Functioning of the stable signal peptide of the pCloDF13‐encoded bacteriocin release protein

Abstract

The pCloDF13-encoded bacteriocin release protein (BRP) is a lipoprotein which is synthesized as a precursor with an amino-terminal signal peptide that appears to be stable after cleavage. The role of the stable signal peptide in the functioning of the BRP was studied with respect to the release of cloacin DF13, 'lysis' and leakage of periplasmic proteins. The BRP gene fragment encoding the stable signal peptide was replaced by a fragment encoding the unstable peptide of the murein lipoprotein (Lpp). The resulting hybrid protein was normally acylated and processed by signal peptidase II, leaving no stable signal peptide in the cells. Expression of the hybrid protein did not result in the specific release of cloacin DF13, whereas 'lysis' and the release of periplasmic enzymes were unaffected. These results indicated a role for the stable BRP signal peptide in the translocation of cloacin DF13 across the cytoplasmic membrane.

Stability and function of the signal peptide of the pCloDF13-derived bacteriocin release protein

“…Mitomycin C was used at 500 ng ml-' to induce cloacin DF13 synthesis. p JL22 (SphI) encoding the wild-type BRP, p JL32-T, encoding a hybrid LppBRP, pJL38 encoding the stable BRP signal peptide alone and pJL40 encoding the unstable Lpp signal peptide alone were described previously (Luirink et al, 1991 ;Van der Wal et al, 1992) and were used either for the construction of mutant BRP genes or as a control in lysis experiments. The expression vector pINIIIAl (Masui et al, 1984) was used for the expression of all mutant BRP constructs.…”

“…The resulting hybrid BRP, correctly targeted by the Lpp signal peptide (Gennity e t al., 1992; Van der Wal e t a/., 1992), did not function in the release of cloacin DF13, but expression did result in quasi-lysis, lethality and leakage of periplasmic proteins (Luirink et al, 1991).…”

Escherichia coli SecB, SecA, and SecY proteins are required for expression and membrane insertion of the bacteriocin release protein, a small lipoprotein

“…The maturation of certain pre-BRPs is unusual in that it occurs very slowly, rendering a signal peptide which is not degraded and a mature, lipid-modified BRP which is located in both membranes (5,18,21,26). The stable signal peptide of these BRPs was shown to play a role in the release of the bacteriocin and in the detrimental effects of high-level expression of the BRP (20).…”

“…The maturation of certain pre-BRPs is unusual in that it occurs very slowly, rendering a signal peptide which is not degraded and a mature, lipid-modified BRP which is located in both membranes (5,18,21,26). The stable signal peptide of these BRPs was shown to play a role in the release of the bacteriocin and in the detrimental effects of high-level expression of the BRP (20).The unusual features of BRP targeting and processing raise the question whether BRPs follow a Sec-dependent or a Sec-independent pathway for transfer across the cytoplasmic membrane. Therefore, the effects of the depletion of Sec proteins on the expression, processing, and functioning of the pCloDF13-encoded BRP were investigated.…”

“…Strong induction of the pCloDF13-encoded BRP results in a sharp decline in culture turbidity, also called quasi-lysis, and cell death due to an accumulation of the stable BRP signal peptide and mature BRP (20). To test whether the BRP-induced quasi-lysis is dependent on the SecB, SecA, and SecY proteins, various E. coli strains defective in one of these proteins were transformed (7) with pJL7 isolated by the method of Birnboim and Doly (1).…”