title: Linker mutagenesis of the Caulobacter crescentus S-layer protein: toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion.
abstract: Linker insertion mutagenesis was used to modify the paracrystalline surface layer (S-layer) protein (RsaA) of the gram-negative bacterium Caulobacter crescentus. Eleven unique BamHI linker insertions in the cloned rsaA gene were identified; at the protein level, these linker insertions introduced 4 to 6 amino acids at positions ranging from the extreme N terminus to the extreme C terminus of the 1,026-amino-acid RsaA protein. All linker-peptide insertions in the RsaA N terminus caused the secreted protein to be shed into the growth medium, suggesting that the RsaA N terminus is involved in cell surface anchoring. One linker-peptide insertion in the RsaA C terminus (amino acid 784) had no effect on S-layer biogenesis, while another (amino acid 907) disrupted secretion of the protein, suggesting that RsaA possesses a secretion signal lying C terminal to amino acid 784, near or including amino acid 907. Unlike extreme N- or C-terminal linker-peptide insertions, those more centrally located in the RsaA primary sequence had no apparent effect on S-layer biogenesis. By using a newly introduced linker-encoded restriction site, a 3' fragment of the rsaA gene encoding the last 242 C-terminal amino acids of the S-layer protein was expressed in C. crescentus from heterologous Escherichia coli lacZ transcription and translation initiation information. This C-terminal portion of RsaA was secreted into the growth medium, confirming the presence of a C-terminal secretion signal. The use of the RsaA C terminus for the secretion of heterologous proteins in C. crescentus was explored by fusing 109 amino acids of an envelope glycoprotein from infectious hematopoietic necrosis virus, a pathogen of salmonid fish, to the last 242 amino acids of the RsaA C terminus. The resulting hybrid protein was successfully secreted into the growth medium and accounted for 10% of total protein in a stationary-phase culture. Based on these results and features of the RsaA primary sequence, we propose that the C. crescentus S-layer protein is secreted by a type I secretion system, relying on a stable C-terminal secretion signal in a manner analogous to E. coli alpha-hemolysin, the first example of an S-layer protein secreted by such a pathway.
authors.affiliation: Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada.
authors.forename: W H
authors.initials: WH
authors.lastname: Bingle
authors.forename: J F
authors.initials: JF
authors.lastname: Nomellini
authors.forename: J
authors.initials: J
authors.lastname: Smit
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D016935:Caulobacter crescentus / Q000235:genetics*; D002462:Cell Membrane / Q000648:ultrastructure; D004252:DNA Mutational Analysis; D005798:Genes, Bacterial; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C104040:IHNVG protein, Infectious hematopoietic necrosis virus; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C055764:S-layer proteins; D014759:Viral Envelope Proteins