Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis

Bacillus subtilis contains three Fur homologs: Fur, PerR, and Zur. Despite significant sequence similarities, they respond to different stimuli and regulate different sets of genes. DNA target site comparisons indicate that all three paralogs recognize operators with a core 7-1-7 inverted repeat. The corresponding consensus sequences are identical at five or more of the seven defined positions. Using site-directed mutagenesis, the Per box at the mrgA promoter was altered to mimic the core 7-1-7 motif of the Fur and Zur boxes. In vitro, the mrgA promoter containing a Zur box was only recognized by Zur, as demonstrated by DNase I footprinting assays. In contrast, both Fur and PerR bound to the mrgA promoter region containing a consensus Fur box. Expression analysis of these promoters is consistent with the in vitro data demonstrating as few as 1 or 2 base changes per half-site are sufficient to alter regulation. Similarly, the Fur box at the feuA promoter can be converted into a Per or a Zur box by appropriate mutations. While both Fur and PerR could recognize some of the same synthetic operator sequences, no naturally occurring sites are known that are subject to dual regulation. However, the PerR-regulated zosA gene is controlled from a regulatory region that contains both Per and Fur boxes. Although purified Fur protein bound to the candidate Fur boxes, Fur has little effect on zosA expression-possibly due to the location of the Fur boxes relative to the zosA promoter. Together, our results identify two nucleotide positions that are important for the ability of PerR, Fur, and Zur to distinguish among the many closely related operator sites present in the B. subtilis genome.     

Regulation of the Bacillus subtilis yciC gene and insights into the DNA-binding specificity of the zinc-sensing metalloregulator Zur

The Bacillus subtilis Zur protein regulates zinc homeostasis by repressing at least 10 genes in response to zinc sufficiency. One of these genes, yciC, encodes an abundant protein postulated to function as a metallochaperone. Here, we used a genetic approach to identify the cis-acting elements and trans-acting factors contributing to the tight repression of yciC. Initial studies led to the identification of only trans-acting mutations, and, when the selection was repeated using a transposon library, all recovered mutants contained insertionally inactivated zur. Using a zur merodiploid strain, we obtained two cis-acting mutations that contained large deletions in the yciC regulatory region. We demonstrate that the yciC regulatory region contains two functional Zur boxes: a primary site (C2) overlapping a sigma(A) promoter approximately 200 bp upstream of yciC and a second site near the translational start point (C1). Zur binds to both of these sites to mediate strong, zinc-dependent repression of yciC. Deletion studies indicate that either Zur box is sufficient for repression, although repression by Zur bound to C2 is more efficient. Binding studies demonstrate that both sites bind Zur with high affinity. Sequence alignment of these and previously described Zur boxes suggest that Zur recognizes a more extended operator than other Fur family members. We used synthetic oligonucleotides to identify bases critical for DNA binding by Zur. Unlike Fur and PerR, which bind efficiently to sequences containing a core 7-1-7 repeat element, Zur requires a 9-1-9 inverted repeat for high-affinity binding.     

Sinorhizobium meliloti fur-like (Mur) protein binds a fur box-like sequence present in the mntA promoter in a manganese-responsive manner

In Sinorhizobium meliloti, the Mur(Sm) protein, a homologue of the ferric uptake regulator (Fur), mediates manganese-dependent regulation of the MntABCD manganese uptake system. In this study, we analyzed Mur(Sm) binding to the promoter region of the S. meliloti mntA gene. We demonstrated that Mur(Sm) protein binds with high affinity to the promoter region of mntA gene in a manganese-responsive manner. Moreover, the results presented here indicate that two monomers, or one dimer, of Mur(Sm) binds the DNA. The binding region was identified by DNase I footprinting analysis and covers a region of about 30 bp long that contains a palindromic sequence. The Mur(Sm) binding site, present in the mntA promoter region, is similar to a Fur box; however, manganese-activated Mur(Sm) binds a canonical Fur box with very low affinity. Furthermore, the data obtained indicate that Mur(Sm) responds to physiological concentrations of manganese.     

The RcnRA (YohLM) system of <Emphasis Type="Italic">Escherichia coli</Emphasis>: A connection between nickel,cobalt and iron homeostasis

The transporter RcnA has previously been implicated in Ni(II) and Co(II) detoxification in E. coli probably through efflux. Here we demonstrate that the divergently described rcnA and rcnR gene products constitute a link between nickel, cobalt and iron homeostasis. Deletion of the rcnA gene resulted in increased cellular nickel, cobalt and iron concentrations. Expression of rcnA was induced by Ni(II) or Co(II). Overproduction of rcnR inhibited induction of rcnA by metal cations but RcnR did not bind to the rcnA promoter in vitro. When rcnR or fur, the gene of the global repressor of iron homeostasis, was deleted, expression of rcnA was also induced by iron. The promoter region of rcnA was positive in a Fur titration (FURTA) in vivo assay indicative of Fur binding. Thus, rcnA is part of the Fur regulon of E.␣coli. The implications of a connection between the homoeostasis of closely related transition metals are discussed.     

Sinorhizobium meliloti Fur-Like (Mur) Protein Binds a Fur Box-Like Sequence Present in the mntA Promoter in a Manganese-Responsive Manner

In Sinorhizobium meliloti, the Mur_Sm protein, a homologue of the ferric uptake regulator (Fur), mediates manganese-dependent regulation of the MntABCD manganese uptake system. In this study, we analyzed Mur_Sm binding to the promoter region of the S. meliloti mntA gene. We demonstrated that Mur_Sm protein binds with high affinity to the promoter region of mntA gene in a manganese-responsive manner. Moreover, the results presented here indicate that two monomers, or one dimer, of Mur_Sm binds the DNA. The binding region was identified by DNase I footprinting analysis and covers a region of about 30 bp long that contains a palindromic sequence. The Mur_Sm binding site, present in the mntA promoter region, is similar to a Fur box; however, manganese-activated Mur_Sm binds a canonical Fur box with very low affinity. Furthermore, the data obtained indicate that Mur_Sm responds to physiological concentrations of manganese.     

Functional specialization within the Fur family of metalloregulators

The ferric uptake regulator (Fur) protein, as originally described in Escherichia coli, is an iron-sensing repressor that controls the expression of genes for siderophore biosynthesis and iron transport. Although Fur is commonly thought of as a metal-dependent repressor, Fur also activates the expression of many genes by either indirect or direct mechanisms. In the best studied model systems, Fur functions as a global regulator of iron homeostasis controlling both the induction of iron uptake functions (under iron limitation) and the expression of iron storage proteins and iron-utilizing enzymes (under iron sufficiency). We now appreciate that there is a tremendous diversity in metal selectivity and biological function within the Fur family which includes sensors of iron (Fur), zinc (Zur), manganese (Mur), and nickel (Nur). Despite numerous studies, the mechanism of metal ion sensing by Fur family proteins is still controversial. Other family members use metal catalyzed oxidation reactions to sense peroxide-stress (PerR) or the availability of heme (Irr).     

Members of the Fur protein family regulate iron and zinc transport in E. coli and characteristics of the Fur-regulated fhuF protein

The regulator Fur represses with Fe2+ as cofactor iron uptake genes. The fhuF gene reacts very sensitive to minor changes of Fe2+ and Fur. It is assumed that FhuF helps in the mobilisation of iron out of the hydroxamate siderophores transported into the cell. Analysis of the protein revealed an unusual [2Fe-2S] cluster bound to a Cys-Cys-X10-Cys-X2-Cys motif in FhuF. suf genes responsible for the synthesis of the iron sulfur center were identified. The Zur protein shows 27% identity to the Fur protein of E. coli. It regulates as a repressor the high affinity uptake system znuACB. Only two additional Zur binding sites in the promoter region of genes with unknown function were found. Properties of Zur and Fur proteins from different bacteria are compared.     

Identification and analysis of a gene encoding a Fur-like protein of Staphylococcus epidermidis

Abstract A gene ( fur ) for a Fur-like protein was identified on a 1.1 kb chromosomal DNA fragment of Staphylococcus epidermidis BN 280; the fur gene is followed by an open reading frame coding for the N-terminus of a putative Superoxide dismutase. Within the − 35 promoter region of both genes, a sequence motif was detected with low similarity to Fur-binding regulatory DNA segments, the so-called Fur boxes. Fur titration in Escherichia coli strain H1717 demonstrated that the E. coli Fur protein binds to the Fur box of the promoter region of the S. epidermidis fur gene. The S. epidermidis Fur protein was expressed in E. coli as indicated by the formation of inactive dimers with the chimeric repressor CI(N)-Fur(C) (Stojiljkovic I. and Hantke. K. (1995) Mol. Gen. Genet. 247, 199–205), but was not able to complement the Fur mutation in E. coli H1681.