Caulobacter crescentus Synthesizes an S-Layer-Editing Metalloprotease Possessing a Domain Sharing Sequence Similarity with Its Paracrystalline S-Layer Protein

Abstract

Strains of Caulobacter crescentus elaborate an S-layer, a two-dimensional protein latticework which covers the cell surface. The S-layer protein (RsaA) is secreted by a type I mechanism (relying on a C-terminal signal) and is unusual among type I secreted proteins because high levels of protein are produced continuously. In efforts to adapt the S-layer for display of foreign peptides and proteins, we noted a proteolytic activity that affected S-layer monomers with foreign inserts. The cleavage was precise, resulting in fragments with an unambiguous N-terminal sequence. We developed an assay to screen for loss of this activity (i.e., presentation of foreign peptides without degradation), using transposon and traditional mutagenesis. A metalloprotease gene designated sap (S-layer-associated protease) was identified which could complement the protease-negative mutants. The N-terminal half of Sap possessed significant similarity to other type I secreted proteases (e.g., alkaline protease of Pseudomonas aeruginosa), including the characteristic RTX repeat sequences, but the C-terminal half which normally includes the type I secretion signal exhibited no such similarity. Instead, there was a region of significant similarity to the N-terminal region of RsaA. We hypothesize that Sap evolved by combining the catalytic portion of a type I secreted protease with an S-layer-like protein, perhaps to associate with nascent S-layer monomers to "scan" for modifications.

Two Outer Membrane Proteins Are Required for Maximal Type I Secretion of the Caulobacter crescentus S-Layer Protein

“…One solution to this problem could be rapid degradation of protein that cannot be exported. We have suggested that the S-layer-associated protease (the product of sapA), which appears poised to recognize defects in the nascent RsaA monomer, might play such a role (47). The data here, however, gave no indication that detectable degradation occurs.…”

“…On this latter point, a preliminary threading of the RsaF a and RsaF b proteins onto the homologous TolC protein (for which the structure has been determined) predicted exposed chargedense regions in the interior of the transporter channel (data not shown). We estimate that this may be the reason why the addition of the furin cleavage site (RKKR) leads to a blockage of RsaA transport, while a variety of other recombinant RsaA proteins with 100 to 200 amino acid insertions have been readily secreted (34,47).…”

S-Layer Anchoring and Localization of an S-Layer-Associated Protease in Caulobacter crescentus

“…5, compare lanes 1 and 4 or lanes 2 and 5). In JS4015, the Sap deficiency derives from a point mutation in the active site (38), which reduces its activity but may not abolish it completely and so may allow for some residual cleavage of RsaA(1-277). This may account for the presence of a small amount of the cleaved version of RsaA(1-277) when JS4015 cells were used as target cells for RsaA(1-277) reattachment (Fig.…”

“…This contrasts with anchoring domains for S-layer proteins of grampositive bacteria, which are usually three repeats of the ϳ55-amino-acid SLH domain (4). Although the native function of Sap is unknown, our previous model of Sap-mediated editing of the recombinant S-layer suggested that because the C terminus of Sap is homologous with the N terminus of RsaA, Sap associates with nascent, but still internal, RsaA monomers and cleaves some mutant versions of RsaA (38). A recombinant S-layer protein subjected to Sap proteolysis would result in the separation of the RsaA C-terminal secretion signal from the rest of RsaA.…”

“…If so, some Sap substrates [such as RsaA:VP2C⌬ (38) and RsaA(1-277)] would be cleaved by Sap on the cell surface rather than in the cytoplasm. Thus far, efforts to examine the culture supernatant have concluded that little or no Sap is found free of cells.…”