Bacillus licheniformis Contains Two More PerR-Like Proteins in Addition to PerR, Fur, and Zur Orthologues

The ferric uptake regulator (Fur) family proteins include sensors of Fe (Fur), Zn (Zur), and peroxide (PerR). Among Fur family proteins, Fur and Zur are ubiquitous in most prokaryotic organisms, whereas PerR exists mainly in Gram positive bacteria as a functional homologue of OxyR. Gram positive bacteria such as Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus encode three Fur family proteins: Fur, Zur, and PerR. In this study, we identified five Fur family proteins from B. licheniformis: two novel PerR-like proteins (BL00690 and BL00950) in addition to Fur (BL05249), Zur (BL03703), and PerR (BL00075) homologues. Our data indicate that all of the five B. licheniformis Fur homologues contain a structural Zn2+ site composed of four cysteine residues like many other Fur family proteins. Furthermore, we provide evidence that the PerR-like proteins (BL00690 and BL00950) as well as PerRBL (BL00075), but not FurBL (BL05249) and ZurBL (BL03703), can sense H2O2 by histidine oxidation with different sensitivity. We also show that PerR2 (BL00690) has a PerR-like repressor activity for PerR-regulated genes in vivo. Taken together, our results suggest that B. licheniformis contains three PerR subfamily proteins which can sense H2O2 by histidine oxidation not by cysteine oxidation, in addition to Fur and Zur.


Introduction
The ferric uptake regulator (Fur) protein is an iron-sensing transcriptional regulator which controls the expression of genes involved in intracellular iron homeostasis [1]. Under ironreplete conditions, Fur mediates the repression of genes involved in intracellular iron increase to prevent iron overload. Since its first recognition in Escherichia coli, Fur family proteins have been found and characterized in a variety of organisms ranging from bacteria to archaea [1,2]. Fur family proteins are not only responsible for the acquisition and storage of iron, but also involved in the oxidative stress response as well as in the acquisition and storage of other metal ions. Now it is appreciated that there are various subgroups of Fur family proteins, which

Construction of E. coli strains overexpressing Fur family proteins
The open reading frames (ORFs) of bl05249, bl03703, bl00075, bl00950 and, bl00690 were PCRamplified with B. licheniformis ATCC14580 chromosomal DNA as template. The PCR fragments of bl05249, bl03703, bl00075, and bl00950 were individually cloned into the NdeI and BamHI sites of expression vector pET-11a (Novagen) resulting in plasmids named pJL303, pJL304, pJL302, and pJL201, respectively. The PCR fragments of bl00690 were cloned into the NcoI and BamHI sites of expression vector pET-16b (Novagen) resulting in plasmid named pJL202. For the purification of N-terminally His-tagged BL00950, the PCR-fragments of bl00950 were cloned into NdeI and BamHI sites of pET-15H-oxyR [16] resulting in plasmids named pJL853. The plasmids were introduced into E. coli BL21 (DE3) pLysS cells for the overexpression of encoded proteins. Each E. coli BL21 (DE3) pLysS strain carrying pJL303, pJL304, pJL302, pJL853, or pJL202 was grown in 1 L of LB medium containing 0.4% (w/v) glucose, chloramphenicol, and ampicillin. At OD 600 of~0.4, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM (with additional final 50 μM ZnSO 4 for cells expressing BL03703), and the cells were allowed to grow for an additional 2 h. The cells were harvested by centrifugation, and lysed by sonication for protein purification. BL00075, BL05249, and BL03703 were purified by heparin-Sepharose and MonoQ chromatography using buffer A (20 mM Tris-HCl, pH 8.0, 0.1 M NaCl, and 5% glycerol (v/v)) containing 10 mM EDTA for BL00075 and BL05249, or 2 mM EDTA for BL03703 with the application of a linear gradient of 0.1-1 M NaCl as described previously [15]. BL00690 was purified by heparin-Sepharose and SP-Sepharose chromatography using buffer A containing 10 mM EDTA with the application of a linear gradient of 0.1-1 M NaCl. Since BL00950 did not bind to heparin-Sepharose resin unlike other Fur family proteins, we used Histagged BL00950 for this study. His-tagged BL00950 was first purified by Ni-NTA chromatography, and subsequently by SP-Sepharose chromatography using buffer A containing 10 mM EDTA with the application of a linear gradient of 0.1-1 M NaCl. All the proteins were further purified using a Superdex 200 HiLoad 16/60 column (GE Healthcare) equilibrated with Chelex-100-treated buffer A. Note that BL00950 was purified as monomer whereas all the other proteins were purified as dimers as judged by elution profiles from Superdex 200 HiLoad gel filtration chromatography. The purities of all of the purified proteins were checked by SDS-PAGE, and their concentrations were determined by measuring A 280 nm using the calculated values of molar extinction coefficient of each protein (BL05249: 11,460 M -1 cm -1 , BL03703: 10,430 M -1 cm -1 , BL00075: 8,940 M -1 cm -1 , BL00690:10,430 M -1 cm -1 , BL00950: 8,940 M -1 cm -1 ).

