Characterization of the Interaction between Fur and the Iron Transport Promoter of the Virulence Plasmid in Vibrio anguillarum

Abstract

The expression of iron transport genes fatDCBA in Vibrio anguillarum strain 775 is negatively regulated by two iron-responsive repressors, the Fur protein and the antisense RNA, RNA␣. Here we report the identification of the promoter for the iron transport genes and studied the interaction between the V. anguillarum Fur protein and this promoter. The iron transport promoter was localized in a region approximately 300 base pairs upstream of fatD by both primer extension and S1 mapping analysis. High activity of the promoter was measured in response to iron depletion in the wild-type strain when a promoter-lacZ fusion was examined, whereas the promoter was constitutive in the Fur-deficient strain. Gel retardation and DNase I footprint analysis showed that Fur binds specifically to two contiguous sites comprising the promoter region and the region downstream of the transcription start site. The identified Fur binding sites showed a low degree of homology to each other as well as to the consensus sequence for the Escherichia coli Fur protein. DNase I footprints pattern suggested a sequential interaction of Fur with these two sites that renders a protection in the template strand and a hypersensitivity to the nuclease in the nontemplate strand.

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Argentina, who was a great mentor and colleague. The GenBank accession number for the sequence reported in this paper is AF130307.

“…Furthermore, at least 10 other units very similar to those found in the aerobactin promoter region (Escolar et al, 2000) can be identified upstream of the adhC1 initiation codon. This region also includes the sequence TAGCATTACAT-TATCTATTTTGCT that matches 19 of the 24 nucleotides found in the non-template strand of the Furbinding site I of the promoter region of the fatDCBA operon of the pJM1 plasmid (Chai et al, 1998). The potential regulatory role of these sequences is further supported by the results obtained using S1 mapping, which showed three adjunct adhC1 transcription initiation sites (Fig.…”

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).…”

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

“…10) are controlled by the concentration of available iron, via at least four plasmid-encoded regulators: two positive regulators, AngR (anguibactin system regulator) and TAF (transacting factor[s]); and two negative regulators, antisense RNA␣ and RNA␤ (19,21,22,25,35,(100)(101)(102)(118)(119)(120)(121). RNA␣ is very stable at high iron concentrations and acts at posttranscriptional level in the repression of fatB and fatA expression, while RNA␤, found under conditions of mild iron limitation, acts on the attenuation of expression of the angR gene in the iron transport-biosynthesis (ITB) operon (19,102 (19,117,133). The promoter for the ITB operon (pITBO) was localized within a region ca.…”

“…In V. anguillarum, Pseudomonas aeruginosa, and other microorganisms, positive regulation by a combination of positive regulatory proteins and the cognate siderophore has also been reported, and the mechanisms are under study (22,26,49,50). Furthermore, it has also been demonstrated in V. anguillarum that negative control of iron uptake gene expression is not only due to repression of transcription initiation by a complex of Fur with iron (19) but also due to posttranscriptional regulation by antisense RNAs acting as a fine-tuning control mechanism (21,26,101). These regulatory strategies enable bacteria, via ironsensing transcriptional activators and repressors, to turn on and off sets of genes that encode proteins for the assembly and export of siderophores and the specific uptake of the ironsiderophore complexes.…”