PROPERTIES OF BACTERIAL PROTEIN SECRETION
Abstract
Several aspects of the secretion of protein into the periplasm of Escherichia coli were studied. Special emphasis was placed on the secretion of the TEM (beta)-lactamase (Bla), which is the product of the plasmid borne gene, bla. Cytoplasmic membrane vesicles were prepared from E. coli by various methods and their physical properties and protein content compared. The inhibition of Bla processing by carbonylcyanide-m-chlorophenylhydrazone (CCCP) in E. coli was observed to be accompanied by a decline in both membrane potential and cellular ATP. It was observed that prior treatment of cells with dicyclohexylcarbodiimide (DCCD) stabilized ATP levels in the presence of CCCP. The inhibition of Bla processing by CCCP was not affected by DCCD treatment, leading to the conclusion that membrane depolarization, not ATP depletion, was responsible for CCCP inhibition of Bla processing. Purified Bla from E. coli was found to contain a single disulfide bond which exhibited extraordinary stability. Presence of the disulfide was found to slightly increase the mobility of the protein in SDS-polyacrylamide electrophoresis. A similar shift in electrophoretic mobility was observed for the in vitro synthesized Bla precursor. The electrophoretic mobilities of samples obtained after pulse-chase labeling of Bla indicated that disfulfide formation occurs concomitant with processing of the precursor to the mature form. Inhibition of secretion by CCCP did not allow disulfide formation with the cell. A plasmid was constructed in which the gene bla is interrupted by an insertion of foreign DNA. The resulting gene product, which lacks the carboxy terminal 36% of Bla, was found to be efficiently processed and secreted by E. coli. The conclusion was drawn that the carboxy terminus is not required for Bla secretion.
Degree
Ph.D.
Subject Area
Biochemistry
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