Characterization of mutants of Caulobacter crescentus defective in surface attachment of the paracrystalline surface layer

Abstract

Strains of Caulobacter crescentus express a paracrystalline surface layer (S-layer) consisting of the protein RsaA. Mutants of C. crescentus NA1000 and CB2, isolated for their ability to grow in the absence of calcium ions, uniformly no longer had the S-layer attached to the cell surface. However, RsaA was still produced, and when colonies grown on calcium-sufficient medium were examined, large two-dimensional arrays of S-layer were found intermixed with the cells. Such arrays were not found in calcium-deficient medium even when high levels of magnesium ions were provided. The arrays could be disrupted with divalent ion chelators and more readily with the calcium-selective ethylene glycol-bis(P-aminoethyl ether)NNN',N'-tetraacetic acid (EGTA).Thus, the outer membrane surface was not needed as a template for self-assembly, but calcium likely was. The cell surface and S-layer gene of assembly-defective mutants of NA1000 were examined to determine the basis of the S-layer surface attachment defect. Mutants had no detectable alteration in the rough lipopolysaccharide (LPS) or a characterized capsular polysaccharide, but another polysaccharide molecule was greatly reduced or absent in all calcium-independent mutants. The molecule was shown to be a smooth LPS with a core sugar and fatty acid complement identical to those of the rough LPS and an 0 polysaccharide of homogeneous length, tentatively considered to be composed of 4,6-dideoxy-4-amino hexose, 3,6-dideoxy-3-amino hexose, and glycerol in equal proportions. This molecule (termed SLPS) was detectable by surface labeling with a specific antiserum only when the S-layer was not present. The rsaA genes from three calcium-independent mutants were cloned and expressed in an S-layer-negative, SLPS-positive strain. A normal S-layer was produced, ruling out defects in rsaA4 in these cases. It is proposed that SLPS is required for S-layer surface attachment, possibly via calcium bridging. The data support the possibility that calcium binding is required to prevent an otherwise lethal effect of SLPS. If true, mutations that eliminate the 0 polysaccharide of SLPS eliminate the lethal effects of calcium-deprived SLPS, at the expense of S-layer attachment.Surface layers (S-layers) are a common feature on the surface of eubacteria and archaebacteria (34). The structures are generally composed of repeated units of a single protein or glycoprotein (44). It is likely that in many bacteria the layer is the outermost layer, directly exposed to the environment (44). In such a position, S-layers may well have roles of protection for the cell or be involved with pathogenesis in the case of pathogenic bacteria (34). Many aspects of this general bacterial structure were summarized in a recent international meeting devoted to such structures (5).Most often, S-layer proteins are assembled on (rather than within) the underlying layer, whether it is a bilayer membrane or a complex of peptidoglycan or pseudomurein (34). Thus, S-layer structural subunits are secreted proteins and must retain b...

Anti-tumor effects of the bacterium caulobacter crescentus in murine tumor models

“…[27][28][29] It is possible that the smooth lipopolysaccharide anchoring the S-layer to the cell surface may have some role in this anti-tumor effects. 16,17 This could be due to nonspecific stimulation of the host immune system. In support of this idea, Caulobacter LPS is known to have adjuvanation properties, stimulating a stronger immune response to an E. coli flagellar antigen.…”

“…Since S layer protein assembles on the outside of the cell, it is a secreted protein that is non-covalently anchored to the cell surface via a specific smooth lipopolysaccharide. 16,17 The gene (rsaA) encoding the C. crescentus S layer subunit protein (RsaA) has been characterized and used to display foreign peptides on the cell surface in a dense, highly ordered manner. 18 Our ongoing studies are directed towards the development of cancer vaccine with Caulobacter-S-layer expressing tandemly repeated copies of the human MUC1 antigen along with additional immune modulating segments.…”

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

“…Analysis of particulate RsaA recovered from the culture fluids of C. crescentus; the particulate material was solubilized with 8 M urea before SDS-PAGE. Lane 1 contains native RsaA (no pilin peptide insertion) released from mutant shedder strain JS3001 (Walker et al, 1994). Lanes 2-9, RsaA with a pilin peptide insertion at: 2, amino acid 69; 3, amino acid 154; 4, amino acid 277; 5, amino acid 353; 6, amino acid 450; 7, amino acid 485; 8 amino acid 551; 9, amino acid 622.…”

Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus The GenBank accession number for the NA1000 rsaADEF and gmd, per, wbqA, wbqR, wbqX and wbqZ sequences reported in this paper is AF06235.

“…The core, a branched chain oligo-Although there is only a limited number of instances identified so far, it appears that in Gram-negative bacteria S-LPS is required for attachment of the S-layer (Boot & Pouwels, 1996 ;Yang et al, 1992). This is the case in C. crescentus where the S-LPS anchors the Slayer to the cell surface (Walker et al, 1994). A region near the N terminus of RsaA appears to be responsible (Bingle et al, 1997b).…”

“…secreted by a Type I secretion mechanism (Awram & Smit, 1998), the RsaA subunits assemble into the S-layer and attach to the outer membrane (Walker et al, 1994).…”