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 ␣-hemolysin, the first example of an S-layer protein secreted by such a pathway.The outer membrane of the dimorphic gram-negative bacterium Caulobacter crescentus is covered by a protein surface layer (S-layer) composed of a single 1,026-amino-acid protein termed RsaA (13). The S-layer is paracrystalline in nature, exhibiting an array of ring-like subunits (each composed of six copies of RsaA) arranged on a lattice with p6 symmetry and interlinked at the threefold rotational axis (57); proper crystallization of the S-layer is dependent on Ca 2ϩ ions (62, 63). The association between RsaA and the outer membrane is not completely understood, but the protein appears to be anchored to the outer membrane via noncovalent interactions with a specific smooth lipopolysaccharide (LPS) molecule (62). So far, the only known function of the C. crescentus S-layer is to protect cells against predation by a Bde...

Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

“…In a previous study we evaluated the effect of four amino acid insertion mutations on the biogenesis of the C. crescentus S-layer (Bingle et al, 1997). Combining the results of this study using pilin peptide insertions (26 amino acids) with the results obtained previously, a clear correlation is evident between the location of an insertion and its effect on S-layer biogenesis (Fig.…”

“…To the precision of restriction mapping, there were clearly a lack of permissive sites in the C-terminus of the protein; this was not unexpected considering RsaA carries a C-terminal secretion signal, the interruption of which abolishes secretion (Bingle et al, 1996(Bingle et al, , 1997. In contrast to the C-terminus, there did appear to be permissive sites in the extreme N-terminal portion of the protein (e.g.…”

“…1C). In a previous study, we demonstrated the probable proteolytic cleavage of a RsaA: hybrid protein C-terminal to a methionyl residue within the 'passenger' portion of the hybrid (Bingle et al, 1997). Because both of these events involve cleavage of the C-terminal to a hydrophobic residue, it is tempting to speculate that C. crescentus possesses a protease with a chymotrypsin-like cleavage specificity.…”

“…For these reasons, an in vitro method has been developed to study crystallization of the C. crescentus S-layer . Although we are aware that the process of in vitro crystallization may differ in some significant aspects from the in vivo process (Bingle et al, 1997), it is currently the most satisfactory of the alternatives.…”

Molecular mechanisms of Campylobacter fetus surface layer protein expression

“…Most organisms with S-layers secrete their SLPs by the general secretory pathway after cleavage of an N-terminal signal sequence . In contrast, Caulobacter crescentus (Bingle et al, 1997) and C. fetus SLPs do not possess N-terminal signal sequences. Analysis of four cloned C. fetus SLP gene cassettes shows potential secretion signals within their C-termini, consistent with an RTXmediated transport pathway, and mutation analyses confirm this hypothesis (S. A.…”