Promoter and operator determinants for fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system

Abstract

The fepA-entD and fes-entF operons in the enterobactin synthesis and transport system are divergently transcribed from overlapping promoters, and both are inhibited by the Fur repressor protein under iron-replete conditions. A plasmid harboring divergentfepA'-'phoA andfes-entF'-'lacZ fusions, both under the control of this bidirectional regulatory region, was constructed for the purpose of monitoring changes in expression of the two operons simultaneously. Deletion analysis, site-directed mutagenesis, and primer extension were employed to define both a single promoter governing the expression of fes-entF and two tandemly arranged promoters giving rise to the opposingfepA-entD transcript. A single Fur-binding site that coordinately regulates the expression of all transcripts emanating from this control region was identified by in vitro protection from DNase I nicking. The substitution of one base pair in the Fur recognition sequence relieved Fur repression but did not change the in vitro affinity of Fur for its binding site. Additional mutations in a limited region outside of the promoter determinants for either transcript inhibited expression of bothfes andfepA. These observations suggest a mechanism of Fur-mediated regulation in this compact control region that may involve other regulatory components.Bacteria adapt to environmental change by dramatic shifts in gene expression and protein production. In response to iron deprivation, most oxygen-metabolizing microorganisms produce and excrete small iron-chelating molecules called siderophores (36) and perform the specific transport functions required for the retrieval of their ferrated derivatives (41). Escherichia coli typically synthesizes and secretes the catecholbased siderophore enterobactin, and some isolates produce the hydroxamate derivative aerobactin, as well.The genes required for enterobactin biosynthesis (ent) and transport (fep) map in a cluster at minute 13 (2) along with fes (for ferric enterobactin esterase); fepE, which encodes a protein of unknown function that is localized on the periplasmic side of the cytoplasmic membrane (13); and two other genes encoding uncharacterized protein products designated P15 (40) and P43 (50) to indicate their approximate sizes. Expression of the enterobactin system emanates from three regulatory regions containing divergently oriented promoters: one located between fepB and the entCEBA(P15) operon (8,44), one between fepD and P43 (50), and a third between fepA and fes (45). Except forfepE, which is constitutively expressed from its own weak promoter (13), the six transcripts arising from these three promoter regions encode all of the genes in the enterobactin gene cluster and are induced when the external iron supply becomes depleted (8,22,45,50). This sensitivity to iron limitation is shared by the aerobactin system and other genes and operons throughout the chromosome (6, 30), many * Corresponding author. Mailing address:

Expression of the Escherichia coli IrgA homolog adhesin is regulated by the ferric uptake regulation protein

“…Iha has 33% identity to IrgA of Vibrio cholerae and 27% identity to FepA of E. coli. Both of these proteins are receptors for the siderophore enterobactin and, like Iha, are directly repressed by Fur [27,[34][35][36]. In fact, Iha from the uropathogen, UCB34, was recently determined to be iron-repressed and to act as both a catecholate siderophore receptor and an adhesin [9].…”

“…This suggests competition for Fur between the binding site on the labeled probe and on the competitor DNA. A second short oligonucleotide containing a 5 bp mutation previously demonstrated to reduce Fur binding to the FepA promoter [27] had diminished ability to inhibit formation of the higher molecular weight complex ( figure 4C). Together, these data demonstrate that Fur binds to the iha promoter at the site predicted by Panina et.…”

Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis ofNeisseria meningitidisgroup B

“…A 21-bp Neisseria consensus that shares Ϸ80% homology to the E. coli consensus has been derived and proposed to be the binding sequence of the Neisseria Fur protein (23). Although the 19-and 21-bp consensuses serve as good indicators to identify a Furregulated gene, the Fur-binding sequence identified in the promoters of several Fur-regulated genes differed from the classical Fur-binding sequence (37)(38)(39)(40)(41)(42). In light of these results, the Fur-binding sequence has been reinterpreted to be a combination of three repeats of 6-bp 5Ј-NAT(A͞T)AT-3Ј rather than a 19-bp palindrome (24).…”

Microcin E492 Amyloid Formation Is Retarded by Posttranslational Modification

“…The observation that in the late stationary phase there is a decrease in the antibacterial activity even when the expression of the maturation genes is augmented may indicate that a factor necessary for posttranslational modification may become limiting. A possible candidate is EntF, a protein that is essential for the production of active MccE492 (9) and that is regulated by iron availability through the Fur repressor (30). The explanation that the decrease in activity may be related to iron availability is supported by the fact that inactive MccE492 is produced under iron-limiting conditions (31).…”