title: Isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters.
abstract: Several methods for isolation of the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A were evaluated. Treatment of cells with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 2 was the most effective means of selectively removing RsaA from cells, and after neutralization, the protein was capable of reassembling into a paracrystalline structure. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid treatment could also be used to extract RsaA and yielded protein capable of reassembly. The success of the methods was likely related to disruption of calcium-mediated bonding; calcium was required for recrystallization, while magnesium and strontium ions were ineffective. Antibody was raised against purified RsaA and, along with the S-layer extraction techniques, was used to evaluate 42 strains of caulobacters isolated from a variety of aquatic and wastewater treatment locations. A single characteristic protein could be isolated from the 35 strains that produced an S layer; with one exception, no proteins were extracted from strains that had no S layer. The presumed S-layer proteins ranged in size from 100 to 193 kDa. All of these proteins specifically reacted with anti-RsaA serum by Western immunoblot analysis. In strain CB15A, a specific S-layer-associated oligosaccharide has been proposed to be involved in a calcium-mediated attachment of the S layer to the cell surface. This molecule was detected by Western immunoblotting with a specific antiserum and on polyacrylamide gels stained for polysaccharides. A comparable band was found in all S-layer-producing strains and for most, S-layer-associated oligosaccharide-specific antibody reacted with them in Western analysis. Overall, in freshwater caulobacters at least portions of their S-layer structures appear to be strongly conserved entities, as well as the means of attachment to the cell surface.
authors.affiliation: Department of Microbiology, University of British Columbia, Vancouver, Canada.
authors.forename: S G
authors.initials: SG
authors.lastname: Walker
authors.forename: S H
authors.initials: SH
authors.lastname: Smith
authors.forename: J
authors.initials: J
authors.lastname: Smit
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D016934:Caulobacter / Q000737:chemistry* / Q000648:ultrastructure; D003461:Crystallography; D005618:Fresh Water; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D011135:Polysaccharides, Bacterial / Q000737:chemistry; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D011135:Polysaccharides, Bacterial