The long range objective of this research is to understand the structure, biogenesis and function of the crystalline surface layer of Caulobacter crescentus. Our work is oriented to discerning fundamental information about the secretion and assembly of this membrane structure and its participation in cellular morphogenesis. Specific objective of this proposal are to 1) Continue structure analysis of this multicomponent structure. Methods of study combine protein purification, antibody labelling, computer- analyzed optical diffraction and advanced electron microscope techniques. 2) Analyze synthesis and export of surface array components using electron microscope immunocytochemical methods. Postembedding thin-section labeling techniques and surface labelling with colloidal gold will be used to determine the morphological pathway of synthesis and secretion. The study focusses on the dominant protein of the surface layer, not only as it is expressed if Caulobacter but also the equivalent process which occurs which occurs when this protein is expressed as a cloned product in E. coli. 3) Begin molecular genetic analysis of the regulation of secretion and self assembly. Using a cloned gene for the major protein of the surface layer we shall combine in vivo and in vitro mutagenesis methods, DNA sequence analysis, and antibody based assays for this protein to determine if and what kind of DNA sequence information for correct secretion, assembly and interaction with other components of the surface array is encoded in the protein molecule and its flanking sequences. Because the major protein is synthesized and exported in E. coli from a cloned gene, it will be possible to analyze molecular genetics in both Caulobacter and E. coli strains. Analysis in E. coli will expedite and augment studies in Caulobacter since there is considerable existing information and mutant strains which are characterized in E. coli.
