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

Abstract

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.

Fig 1. Sequence analysis of five Fur family proteins from B. licheniformis.

(A) Phylogenetic tree of Fur-like proteins. The tree was constructed based on a multiple sequence alignment of Fur-like proteins from Bacillus subtilis (BS), Bacillus licheniformis (BL), Listeria monocytogenes (LM), and Staphylococcus aureus (SA), using CLUSTALW. Note that BL00690 and BL00950 cluster with PerR proteins. The scale bar represents an estimated distance of 0.1 amino acid substitution/site. (B) Amino acid identity matrix for BL00690 and BL00950. The amino acid sequences of BL00690 and BL00950 were compared with other Fur family proteins. The sequence identity values were shown as %. (C) Sequence alignment of Fur family proteins. The predicted structural Zn²⁺-binding site (blue) and regulatory metal binding site (red) are highly conserved in all five Fur-like proteins from B. licheniformis. The numbers above each sequence alignment group correspond to the sequence numbers of PerR_BS, Zur_BS, and Fur_BS, respectively.

Fig 2. Zn²⁺-contents of Fur family proteins from B. licheniformis.

(A) Purified Fur family proteins from B. licheniformis. B. licheniformis Fur-like proteins were purified after overexpression in E. coli, and analyzed by SDS-PAGE after alkylation by iodoacetamide. (B) H₂O₂-dependent Zn²⁺-release. Release of Zn²⁺ from proteins (5 μM) was measured by monitoring Zn²⁺-PAR complex at 494 nm every 1 sec for 30 min after treatment of 0, 1, 10, and 100 mM H₂O₂. Data for experiments with 100 mM H₂O₂ are only shown for clarity. The Zn²⁺-content of proteins was calculated using a molar extinction coefficient of 85,000 M^-1cm^-1 at 494 nm for Zn²⁺-PAR complex.

Fig 3. H₂O₂-dependent oxidation of Fur family proteins from B. licheniformis.

Oxidation of PerR_BL (A), PerR2 (B), PerR3 (C), Fur_BL (D), and Zur_BL (E) before and after H₂O₂ treatment. E. coli cells expressing PerR_BL (LE0008), PerR2 (LE0002), PerR3 (LE0001), Fur_BL (LE0009), or Zur_BL (LE0010), were treated with or without 1 mM H₂O₂ for 1 min. Oxidation status of proteins was analyzed by MALDI-TOF MS after SDS-PAGE fractionation and in-gel tryptic digestion. Asterisks represent peptides containing one (PerR_BL and PerR3), two (PerR2 and Fur_BL), or three (Zur_BL) carboxyamidomethylated cysteine residues.

Fig 4

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 H₂O₂ 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 H₂O₂ 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 H₂O₂ for 30 min, and β-galactosidase 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 FeSO₄, 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 ZnCl₂, and β-galactosidase activities were measured using P_yciC-lacZ reporter fusion.

Fig 5. Repressor activity of PerR2.

(A) Western blot analysis of FLAG-fused Fur family proteins expressed from xylA promoter. B. subtilis perR fur zur triple mutant cells expressing no Fur family protein (LB1227), PerR3 (LB1287), PerR2 (LB1288), PerR_BS (LB1490), Fur_BS (LB1491), and Zur_BS(LB1493) were used. (B) Repressor activities of PerR2 and PerR3. 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). (C) Effects of PerR2 and PerR3 on the growth of the B. subtilis perR deletion mutant strain. The B. subtilis perR deletion mutant cells expressing no PerR (LB1010), PerR_BS (LB2128) PerR2 (LB4034), or PerR3 (LB4106) were grown on LB agar plate for 1 day. (D) DNA binding activities of PerR2 and PerR3. DNA binding activities of PerR2 and PerR3 were measured by EMSA in the presence of 100 μM MnCl₂.