Transport and anchoring of beta-lactamase to the external surface of Escherichia coli.
The outer membrane of Gram-negative bacteria presents an effective barrier that restricts the release of proteins from the cell. Virtually all extracellular proteins of Gram-negative bacteria are exported by specialized systems requiring the action of several gene products. We have constructed a tripartite fusion consisting of (i) the signal sequence and first nine N-terminal amino acids of the mature major Escherichia coli lipoprotein, (ii) amino acids 46-159 of the outer membrane protein OmpA, and (iii) the complete mature beta-lactamase (EC 3.5.2.6) sequence. This protein had an enzymatically active beta-lactamase and was found predominantly in the outer membrane. Immunofluorescence microscopy, the accessibility of the fusion protein to externally added proteases, and the rates of hydrolysis of nitrocefin and penicillin G by whole cells demonstrated that a substantial fraction (20-30%) of the beta-lactamase domain of the fusion protein was exposed on the external surface of E. coli. In cells grown at 24 degrees C the localization of beta-lactamase on the cell surface was almost quantitative (greater than 80% of the enzymatically active protein was exposed to the extracellular fluid) as determined by nitrocefin and penicillin G hydrolysis and trypsin accessibility. These results demonstrated that a soluble protein, beta-lactamase, can be transported through--and become anchored on--the outer membrane by fusion to the proper targeting and localization signals.
Francisco
J A
JA
Department of Chemical Engineering, University of Texas, Austin 78712.
Earhart
C F
CF
Georgiou
G
G
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D002451:Cell Compartmentation; D004926:Escherichia coli / Q000378:metabolism*; D005818:Genetic Engineering; D008074:Lipoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases