ANALYSIS OF THE SODIUM DODECYL SULFATE-STABLE PEPTIDOGLYCAN AUTOLYSINS OF SELECT GRAM-NEGATIVE PATHOGENS BY USING RENATURING POLYACRYLAMIDE-GEL ELECTROPHORESIS

For the first time, peptidoglycan autolysins from cellular fractions d erived from sonicated cultures of Pseudomonas aeruginosa PAO1, Escheri chia coli W7, Klebsiella pneumoniae CWK2, and Proteus mirabilis 19 wer e detected and partially characterized by zymogram analysis. Purified murein sacculi from P. aeruginosa PAO1 were incorporated into a sodium dodecyl sulfate (SDS)-polyacrylamide gel at a concentration of 0.05% (wt/vol) to serve as a substrate for the separated autolysins. At leas t 11 autolysin bands of various intensities with M(r)s ranging between 17,000 and 122,000 were detected in each of the homogenated cultures. Some of the autolysins of the four bacteria had similar M(r)s. The zy mogram analysis was used to show that a number of the autolysins from E. coli were inhibited by the heavy metals Hg2+ and CU2+, at 1 and 10 mM, respectively, high ionic strengths, and reagents known to affect t he packing of lipopolysaccharides. The activity of an autolysin with a n M(r) of 65,000 was also impaired by penicillin G, whereas it was enh anced by gentamicin. A preliminary screen to determine the relationshi p between penicillin-binding proteins (PBPs) and autolysins was carrie d out by using a dual assay in which radiolabelled penicillin V bands were visualized an an autolysin zymogram. Radiolabeled bands correspon ding to PBPs 3, 4, 5, and 6 from E. coli and P. aeruginosa; PBPs 3, 4, and 6 from Proteus mirabilis; and PBP 6 from K. pneumoniae degraded t he murein sacculi in the gels and were presumed to have autolytic acti vity, although the possibility of two distinct enzymes, each with one of the activities, comigrating in the SDS-polyacrylamide gels could no t be excluded. Some radiolabelled bands possessed an M(r) of <34,000 a nd coincided with similar low-M(r) autolysin bands.