Analysis of the sodium dodecyl sulfate-stable peptidoglycan autolysins of select gram-negative pathogens by using renaturing polyacrylamide gel electrophoresis

Abstract

For the first time, peptidoglycan autolysins from cellular fractions derived from sonicated cultures of Pseudomonas aeruginosa PAO1, Escherichia coli W7, Klebsiella pneumoniae CWK2, and Proteus mirabilis 19 were 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 least 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 zymogram 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 the packing of lipopolysaccharides. The activity of an autolysin with an M(r) of 65,000 was also impaired by penicillin G, whereas it was enhanced by gentamicin. A preliminary screen to determine the relationship between penicillin-binding proteins (PBPs) and autolysins was carried out by using a dual assay in which radiolabelled penicillin V bands were visualized on an autolysin zymogram. Radiolabelled bands corresponding 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 the murein sacculi in the gels and were presumed to have autolytic activity, although the possibility of two distinct enzymes, each with one of the activities, comigrating in the SDS-polyacrylamide gels could not be excluded. Some radiolabelled bands possessed an M(r) of <34,000 and coincided with similar low-M(r) autolysin bands.