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.
Département de Génétique et Microbiologie, Centre Médical Universitaire, Genève, Suisse.
M
M
Michéa-Hamzehpour
J C
JC
Sanchez
S F
SF
Epp
N
N
Paquet
G J
GJ
Hughes
D
D
Hochstrasser
J C
JC
Pechère
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002442:Ceftazidime / Q000633:toxicity; D004351:Drug Resistance; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D015378:Imipenem / Q000633:toxicity; D008969:Molecular Sequence Data; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D001425:Bacterial Outer Membrane Proteins; D015378:Imipenem; D002442:Ceftazidime