Cloning, expression and release of native and mutant cloacin DF13 immunity protein.

ABSTRACT

The pCloDF13 encoded immunity protein gene was subcloned in the expression vector pINIIIA1 and several deletion, insertion and point mutations were constructed in the amino-terminal and carboxyl-terminal regions of the protein. The expression, stability, BRP-dependent export and protective capacity of the native and mutant immunity proteins were studied by SDS-PAGE, immunoblotting and an in vivo activity assay. In the absence of cloacin the unbound, native immunity protein was stable produced by E. coli cells and released after BRP induction. The expression of most of the mutant immunity proteins was strongly reduced and non of the proteins were found to be released. All mutations in the carboxyl-terminal region strongly affected expression of the proteins, probably by causing protein instability and proteolytic degradation. One of these mutant immunity proteins, with an insertion mutation in its carboxyl-terminal region, still caused an intermediate immunity of susceptible cells against extracellularly added cloacin DF13. Mutations in the amino-terminal region of the immunity protein had less effect on its expression and did not affect the protective capacity of the protein.