Electrophoretic mobility shift assay
The 431 bp DNA fragment containing B. subtilis mrgA promoter region was generated by PCR, and subsequently digested with EcoRI, resulting in a 273 bp fragment containing PerR box and a 154 bp fragment used for a non-specific control. The DNA fragments were endlabelled with [γ-32 P] ATP using T4 polynucleotide kinase (NEB) and unincorporated labels were removed using nucleotide removal kit (Qiagen). Protein (BL00690 or BL00950) and a labelled probe were mixed in binding buffer (20 mM Tris-HCl pH 8.0, 50 mM KCl, and 5% glycerol (v/v), 50 μg/ml BSA and 100 μM MnCl 2 ), and separated by 6% PAGE with a 45 mM Tris-borate buffer containing 100 μM MnCl 2 . After 2 h at 120 V, the gel was dried and exposed to X-ray film with an intensifying screen (Kodak) at -80°C.

Measurement of Zn 2+ release by H 2 O 2 using PAR
Measurement of Zn 2+ release by H 2 O 2 was performed as described previously [15,17]. 5 μM protein in buffer A was treated with 0, 1, 10, or 100 mM H 2 O 2 in the presence of 100 μM 4-(2-pyridylazo)resorcinol (PAR), and Zn 2+ -release was measured by monitoring the Zn 2+ -PAR complex at 494 nm every 1 s for 30 min. The Zn 2+ content of purified proteins by PAR assay was determined using a molar extinction coefficient of 85,000 M -1 cm -1 at 494 nm for Zn 2+ -PAR complex.

MALDI-TOF MS and LC-ESI MS/MS analysis
The analysis of protein oxidation after overexpression in E. coli was performed as previously described [17,18]. Briefly, aliquots of E. coli cells (1.8 ml of culture of LE0001, LE0002, LE0008, LE0009, or LE0010) were either treated with 1 mM H 2 O 2 (final concentrations) for 1 min or not. Cells harvested by centrifugation after the addition of 200 μM of trichloroacetic acid (TCA) were sonicated in 500 μl of 10% TCA. The pellets obtained by centrifugation were resuspended with 20 μl IA buffer (50 mM iodoacetamide, 0.5 M Tris pH 8.0, 5% glycerol, 100 mM NaCl, 1 mM EDTA, 2% SDS) and incubated for 1 h in the dark to alkylate free thiols. After separation on 13.3% Tris-Tricine SDS-PAGE and staining with Coomassie Brilliant Blue R-250, protein bands were cut and analyzed by MALDI-TOF MS using a Voyager-DE STR instrument (Applied Biosystems) after in-gel tryptic digestion. The sites of oxidation were identified by LC-MS/MS analyses using an Agilent nanoflow-1200 series HPLC system connected to a linear ion trap mass spectrometer (Thermo Scientific).

Construction of deletion mutant, complementation, and reporter fusion strains
The B. subtilis fur deletion mutant strain (HBL100) was constructed using long-flanking homology PCR as described previously [19]. The fur zur double mutant strain (HBL112) was generated by transformation of HBL100 with zur::tet cassette, and the perR fur zur triple mutant strain (LB1066) was generated by transformation of HBL112 with perR::cat cassette.
