Secretion of the Caulobacter crescentus S-Layer Protein: Further Localization of the C-Terminal Secretion Signal and Its Use for Secretion of Recombinant Proteins

Abstract

The secretion signal of the Caulobacter crescentus S-layer protein (RsaA) was localized to the C-terminal 82 amino acids of the molecule. Protein yield studies showed that 336 or 242 C-terminal residues of RsaA mediated secretion of >50 mg of a cellulase passenger protein per liter to the culture fluids.The gram-negative bacterium Caulobacter crescentus possesses a paracrystalline protein surface (S) layer covering the outer membrane (24). The S-layer protein (RsaA) is secreted by a dedicated three-component ABC transporter (type I) secretion system (1, 23). Proteins secreted by type I systems typically exhibit two features: (i) an extreme C-terminal secretion signal located within the last 60 amino acids (aa) which is not cleaved as part of the secretion process and (ii) more-Nterminal glycine-rich ("RTX") motifs, nine-residue sequences which include the consensus sequence GGXGXD. These motifs are responsible for Ca 2ϩ binding and are also thought to be important for proper presentation of the secretion signal to the secretion machinery (3,10,18,19). RsaA possesses six RTX motifs which exactly match the consensus sequence, but the exact location of the secretion signal is unclear (13). It is known that the signal must lie within the 242 C-terminal aa because this portion of RsaA is capable of autonomous secretion (5).In 1996, no S-layer protein was known to be secreted by a type I secretion system (9). Since that time, the putative S-layer protein of Serratia marcescens (17) and the S-layer proteins of two Campylobacter species (see, e.g., reference 26) have been shown or are suspected to be secreted by type I systems. Problems with defining the C-terminal secretion signals of proteins secreted by type I systems are that these sequences are not cleaved as part of the secretion process and that, as a group, the C termini of such proteins do not display a high degree of primary structural homology (4). Families of type I secreted proteins which exhibit similar C-terminal sequences have been recognized (4), but it is not clear to which current family, if any, the C. crescentus S-layer protein belongs (1,17,26).The experiments reported in this study were undertaken primarily to define the location of the RsaA secretion signal. A secondary goal was of a more applied nature. In a previous report (5), we demonstrated that the last 242 C-terminal aa of RsaA could be used to secrete a 109-aa segment of the infectious hematopoietic necrosis virus surface glycoprotein from C. crescentus. The hybrid protein failed to anchor to the cell surface but formed macroscopic aggregates in the culture fluids which could be recovered in highly purified form by simple coarse filtration of the culture through nylon mesh. This result suggested that the C. crescentus S-layer protein secretion system could be an attractive one for low-cost protein production. For this reason, it was of interest to determine whether other passenger proteins could be secreted when they were linked to the RsaA C terminus and whether smaller portions of the R...

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

“…Mutations (8) and truncations (9) in the RsaA extreme N terminus lead to an S-layer shedding phenotype. A species of smooth lipopolysaccharide (SLPS), whose O side chain is composed at least in part of N-acetylperosamine, is found on the outer membrane of C. crescentus (2).…”

S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface ofCaulobacter crescentus: Development of an Immunoactive Reagent

“…All of these considerations have implications for the development of biotechnological applications based on the expression of foreign proteins using the Caulobacter S-layer protein. Indeed, we have exploited the S-layer secretion apparatus in order to express heterologous proteins as large as 600 aa in size (23) by fusing them to portions of RsaA containing the C-terminal secretion signal (5)(6)(7). The resulting protein products are secreted from C. crescentus at yields ranging up to 250 mg/liter (7).…”

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

“…Prior work has shown that C. crescentus, a Gram-negative bacterium, has an S-layer subunit composed of a single highly expressed (27) protein (RsaA), secreted by a type I mechanism, such that there is no cleaved N-terminal signal leader but there is an uncleaved C-terminal secretion signal (4,11). Six RsaA monomers (12) form the characteristic hexagonal core with p6 symmetry seen by image analysis (47,48), and the 2D lattice is completed by hexagonal cores connected at junction points with p3 symmetry (Fig.…”