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

Linker insertion mutagenesis was used to modify the paracrystalline su rface layer (S-layer) protein (RsaA) of the gram-negative bacterium Ca ulobacter crescentus, Eleven unique BamHI linker insertions in the clo ned rsaA gene were identified; at the protein level, these linker inse rtions introduced 4 to 6 amino acids at positions ranging from the ext reme N terminus to the extreme C terminus of the 1,026-amino-acid RsaA protein,;yl linker peptide insertions in the RsaA N terminus caused t he 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 effe ct on S-layer biogenesis, while another (amino acid 907) disrupted sec retion of the protein, suggesting that RsaA possesses a secretion sign al lying C terminal to amino acid 784, near or including amino acid 90 7, Unlike extreme N- or C-terminal linker-peptide insertions, those mo re centrally located in the RsaA primary sequence had no apparent effe ct on S-layer biogenesis. By using a newly introduced linker-encoded r estriction 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. cres centus from heterologous Escherichia coli lacZ transcription and trans lation initiation information, This C-terminal portion of RsaA was sec reted 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 amin o acids of an envelope glycoprotein from infectious hematopoietic necr osis virus, a pathogen of salmonid fish, to the last 242 amino acids o f the RsaA C terminus, The resulting hybrid protein was successfully s ecreted 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 p rotein is secreted by a type I secretion system, relying on a stable C -terminal secretion signal in a manner analogous to E. coli alpha-hemo lysin, the first example of an S-layer protein secreted by such a path way.