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We have used Escherichia coli alkaline phosphatase to show the interplay among the characteristics of two amino-terminal domains in the preprotein (the signal peptide and the early mature region), the efficiency with which this protein is transported, and its requirement for SecB to accomplish the transport process. The results suggest that although alkaline phosphatase does not normally require SecB for transport, it is inherently able to utilize SecB, and it does so when its ability to interface with the transport machinery is compromised.

The SecB, SecA, and SecY dependency of a small outer membrane lipoprotein in Escherichia coli, the bacteriocin release protein (BRP), was studied. The detrimental effect of BRP expression on the culture turbidity (quasi-lysis) was strongly reduced in the sec mutants. Immunoblotting and radioactive labeling experiments showed that the expression, membrane insertion, and processing of the BRP precursor are dependent on SecB, SecA, and SecY. Labeling experiments with hybrid BRP gene constructs revealed that the mature part of the BRP precursor and not its stable signal sequence is important for its SecB dependency.

A plasmid containing a pColV-K30 fragment that encoded only for the cloacin DF13/aerobactin receptor protein was constructed. Escherichia coli cells harboring this plasmid were sensitive to cloacin DF13 but were unable to take up ferric-aerobactin. Another pColV-K30-determined polypeptide (molecular weight, 50,000), localized in the membrane fraction, was essential for the uptake of ferric-aerobactin.

By oligonucleotide-directed mutagenesis, stop codon mutations were introduced at various sites in the pCloDF13-derived bacteriocin release protein (BRP) structural gene. The expression, lipid modification (incorporation of [3H]palmitate), and processing (in the presence and absence of globomycin) of the various carboxyl-terminal shortened BRPs were analyzed by a special electrophoresis system and immunoblotting with an antiserum raised against a synthetic BRP peptide, and their functioning with respect to release of cloacin DF13, lethality, and apparent host cell lysis were studied in Sup-, supF, and supP strains of Escherichia coli. All mutant BRPs were stably expressed, lipid modified, and processed by signal peptidase II, albeit with different efficiencies. The BRP signal peptide appeared to be extremely stable and accumulated in induced cells. Full induction of the mutant BRPs, including the shortest containing only 4 amino acid residues of the mature polypeptide, resulted in phospholipase A-dependent and Mg2+-suppressible apparent cell lysis. The extent of this lysis varied with the mutant BRP used. Induction of all mutant BRPs also prevented colony formation, which appeared to be phospholipase A independent. One shortened BRP, containing 20 amino acid residues of the mature polypeptide, was still able to bring about the release of cloacin DF13. The results indicated that the 8-amino-acid carboxyl-terminal segment of the BRP contains a strong antigenic determinant and that a small segment between amino acid residues 17 and 21, located in the carboxyl-terminal half of the BRP, is important for release of cloacin DF13. Either the stable signal peptide or the acylated amino-terminal BRP fragments (or both) are involved in host cell lysis and lethality.