Studies of the cell surface of caulobacter crescentus

Caulobacter crescentus is a Gram-negative eubacteria which produces a surface layer (S-layer). S-layers are paracrystalline assemblies of protein that cover the outer surface of some eubacteria and archaebacteria cells. The method by which the protein subunits composing the S-layer of C. crescentus, RsaA, interact to form the array and attach to the cell was examined in this thesis. The S-layer was extracted from the cell surface of C. crescentus NA1000 by treating cells with a pH 2 solution or a solution containing 10 mM ethylene glycol-bis(ß-aminoethylether)- N,N,N’,N’-tetraacetic acid (EGTA). The extracted extract was examined by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE) and found to consist of nearly pure RsaA. The isolated S-layer was amorphous in structure but could reassemble in vitro into a crystalline array in the presence of calcium ions. Two mutants of C. crescentus NA1000, JS1001 and JS1002, selected for the ability to grow in the absence of calcium had the additional phenotype of being unable to attach the S-layer to the cell surface although they produced a wild-type RsaA. These mutants shed the S-layer into the surrounding medium during growth. Methods were developed to identify, isolate and purify the cell surface molecules of the wild-type and S-layer attachment-defective strains. It was determined that the mutant strains did not produce a smooth lipopolysaccharide (LPS) although they produced the wild type rough LPS and extracellular polysaccharide. The smooth LPS (termed the S-layer associated oligosaccharide or SAO) was very homogeneous in length as determined by SDS-PAGE and silver staining. RsaA negative strains that could form and attach an S-layer on the cell surface if RsaA was expressed on a plasmid vector were also shown to produce SAO. All S-layer attachment-defective mutants examined did not produced SAO. Two cosmids were identified that partially restored the production of SAO in the mutant strain JS1001 however restoration of S-layer attachment did not occur.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat