Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of korB

Abstract

The korB gene of broad host-range plasmid RK2 prevents host-cell lethality by kilB and negatively controls RK2 replication. We precisely mapped the limits of korB to a region near korA, an autoregulated gene involved in control of several RK2 genes. The following results show that korA and korB are cotranscribed from the korA promoter: Mutants deleted for the korA promoter fail to express korB, even with korA function supplied in trans; the korA promoter is nonessential to korB if a heterologous promoter is present; and RNA produced in vivo has both korA- and korB-specific sequences. Analysis of polypeptides synthesized from wild-type and mutant korB plasmids in maxicells revealed that korB encodes a 52-kDa polypeptide, whose activity is extremely sensitive to changes in its carboxyl terminus but relatively unaffected by replacement of its amino terminus. The minimal korB-encoding region allowed us to identify two new regulatory functions, both of which duplicate previously known functions of korA. First, korB alone was found to control the kilB1 component of kilB, thus resolving the paradox of korA-independent control of kilB. Second, analysis of polypeptides from the korA-korB region in the presence and absence of korB, and studies with the korA promoter fused to the chloramphenicol acetyltransferase structural gene (cat) showed that korB, like korA, autoregulates expression of the korA-korB operon. We suggest that korA and korB gene products act as co-repressors in the control of certain RK2 genes.