For the expression of FLAG fusion proteins from their own promoter in B. subtilis, the PCR fragments containing ORF and about 200 bp upstream region (bl00075, bl05249, bl03703, bl00690, bl00950, fur BS , zur BS ) were individually cloned into BamHI and EagI sites of pJL070. For the expression of FLAG fusion proteins from xylA promoter in B. subtilis, the pXT plasmid which can fuse a xylose-inducible promoter to the gene of interest was used. The PCR fragments containing ribosome binding sequence and perR ORF from pJL070 were cloned into BamHI and EcoRI sties of pXT, generating pJL240. Then, the PCR fragments containing consensus ribosome binding sequence and ORF (perR BS , fur BS , zur BS , bl00690, and bl00950) were each cloned into BamHI and EagI sites of pJL240. For the expression of PerR BS -FLAG, BL00690-FLAG, and BL00950-FLAG from B. subtilis perR promoter in B. subtilis (for the construction of LB2128, LB4034, and LB4106 strains), NdeI site was introduced at the beginning of perR ORF in pJL070 by QuikChange site-directed mutagenesis (Stratagene) generating pJL448. Then, the PCR amplified bl00690 and bl00950 ORFs were each cloned into NdeI and EagI sites of pJL448. The ScaI digest of each plasmid was introduced to the corresponding B. subtilis strain to generate a transformant containing FLAG-fused gene in the amyE (pJL070-derived plasmids) or thrC (pJL240-derived plasmids) locus. The reporter fusion strains were constructed by transduction with SPβ phages, and β-galactosidase assays were performed, as described previously [15].

Identification of five Fur family proteins in B. licheniformis
Many Gram positive bacteria such as B. subtilis, L. monocytogenes and S. aureus encode three Fur family proteins: Fur, Zur, and PerR [20][21][22]. Interestingly, the BLAST homology searches of the B. licheniformis ATCC14580 genome sequence [13] with each one of the B. subtilis Fur family proteins revealed the presence of five putative genes encoding Fur family proteins. BL00075, BL03703, and BL05249 of B. licheniformis show the highest similarity to PerR BS , Zur BS , and Fur BS from B. subtilis, respectively, and all these proteins cluster with their homologues from L. monocytogenes and S. aureus as well as B. subtilis (Fig 1A). Although the sequence identity between BL00690 and BL00950 is not high (33%), both proteins cluster with PerR proteins with sequence identities ranging between 41 and 44% for BL00690 and between 41 and 46% for BL00950 ( Fig 1B). In comparison, BL00690 and BL00950 exhibit sequence identities of~25% to Fur and Zur proteins (Fig 1B), which are comparable to those between PerR and Fur or between PerR and Zur [1,2,22,23].
As shown in Fig 1C, all the five Fur family proteins from B. licheniformis retain four highly conserved Cys residues corresponding to Cys96, Cys99, Cys136, and Cys139 of B. subtilis PerR. These four Cys residues arranged in two CXXC motifs are involved in high affinity structural Zn 2+ -binding in most Fur family proteins including PerR BS [9,15]. In addition to this structural Zn 2+ -binding site, Fur family proteins also have a regulatory metal binding site. For PerR BS , this site is composed of His37, Asp85, His91, His93 and Asp104 [9,11]. These five residues are conserved in BL00690 as well as PerR proteins including BL00075. Although Fur proteins and BL00950 also have conserved N-donor ligands (corresponding to His37, His91, and His93 of PerR BS ), these proteins have a Glu residue in place of Asp104 (for Fur proteins) or Asp85 (for BL00950) as an O-donor ligand. Zur proteins are known to use S-donor ligand corresponding to Cys84 of Zur BS instead of O-donor ligand corresponding to Asp85 of PerR BS for regulatory Zn 2+ -binding, and do not have a conserved N-donor ligand corresponding to His37 of PerR BS [3,5,7]. Based on their repressor activities as described below as well as their sequence similarity and conserved amino acid residues involved in putative structural and regulatory metal binding, we functionally annotate BL00075, BL03703, and BL05249 as PerR BL , Zur BL , and Fur BL , respectively. And, the new Fur homologues, BL00690 and BL00950, were annotated as PerR2 and PerR3, respectively, based on their sequence similarity to the PerR proteins and their ability to sense peroxide by histidine oxidation as described below.
All the five Fur family proteins from B. licheniformis contain structural Zn 2+ The sequence analysis indicates that all the Fur family proteins from B. licheniformis have conserved cysteine residues putatively involved in structural Zn 2+ -binding. To investigate the involvement of cysteine residues in Zn 2+ coordination, we purified all the five Fur family proteins after overexpression in E. coli (Fig 2A), and measured Zn 2+ -release from each protein upon H 2 O 2 treatment by monitoring the formation of PAR-Zn 2+ complex ( Fig 2B) as described previously [15,17]. Interestingly, unlike other Fur family proteins PerR3 did not bind to heparin-Sepharose (which is widely used for the purification of DNA-binding proteins). Furthermore, PerR3 was purified as monomeric protein by a gel filtration chromatography, whereas the other four Fur family proteins were purified as dimeric proteins (see Materials and Methods).
PAR-Zn 2+ complex formation was not detected for 30 min without H 2 O 2 treatment, and the rate of Zn 2+ -release was dependent on added H 2 O 2 concentrations (Fig 2B). These results indicate that all the Fur family proteins from B. licheniformis, including PerR3 purified as monomers, have stably bound Zn 2+ which cannot easily be removed by high affinity Zn 2+ -chelator PAR (K app~1 0 13 M -2 for PAR 2 -Zn 2+ complex, [24]) in the absence of H 2 O 2 . Furthermore, the dependence of Zn 2+ -release on H 2 O 2 strongly suggests that Zn 2+ is coordinated by conserved cysteine residues as observed with PerR proteins [15,17]. The second-order rate constants of Zn 2+ release by H 2 O 2 were determined to be~0.03 M -1 s -1 for PerR2, PerR3, and Fur BL ,~0.04 M -1 s -1 for PerR BL , and~0.01 M -1 s -1 for Zur BL . The slow rates of H 2 O 2 -mediated Zn 2+ release for Fur family proteins from B. licheniformis, which are comparable to those observed with B. subtilis and S. aureus PerR proteins (~0.05 M -1 s -1 ) [15,17], suggest that the Zn 2+ sites play a structural rather than a H 2 O 2 sensing role. The Zn 2+ contents of the purified proteins per monomer were determined to be~0.8 for PerR2,~0.9 for PerR3,~0.5 for Fur BL ,~0.7 for PerR BL , and 0.5 for Zur BL . The retention of~0.5-0.9 Zn 2+ per monomer, despite the use of strong metal chelator EDTA during protein purification (see Materials and Methods), also supports the notion that all the Fur family proteins from B. licheniformis have a structural Zn 2+ site. Altogether, these data indicate that all the five Fur family proteins from B. licheniformis contain a structural Zn 2+ presumably coordinated by conserved cysteine residues like many other Fur proteins.

PerR2 (BL00690) and PerR3 (BL00950) as well as PerR BL can sense H 2 O 2 by protein oxidation
Previously we have shown that the oxidation of PerR proteins can be easily and efficiently evaluated using E. coli system [17,18]. To investigate the oxidation of Fur family proteins from B. licheniformis, we analyzed protein oxidation by MALDI-TOF MS after overexpression in E. coli with or without H 2 O 2 treatment (Fig 3) as described previously [17,18]. As noted for PerR BS , PerR BL showed H 2 O 2 -dependent oxidation at two tryptic peptides, T5 (His25 to Lys45, m/z = 2401. 19) containing His37 and T11 Ã (Phe84 to Arg98, m/z = 1910.85) containing His91, befitting its role as PerR (Fig 3A). In contrast, Fur BL and Zur BL displayed no detectable changes in tryptic peptide peaks after H 2 O 2 treatment (Fig 3D and 3E). Interestingly, PerR2 exhibited significant degree of oxidation at T8 peptide (Asn38 to Arg50, m/z = 1506.80) containing His39 (corresponding to His37 in PerR BS ) even without H 2 O 2 treatment, and further oxidation at T8 peptide and T13 Ã peptide (Phe85 to Lys102, m/z = 2170.99) containing His92 (corresponding to His91 in PerR BS ) after H 2 O 2 treatment (Fig 3B). PerR3 also displayed H 2 O 2 -dependent oxidation, although less when compared with PerR BL and PerR2, at T7 peptide (Thr27 to Arg42, m/z = 1660.84) containing His34 (corresponding to His37 in PerR BS ) and T13 Ã peptide (Tyr77 to Lys90, m/z = 1824.87) containing His84 (corresponding to His91 in PerR BS ) ( Fig 3C). As expected, the sites of oxidation responsible for the 16 Da mass increase were mapped to be His37 and His91 for PerR BL , His39 and His92 for PerR2, and His34 for PerR3 (S1-S5 Figs). The site of oxidation for T13 Ã +16 from PerR3 could not be exactly mapped partially due to the weak signal intensity. The presence of significantly oxidized T8 peptide (T8+16) from PerR2 as compared to that (T5+16) from PerR BL in the absence of H 2 O 2 treatment suggests that PerR2 is more sensitive than PerR BL to oxidation by H 2 O 2 encountered during aerobic growth of E. coli [17]. In addition, no detectable oxidation without external H 2 O 2 treatment and the inefficient oxidation by H 2 O 2 treatment for PerR3 suggest that PerR3 is less sensitive to oxidation by H 2 O 2 than PerR BL or PerR2.
