , fracture (geology)
The paracrystalline surface (S)‐layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C . crescentus cell surface as a part of the S‐layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S‐layers can be readily exploited as carrier proteins to display ‘epitope‐size’ heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules 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 proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S‐layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized 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 RsaA N‐terminus; the C‐terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S‐layer biogenesis: (i) insertions in the extreme N‐terminus of RsaA either produce no apparent effect on S‐layer biogenesis or disrupt surface‐anchoring of the protein; (ii) insertions in the extreme C‐terminus either produce no apparent effect on S‐layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S‐layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N‐ and C‐terminus as regions that are important for surface anchoring and secretion respectively.