Characterization of the cvaA and cvi promoters of the colicin V export system: iron-dependent transcription of cvaA is modulated by downstream sequences.

Secretion of the Escherichia coli toxin colicin V was previously determined to be iron regulated via the Fur (ferric uptake regulator) protein, based on studies in fur mutants. The iron dependence of transcription and expression of cvaA, which encodes a transporter accessory protein, and cvi, encoding the colicin V immunity protein, was assessed under conditions of iron excess or depletion. Immunoblots showed that production of both Cvi and CvaA is iron dependent. The iron-dependent transcriptional start for cvaA identified by primer extension and S1 nuclease analysis, P1, lies 320 bp upstream of the translational start and is associated with a newly identified Fur binding site. Beta-galactosidase activity in transcriptional lacZ fusions with the P1 promoter alone is higher than with downstream sequences present and is induced 10-fold by iron depletion. Including immediate downstream regions with P1 enhances activity from P1 even more but reduces the induction by iron depletion fivefold. Including subsequent downstream sequences, however, down-modulates overall transcription from P1 almost fourfold. Deletion of a long stem-loop structure in this region alleviates the down-modulation by increasing transcription, indicating that the sequences or structure of this element may contribute to this down-regulation. Characterization of the cvi promoter by primer extension showed that it resides where predicted, about 50 bp upstream of cvi associated with a previously identified Fur binding site. The cvi promoter is also inducible by iron depletion. The modulating sequences from cvaA were placed downstream of the cvi promoter to test their effects in transcriptional fusions of the cvi promoter to lacZ. The fusion results showed that these sequences also modulate transcription of the cvi promoter in a manner similar to that of the cvaA promoter. The potential for up- and down-regulation within the long untranslated region downstream of the cvaA promoter suggests a novel mechanism that fine-tunes expression of the colicin V secretion genes.