All the peptides (T11 Ã peptide of PerR BL , T13 Ã peptide of PerR2, and T13 Ã peptide of PerR3) containing putative Zn 2+ -binding motif CXXC motif (corresponding to C 96 XXC 99 in PerR BS ) were detected in their fully alkylated form (Fig 3, S2 and S4 Figs). Note that the  small amount of T13 peptide of PerR2, which is detected without alkylation even in the absence of H 2 O 2 treatment, underwent no further oxidation after H 2 O 2 treatment (Fig 3B). This observation that the cysteine residues are refractory to oxidation by H 2 O 2 treatment is consistent with the idea that these cysteine residues are involved in structural Zn 2+ -binding. All these data together suggest that PerR2 and PerR3 as well as PerR BL can sense H 2 O 2 with differential sensitivity, by histidine oxidation but not by cysteine oxidation. -FLAG was expressed from its own promoter (with~200 nucleotide sequence upstream of ORF) in a B. subtilis strain lacking a functional perR, zur, or fur gene, respectively (Fig 4). Since the FLAG epitope-tagged B. subtilis Fur family proteins are fully functional and the epitope tag provides a convenient means of monitoring protein levels in vivo, C-terminal FLAG-tagged proteins were used for activity analyses in vivo [11,17,26,29]. The repressor activity of PerR BL -FLAG was monitored using a B. subtilis mrgA promoter-lacZ reporter fusion (P mrgA -lacZ) which is under the control of PerR BS . As reported previously [11], the P mrgA -lacZ was repressed in cells expressing PerR BS -FLAG but derepressed in the perR null mutant cells. The P mrgA -lacZ was also fully repressed by PerR BL -FLAG, and the repression was relieved upon H 2 O 2 treatment as observed with PerR BS -FLAG (Fig 4A). Fur BL showed a full repressor activity for Fur BS -regulated feuA promoter-lacZ reporter fusion (P feuA -lacZ) (Fig 4B). Zur BL -FLAG exhibited a full repressor activity for B. subtilis yciC promoter-lacZ reporter fusion (P yciC -lacZ) which is under the control of Zur BS , despite the lower levels of expression when compared to Zur BS -FLAG (Fig 4C). We also examined the metal-dependent repressor activities of Fur BL and Zur BL using a metal-limited minimal medium (MLMM). As expected, Fur BL fully repressed the P feuA -lacZ in the presence of Fe like Fur BS , and Zur BL fully repressed the P yciC -lacZ in the presence of Zn like Zur BS (Fig 4G and 4H).
These results imply that PerR BL (BL00075), Fur BL (BL05249), and Zur BL (BL03703) may function as PerR, Fur, and Zur, respectively, in B. licheniformis, and that each protein can be expressed from its own promoter located in~200 nucleotide sequence upstream of each ORF.

