Differentiation of Bacterial Autolysins by Zymogram Analysis

Abstract

The use of zymograms in which the bacterial cell wall heteropolymer peptidoglycan is incorporated into the resolving gel of SDS–PAGE has led to the identification of various SDS stable peptidoglycan hydrolases (autolysins). To examine the specificity of autolysins with respect to O-acetylated peptidoglycan, a discontinuous SDS–PAGE system has been developed that operates under neutral conditions. [Bis(2-hydroxyethyl)imino]tris(hydroxymethyl)methane (Bis-Tris) buffers are employed with pH 6.8 and 6.3 for the separating and stacking gels, respectively, while the anode buffer N-2-acetamido-2-hydroxyethanesulfonic acid (Aces)-HCl and the Bis-Tris cathode buffer both had a pH of 6.8. These conditions resulted in a relative trailing ion mobility of 0.349 and 0.137 in the resolving and staking gel, respectively, under room temperature conditions. Peptides and proteins were resolved in the 3–100 kDa range with a 10% acrylamide resolving gel. Comparison of zymograms that incorporated unacetylated or chemically O-acetylated peptidoglycan revealed the specificity of hen egg-white lysozyme for the unacetylated material. A preliminary analysis of the autolysins produced by the urinary tract pathogen Proteus mirabilis indicated that some enzymes were specific for either O-acetylated or non-O-acetylated peptidoglycan while others displayed no clear preference toward either of the two substrates.