ISOLATION, PURIFICATION, AND CHARACTERIZATION OF THE MAJOR AUTOLYSIN FROM PSEUDOMONAS-AERUGINOSA

The major (26 kDa) autolysin from Pseudomonas aeruginosa was purified to apparent homogeneity by a combination of preparative electrophoresi s, ion-exchange, and dye-ligand chromatographies. This purification wa s facilitated by the development of a spot-assay that involved the spo tting and subsequent incubation of autolysin samples on polyacrylamide gels containing peptidoglycan. The pI of the 26-kDa autolysin was det ermined to be between 3.5 and 4 and disulfide bonds within the enzyme were essential for activity. The autolysin catalyzed the release of re ducing sugars from the peptidoglycans of Pseudomonas aeruginosa and Es cherichia coli indicating it to be a beta-glycosidase. It was ineffect ive at hydrolysing the peptidoglycan from Gram-positive bacteria and t he O-acetylated peptidoglycans from either Proteus mirabilis or Staphy lococcus aureus. The N-terminal sequence of the purified autolysin was determined to be His-Glu-Pro-Pro-Gly. The 26-kDa autolysin together w ith a 29-kDa autolysin was determined to be secreted into the medium b y a mechanism that involves the production and release of surface memb rane vesicles during normal growth, but the enzymes were not found fre e and active in culture broth supernatants.