PerR2 (BL00690), but not PerR3 (BL00950), has a PerR-like repressor activity
PerR2-FLAG and PerR3-FLAG could not be expressed with~200 nucleotide sequence upstream of their ORFs, thus it is likely that the genes encoding these proteins do not have their own promoters. To express PerR2-FLAG and PerR3-FLAG and investigate the roles of Characterization of Fur Family Proteins from Bacillus licheniformis these proteins in vivo, we used the pXT system which fuses a xylose-inducible promoter to the gene of interest, and a triple mutant B. subtilis strain which lacks all of the three fur family genes (Fig 5). Despite the use of same xylA promoter along with the consensus ribosome binding site, the expression levels of Fur family proteins were not identical possibly by differences in mRNA and/or protein stability (Fig 5A). However, as observed with single mutant background with their own promoters (Fig 4), PerR BS -FLAG, Fur BS -FLAG, and Zur BS -FLAG expressed from xylA promoter fully repressed the P mrgA -lacZ, P feuA -lacZ, and P yciC -lacZ, respectively (Fig 5B). Although PerR3-FLAG was highly expressed under the control of xylA promoter, PerR3-FLAG showed no repressor activity for PerR-regulated reporter fusion as well as for Fur-and Zur-regulated reporter fusions. Interestingly, PerR2-FLAG exhibited repressor activity for the PerR-regulated P mrgA -lacZ, but no repressor activity for the Fur-regulated P feuA -lacZ nor the Zur-regulated P yciC -lacZ. This specific repressor activity of PerR2 for the known PerR-regulated promoters, along with its H 2 O 2 -dependent histidine oxidation, suggest that PerR2 may act as a second PerR in B. licheniformis.
It is known that a B. subtilis perR null mutant strain grows very poorly in nonstressed conditions due to Fe deficiency resulting from elevated levels of Fur BS and KatA [19]. To examine whether PerR2 can complement the perR null mutant strain and rescue the small colony phenotype, complementation experiments were performed ( Fig 5C). As expected, the perR null mutant strain expressing PerR BS -FLAG showed a wild-type like colony phenotype. The perR null mutant strain expressing PerR2 also exhibited significantly increased colony size, indicating that PerR2 can rescue the Fe-deficiency presumably by reducing the levels of KatA and/or Fur BS . In contrast, the perR null mutant strain expressing PerR3 still exhibited the small colony phenotype (Fig 5C) consistent with the lack of repressor activity for the PerR-regulated gene (Fig 5B).
To investigate the interaction of PerR2 with DNA, we performed electrophoretic mobility shift assays using the B. subtilis mrgA promoter regions as probe. As shown in Fig 5D, PerR2 specifically shifted the DNA fragment containing PerR box but not the DNA fragment lacking PerR box. This result indicates that the repressor activity of PerR2 observed with the PerR-regulated promoter fusion is due to direct interaction of PerR2 with PerR box. However, it should be noted that the apparent K d value of PerR2 for DNA binding was measured to be~70 nM. This rather weak DNA binding activity of PerR2, as compared to that of PerR BS (K d~1 0 nM) [29], is likely to reflect the higher oxidation (inactivation) levels of PerR2 as shown in Fig 3B. In contrast, consistent with the lack of repressor activity for the PerR regulated promoter fusion, PerR3 showed no DNA binding activity (Fig 5D). In vivo repressor activities of PerR BL , Fur BL , and Zur BL (A) Repressor activities of PerR BS and PerR BL for P mrgA -lacZ reporter fusion. B. subtilis cells expressing no PerR orthologue (LB1532), PerR BS -FLAG (HB9738), or PerR BL -FLAG (LB1023) were treated without or with 100 μM H 2 O 2 for 30 min, and β-galactosidase activities were measured using P mrgA -lacZ reporter fusion. (B) Repressor activities of Fur BS and Fur BL for P feuA -lacZ reporter fusion. B. subtilis cells expressing no Fur orthologue (LB1040), Fur BS -FLAG (LB1041), or Fur BL -FLAG (LB1042) were treated without or with 100 μM H 2 O 2 for 30 min, and β-galactosidase activities were measured using P feuA -lacZ reporter fusion. (C) Repressor activities of Zur BS and Zur BL for P yciC -lacZ reporter fusion. B. subtilis cells expressing no Zur orthologue (LB1034), Zur BS -FLAG (LB1035), or Zur BL -FLAG (LB1036) were treated without or with 100 μM H 2 O 2 for 30 min, and βgalactosidase activities were measured using P yciC -lacZ reporter fusion. (D-F) Western blot analyses of FLAG-fused PerR orthologues (D), Fur orthologues (E), and Zur orthologues (F). The FLAG-fused proteins were probed by anti-FLAG antibody. (G) Fe-dependent repressor activities of Fur BS and Fur BL for P feuA -lacZ reporter fusion. B. subtilis cells expressing no Fur orthologue (LB1040), Fur BS -FLAG (LB1041), or Fur BL -FLAG (LB1042) were grown in MLMM supplemented with or without 10 μM FeSO 4 , and β-galactosidase activities were measured using P feuA -lacZ reporter fusion. (H) Zn-dependent repressor activities of Zur BS and Zur BL for P yciC -lacZ reporter fusion. B. subtilis cells expressing no Zur orthologue (LB1034), Zur BS -FLAG (LB1035), or Zur BL -FLAG (LB1036) were grown in MLMM supplemented with or without 10 μM ZnCl 2 , and β-galactosidase activities were measured using P yciC -lacZ reporter fusion.

Discussions
Proteins with Fur-like domain architecture are widespread in prokaryotes with~20,000 homologues in EMBL-EBI InterPro database (IPR002481). Depending on signals they respond, Fur family proteins are classified as Fur (Fe), Zur (Zn), Mur (Mn), Nur (Ni), PerR (peroxide), and Irr (heme) [1,2]. Among these, Fur is the most ubiquitous, and Zur, albeit not as ubiquitous as Fur, is also widespread in Gram negative and Gram positive bacteria. In contrast, PerR is mainly found in Gram positive bacteria as a functional substitute for OxyR, although it is also found in some Gram negative bacteria, and, in some cases, coexists with OxyR [1,10]. Mur and Irr have been found in some α-proteobacteria including Rhizobiales and Rhodobacterales [30], and Nur has been only found in Streptomyces genus [31]. Although four Fur paralogues (Fur orthologue FurA, PerR orthologue CatR, Zur, and Nur) have been found and characterized in S. coelicolor [3,[31][32][33], many bacteria contains up to three Fur family proteins, usually two or three. For example, Gram negative bacteria E. coli and V. cholerae contain two (Fur and Zur) [4,7], and Gram positive bacteria B. subtilis and S. aureus contain three (Fur, Zur, and PerR) [20,22]. In this study, we found that B. licheniformis, a close relative of B. subtilis, contains five Fur family proteins. Like many other Fur family proteins, all these proteins retain a tightly bound Zn 2+ presumably coordinated by highly conserved cysteine residues. Three of them were identified as Fur, Zur, and PerR orthologues of B. subtilis based on their repressor activity. The other two were identified as PerR-like proteins based on their sequence similarity to PerR proteins and their H 2 O 2 -dependent oxidation of histidine residues.
The H 2 O 2 -sensing mechanism of PerR has only been extensively studied in B. subtilis and S. aureus, despite its wide distribution in most Gram positive bacteria and in some Gram negative bacteria [10,11,17,25]. Unlike OxyR which utilizes the oxidation of cysteine thiol, PerR uses a distinct Fe-dependent histidine oxidation mechanism for H 2 O 2 sensing, where H 2 O 2 oxidizes the histidine ligands of the Fe 2+ at the regulatory site to 2-oxo-histidine. Our results indicate that PerR BL also uses a histidine oxidation mechanism for H 2 O 2 sensing. Furthermore, we found that H 2 O 2 can also oxidize the two other PerR-like proteins, PerR2 and PerR3, but not Fur BL and Zur BL . MALDI-TOF MS and ESI-MS/MS analyses of the tryptic peptides, along with sequence analyses, of PerR2 and PerR3 indicate that the oxidation events occur at histidine residues rather than cysteine residues. Despite the high similarity between the regulatory metal binding sites of PerR and Fur, Fur does not react with H 2 O 2 under conditions where PerR does [11]. Recently, it has been suggested that O-donor ligand corresponding to Asp104 of PerR BS or Glu108 of Fur BS is the key residue which determines the accessibility of H 2 O 2 to Fe 2+ -coordination site [12]. It is noteworthy that PerR2 and PerR3, as well as other PerR proteins, also contain a conserved Asp at this position, whereas Fur proteins have a Glu (Fig 1C).
