FACTORS CONTROLLING IN-VITRO RECRYSTALLIZATION OF THE CAULOBACTER-CRESCENTUS PARACRYSTALLINE S-LAYER

The S-layer of Caulobacter is a two-dimensional paracrystalline array on the cell surface composed of a single protein, RsaA. We have establ ished conditions for preparation of stable, soluble protein and then e fficient in vitro recrystallization of the purified protein. Efficient recrystallization and long range order could not be obtained with pur e protein only, though it was apparent that calcium was required for c rystallization. Recrystallization was obtained when lipid vesicles wer e 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 surf ace. The specific type of phospholipids did not appear critical; phosp holipids rather different from those present in Caulobacter membranes or archaebacterial tetraether lipids worked equally well. The source o f LPS was critical; rough and smooth variants of Salmonella typhimuriu m LPS as well as the rough form of Caulobacter LPS were ineffective. T he requirement for calcium ions for recrystallization was further eval uated; strontium ions could substitute for calcium, and to a lesser ex tent, cobalt, barium, manganese and magnesium ions also stimulated cry stallization. On the other hand, nickel and cadmium provided only weak crystallization stimulation, and zinc, copper, iron, aluminum ions, a nd the monovalent potassium, sodium, and lithium ions were ineffective . The recrystallization could also be reproduced with Langmuir-Blodget t lipid monolayers at an air-water interface. As with the vesicle expe riments, 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, al beit at lower efficiency, using RsaA protein solubilized by 8 M urea, a method which allows retrieval of protein from inclusions, when expre ssed as heterologous protein in Escherichia coli or when retrieved as shed, precipitated protein from certain mutant caulobacters. In summar y, the clarification of recrystallization methods has confirmed the re quirement of SLPS as a surface attachment component and suggests that its presence in a membrane-like structure greatly stimulates the exten t and quality of S-layer formation. The in vitro approach allowed the demonstration that specific ions are capable of participating in cryst allization 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.