Factors controlling in vitro recrystallization of the Caulobacter crescentus paracrystalline S-layer

Abstract

The S-layer of Caulobacter is a two-dimensional paracrystalline array on the cell surface composed of a single protein, RsaA. We have established conditions for preparation of stable, soluble protein and then efficient in vitro recrystallization of the purified protein. Efficient recrystallization and long range order could not be obtained with pure protein only, though it was apparent that calcium was required for crystallization. Recrystallization was obtained when lipid vesicles were provided, but only when the vesicles contained the specific species of Caulobacter smooth lipopolysaccharide (SLPS) that previous studies implicated as a requirement for attaching the S-layer to the cell surface. The specific type of phospholipids did not appear critical; phospholipids rather different from those present in Caulobacter membranes or archaebacterial tetraether lipids worked equally well. The source of LPS was critical; rough and smooth variants of Salmonella typhimurium LPS as well as the rough form of Caulobacter LPS were ineffective. The requirement for calcium ions for recrystallization was further evaluated; strontium ions could substitute for calcium, and to a lesser extent, cobalt, barium, manganese and magnesium ions also stimulated crystallization. On the other hand, nickel and cadmium provided only weak crystallization stimulation, and zinc, copper, iron, aluminum ions, and the monovalent potassium, sodium, and lithium ions were ineffective. The recrystallization could also be reproduced with Langmuir-Blodgett lipid monolayers at an air-water interface. As with the vesicle experiments, this was only successful when SLPS was incorporated into the lipid mix. The best method for RsaA preparation, leading to apparently monomeric protein that was stable for many months, was an extraction with a low pH aqueous solution. We also achieved recrystallization, albeit at lower efficiency, using RsaA protein solubilized by 8 M urea, a method which allows retrieval of protein from inclusions, when expressed as heterologous protein in Escherichia coli or when retrieved as shed, precipitated protein from certain mutant caulobacters. In summary, the clarification of recrystallization methods has confirmed the requirement of SLPS as a surface attachment component and suggests that its presence in a membrane-like structure greatly stimulates the extent and quality of S-layer formation. The in vitro approach allowed the demonstration that specific ions are capable of participating in crystallization and now provides an assay for the crystallization potential of modified S-layer proteins, whether they were produced in or can be secreted by caulobacters.Caulobacter crescentus and strains related to this species produce S-layers of similar appearance and with comparable structural and biochemical properties (3,14,27,30). The structure is a two-dimensional highly ordered array that is attached to the outer membrane, has no portion that penetrates the outer membrane, and completely covers the bacterium. It is composed of a single ...

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

“…These findings were anticipated since C. crescentus is known to be unable to grow at 37˚C or in the levels of sodium chloride present in environment. 24 Fluorescence activated cell sorting (FACS) of LLC-MUC1. This experiment was designed to evaluate the extent of expression of MUC1 antigen by the LLC-MUC1 transfected cell line.…”

Inactivation of swmA Results in the Loss of an Outer Cell Layer in a Swimming Synechococcus Strain

“…In addition, SwmA is one of the most abundant proteins in motile cells (5), which is a characteristic shared by other S-layer proteins (20) and is consistent with the hypothesis that this protein forms a layer covering the entire cell surface. Also, divalent cations, specifically Ca 2ϩ , have been shown to be important for the integrity, attachment, and recrystallization of S-layers in various bacteria (9,10,21,37), and treatment with divalent cation chelators causes some S-layers to dissociate from the cell surface. Treatment of Synechococcus sp.…”

S-Layer Proteins

“…Whole-cell preparations or partially purified cell products are currently used as attenuated vaccines against fish pathogens (57,143). Another broad application for S-layers is based on the ability of subunits to recrystallize into coherent lattices on functional lipid membranes (105,135), including liposomes (68,75,94). These S-layer-stabilized lipid membranes mimic the supramolecular structure of those archaeal envelopes which possess S-layers as exclusive wall components or virus envelopes.…”