Jean-Charles Sanchez

Two-dimensional polyacrylamide gel electrophoresis isolation and microsequencing of Pseudomonas aeruginosa proteins.

Outer membrane (OM) proteins of beta-lactam-susceptible and -resistant strains of Pseudomonas aeruginosa were analyzed by 2-D polyacrylamide gel electrophoresis. Carrier ampholytes, pH 4-8, and immobilized pH gradient (IPG), pH 3.5-10.0, procedures were used. An acidic-protein spot (pI = 5.2) detected in susceptible but not in an imipenem-resistant strain was sequenced and twenty-five N-terminal amino acids had total homology with the OM protein D, the imipenem-specific porin of P. aeruginosa. A basic-protein spot (pI = 9.0) detected in ceftazidime-resistant, but not in a susceptible strain was sequenced and fourteen N-terminal amino acids had homology with a beta-lactamase encoded by the ampC gene of P. aeruginosa. The IPG procedure allows identification of more than one hundred proteins of the OM fraction from a single gel. Detection of beta-lactamase in OM fractions might reflect a periplasmic contamination, but its anchorage within the OM cannot be ruled out.