Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Serratia marcescens LRL781, a gram-negative bacterium, produces an extracellular protease. Evidence is provided for an outer membrane-associated, larger molecular weight, enzymatically inactive precursor of the protease. Antiserum was prepared against the extracellular protease, which was purified from the supernatant fraction of S. marcescens LRL781 cultures. Column adsorption of the antiprotease serum was performed using a slurry of live cells and cellulose. The resulting effluent was serologically inactive against protease preparations and chromium chloride-coupled protease-rabbit erythrocytes. Whole washed cells agglutinated in the presence of the antiserum. Cells were uniformly labeled with ferritin conjugated goat anti-rabbit IgG, indicating that the protease precursor is a surface antigen. In addition, agglutination titers determined on S. marcescens LRL781 cultures of different ages showed the same titer, suggesting the surface antigen was present on all cultures. Cell surface fractions of S. marcescens LRL781 were prepared using a lithium chloride-lithium acetate extraction buffer. The precursor molecule co-purified with the outer membrane fraction. An enzyme-linked immunosorbent assay (ELISA) was used to quantitate the antigen present in each cell fraction. The ELISA indicated that the outer membrane fraction had a higher specific activity than any other cell fraction. The precursor was associated with a carbohydrate moiety. The precursor occurs in a large insoluble aggregate as indicated by lack of penetration into polyacrylamide gels and by a high specific activity in the void volume of a Sepharose 6B-C1 column. Western blot electrophoresis yielded results similar to that of the ELISA (ie. the antigen remained at the top of the gel). O'Farrell (1975) gel electrophoresis revealed that the precursor is acidic in nature, as is the protease, and that the precursor has an apparent molecular weight of 71,000 (molecular weight of protease 47,000). The outer membrane fraction could not be solubilized with detergents, enzymes, or organic solvents. Peptide mapping of protein spots excised from O'Farrell gels was performed, and revealed that nine peptides are shared by the protease and the precursor. Cleveland (1977) gel analysis was used to detect four serologically-related peptides shared between the protease and its precursor.