Despite the presence of bona fide PerR BL , PerR2 also showed specific repressor activity on the representative PerR-regulated gene but not on Fur-or Zur-regulated gene, and the perR null mutant small colony phenotype could be rescued by PerR2 (Fig 5). Thus, it is reasonable to speculate that PerR regulon in B. licheniformis is under the control of both PerR BL and Repressor activities of PerR3 and PerR2 were measured using P mrgA -lacZ, P feuA -lacZ, and P yciC -lacZ reporter fusions. As a control, repressor activities of PerR BS , Fur BS , and Zur BS were also measured using P mrgA -lacZ, P feuA -lacZ, and P yciC -lacZ reporter fusions, respectively. The reporter fusion strains were constructed from the strains used in Fig 5A ( Table 1) PerR2. In the simplest scenario, the two proteins would exert influence on the PerR regulon genes simultaneously. Alternatively, each protein may regulate genes under different conditions. The higher sensitivity of PerR2 than PerR BL seems to suggest the differential role of these proteins under different oxidation conditions. Unlike PerR2, no repressor activity of PerR3 was observed for any genes under the control of PerR, Fur, and Zur using B. subtilis reporter fusion assays. And, PerR3 was purified as monomer after overexpression in E. coli, whereas all the other four Fur family proteins from B. licheniformis were purified as dimer. Considering that all the biochemically characterized Fur family proteins are dimeric DNA binding proteins, PerR3 may not be a canonical Fur family protein. However, the oxidation of PerR3 by H 2 O 2 , albeit less sensitive as compared to PerR BL or PerR2, suggests that PerR3 may play a role as a H 2 O 2 sensor in B. licheniformis. It has been previously reported that the transcription of perR3 (bl00950, bli04114) is massively induced after H 2 O 2 treatment [34]. Interestingly, the genes encoding for PerR3 and BL00949 (BLi04115, putative ferrochelatase) are located directly downstream of katA gene (bl00951, bli04113). Furthermore, in contrast to B. subtilis katA gene which is monocistronically transcribed under the control of PerR BS , B. licheniformis katA gene is cotranscribed with perR3 and bl00949 after H 2 O 2 treatment [34]. These imply that PerR3 may have some role especially under conditions of H 2 O 2 stress.
In summary, we have shown that B. licheniformis contains a total of five Fur family proteins: two novel PerR-like proteins in addition to the canonical PerR, Fur, and Zur. The presence of two additional Fur family proteins in B. licheniformis, in contrast to its close relative B. subtilis, may indicate that the metal ion regulation and peroxide stress response under the control of Fur family proteins are far more complex than previously reported for B. subtilis. Further study is required to identify distinct roles of PerR2 and PerR3 along with their relevance to other Fur family proteins in B. licheniformis. The b-and y-ions are shown in purple and blue, respectively. The y-ions not containing His37 (y4-y8) appear at the predicted m/z values, whereas the subsequent y-ions containing His37 (y9-y20) have a +16 Da mass shift. Note that almost all the y9-and y10-ions (containing His37 but not Met35) have a +16 Da mass shift. The b-ions not containing His37 (b5-b12) appear at the predicted m/z values, whereas the subsequent b-ions containing His37 (b13-b20) have a +16 Da mass shift. Note that almost all the b10-and b11-ions (containing Met35 but not His37) appear at the predicted m/z values. Taken together, these data indicate that most of the oxidation in T5+16 peptide occurred at His37 rather than Met35.  29, shown in green) was analyzed by tandem MS. The b-and y-ions are shown in purple and blue, respectively. The y-ions not containing His34 (y3-y8) appear at the predicted m/z values, whereas the subsequent y-ions containing His34 (y9-y15) has a +16 Da mass shift. The b-ions not containing His34 (b5-b7) appear at the predicted m/z values, whereas the subsequent b-ions containing His34 (b8-b16) has a +16 Da mass shift. These data indicate that the oxidation in T7+16 peptide occurred at His34. (TIF)