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 deter mined to be iron regulated via the Fur (ferric uptake regulator) prote in, based on studies in fur mutants. The iron dependence of transcript ion and expression of cvaA, which encodes a transporter accessory prot ein, and cvi, encoding the colicin V immunity protein, was assessed un der conditions of iron excess or depletion. Immunoblots showed that pr oduction of both Cvi and CvaA is iron dependent. The iron-dependent tr anscriptional start for cvaA identified by primer extension and S1 nuc lease analysis, P1, lies 320 bp upstream of the translational start an d is associated with a newly identified Fur binding site. beta-Galacto sidase activity in transcriptional lacZ fusions with the P1 promoter a lone is higher than with downstream sequences present and is induced 1 0-fold by iron depletion. Including immediate downstream regions with P1 enhances activity from P1 even more but reduces the induction by ir on depletion fivefold. Including subsequent downstream sequences, howe ver, down-modulates overall transcription from P1 almost fourfold. Del etion 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 i t resides where predicted, about 50 bp upstream of cvi associated with a previously identified Fur binding site. The cvi prompter is also in ducible by iron depletion. The modulating Sequences from cvaA were pla ced downstream of the cvi promoter to test their effects in transcript ional fusions of the cvi promoter to lacZ. The fusion results showed t hat these sequences also modulate transcription of the cvi promoter in a manner similar to that of the cvaA promoter, The potential for up-a nd down-regulation within the long untranslated region downstream of t he cvaA promoter suggests a novel mechanism that fine-tunes expression of the colicin V secretion genes.