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 electrophoresis, ion-exchange, and dye-ligand chromatographies. This purification was facilitated by the development of a spot-assay that involved the spotting and subsequent incubation of autolysin samples on polyacrylamide gels containing peptidoglycan. The pl of the 26-kDa autolysin was determined to be between 3.5 and 4 and disulfide bonds within the enzyme were essential for activity. The autolysin catalyzed the release of reducing sugars from the peptidoglycans of Pseudomonas aeruginosa and Escherichia coli indicating it to be a beta-glycosidase. It was ineffective at hydrolysing the peptidoglycan from Gram-positive bacteria and the O-acetylated peptidoglycans from either Proteus mirabilis or Staphylococcus aureus. The N-terminal sequence of the purified autolysin was determined to be His-Glu-Pro-Pro-Gly. The 26-kDa autolysin together with a 29-kDa autolysin was determined to be secreted into the medium by a mechanism that involves the production and release of surface membrane vesicles during normal growth, but the enzymes were not found free and active in culture broth supernatants.
Watt
S R
SR
Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Canada.
Clarke
A J
AJ
D000596:Amino Acids / Q000032:analysis; D002852:Chromatography, Ion Exchange; D017077:Culture Media, Conditioned / Q000737:chemistry; D003553:Cystine / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008970:Molecular Weight; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000302:isolation & purification* / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D017421:Sequence Analysis; D013045:Species Specificity
D000596:Amino Acids; D017077:Culture Media, Conditioned; D010457:Peptidoglycan; D003553:Cystine; D009238:N-Acetylmuramoyl-L-alanine Amidase