orf4 of the Bacillus cereus sigB gene cluster encodes a general stress-inducible Dps-like bacterioferritin

Abstract

The function of orf4 in the sigB cluster in Bacillus cereus ATCC 14579 remains to be explored. Amino-acid sequence analysis has revealed that Orf4 is homologous with bacterioferritins and Dps. In this study, we generated an orf4-null mutant and produced recombinant protein rOrf4 to establish the role of orf4. In vitro, the purified rOrf4 was found to exist in two distinct forms, a dimeric form and a polymer form, through size exclusion analysis. The latter form exhibited a unique filament structure, in contrast to the typical spherical tetracosamer structure of bacterioferritins; the former can be induced to form rOrf4 polymers immediately after the addition of FeCl(2). Catalysis of the oxidation of ferrous irons by ferroxidase activity was detected with rOrf4, and the mineralized irons were subsequently sequestered only in the rOrf4 polymer. Moreover, rOrf4 exerted DNA-protective activity against oxidative damage via DNA binding in a nonspecific manner, as is seen with Dps. In vivo, deletion of orf4 had no effect on activation of the alternative sigma factor sigma(B), and therefore, orf4 is not associated with sigma(B) regulation; however, orf4 can be significantly upregulated upon environmental stress but not H(2)O(2) treatment. B. cereus strains with constitutive Orf4 expression exhibited a viability higher than that of the orf4-null mutant, under specific oxidative stress or heat shock. Taken together, these results suggest that Orf4 functions as a Dps-like bacterioferritin in response to environmental stress and can provide cell protection from oxidative damage through iron sequestration and DNA binding.

FIG. 1.

Confirmation of orf4 deletion and orf4 not being involved in σ^B regulation. (A) Schematic diagram of genetic organization of sigB cluster. (B) Construction of the orf4 mutant by allelic exchange. The orf4 gene was replaced by a 1.2-kb spectinomycin resistance cassette. DNA was introduced into B. cereus by electroporation. BamHI restriction enzyme sites were present in the spectinomycin resistance cassette. The thick line indicates the fragment from the orf4 upstream region used as the probe for Southern blotting. The predicted hybridization sizes are shown. (C) Southern blotting confirming the disruption of orf4. Chromosomal DNA from the wild-type strain and the orf4 mutant were digested with BamHI and probed with the DNA upstream of orf4; the expected DNA sizes are indicated by the arrow symbol. Lane 1, wild-type (WT) genomic DNA; lane 2, orf4 mutant genomic DNA. The sizes of the molecular weight markers are indicated on the left. (D) Orf4 deletion did not affect σ^B activation. Cell lysates harvested from the wild-type and orf4 mutant strains at the indicated times were measured by Western blotting using anti-σ^B antibody.

FIG. 2.

Expression of Orf4 in B. cereus. (A) Detection of Orf4 expression with Western blotting. Cells were harvested at different times under various stresses or at different growth stages. Cell extracts (20 μg) were used for SDS-PAGE and then Western blotting with anti-Orf4 polyclonal antibody. Orf4 is depicted by the arrows. (B) Real-time PCR quantification of orf4 transcript levels. Cultures of wild-type strain B. cereus were grown until the mid-exponential growth phase and exposed to the stresses indicated in panel A for 10 min. RNA was extracted for real-time PCR. Relative expression levels of orf4 are shown in comparison with that of mid-exponential phase, which is shown as onefold. Means and error bars of three independently stress-treated cultures are displayed. The numbers 1, 2, 3, 4, and 5 represent mid-exponential phase, 42°C heat, 2.5% NaCl, 4% ethanol, and 50 μM H₂O₂, respectively.

FIG. 3.

Characterization of the rOrf4 protein. (A) His6-tag rOrf4 was purified using a nickel column followed by Sephacryl 200 gel filtration chromatography. The fractions were analyzed by 12% SDS-PAGE. M represents protein markers and the labels on the left indicate the molecular masses (in thousands). (B) Native Orf4 was analyzed using Superose 12 following DEAE chromatography. The molecular masses (in thousands) of the gel filtration fractions are denoted by the arrows on the top of the SDS-PAGE results. (C) Negatively stained electron micrographs of the automated six-His-tagged rOrf4 polymer. The sample was placed on a carbon film mounted on a 300-mesh copper grid, immediately removed, and stained with 2% phosphotungstic acid (pH 7.2). Micrographs were taken using a Hitachi 7100 electron microscope operating at 75 kV. The scale bar corresponds to 200 nm.

FIG. 4.

Kinetics of iron oxidation in B. cereus Orf4. Iron oxidation kinetics were monitored spectrophotometrically at 310 nm after the addition of 300 μM Fe(II) to 1 μM in deoxygenated MOPS buffer. Absorbance values with Fe(II) (solid line) or without Fe(II) (dashed line) are shown.

FIG. 5.

Molecular mass determination of recombinant Orf4 proteins. (A) Size exclusion analysis. A total of 50 μM Orf4 was incubated with or without 500 μM FeCl₂ and then subjected to Superose 12 gel permeation chromatography. Blue line and green line denote six-His-tagged rOrf4 dimer and native Orf4 dimer incubation with FeCl₂, respectively. Red line represents six-His-tagged rOrf4 dimer incubation without FeCl₂. Arrows indicate the molecular mass and corresponding elution volume for Orf4 dimer and iron-induced polymer. mAU, absorption units at 280 nm. (B) SDS-PAGE analysis. Fractions represented in panel A were analyzed by 12% PAGE. Six-His-tagged rOrf4 dimer was incubated without FeCl₂ (top) or with FeCl₂ (bottom). Molecular masses (in thousands) are shown. (C) Detection of ferric iron. The fractions represented in panel B were assessed at a λ310 nm. The solid line represents His₆-tagged rOrf4 incubation with ferrous iron; the dashed line indicates incubation without ferrous iron.

FIG. 6.

rOrf4 interaction with DNA. (A) DNA binding of rOrf4. Plasmid DNA pGEX-6p-3 (0.2 μg) was incubated with different amounts of rOrf4 and subsequently loaded onto 1% agarose gel for electrophoresis. Lane 1, 14-kb DNA marker; lane 2, pGEX-6p-3; lanes 3 to 6, pGEX-6p-3 incubation with 5, 10, 30, and 45 μg rOrf4; lanes 7 to10, pGEX-6p-3 incubation with 5, 10, 30, and 45 μg BSA. (B) DNA protection assay of rOrf4. Plasmid DNA pGEX-6p-3 (0.2 μg) was added to 10 mM H₂O₂ in the presence of FeSO₄ after incubation with or without proteins. Lane 1, 14-kb DNA marker; lane 2, pGEX-6p-3; lanes 3 to 7, pGEX-6p-3 + 1 to 5 μg rOrf4+FeSO₄ + H₂O₂; lane 8, pGEX-6p-3 + FeSO₄ + H₂O₂.

FIG. 7.

Orf4 protects cells from oxidative stress. (A) Constitutive expression of Orf4. Four B. cereus strains including the wild-type strain, WT708 (orf4-null mutant), WT710 (wild-type strain harboring pRF305), and WT711 (orf4-null mutant harboring pRF305) were measured for Orf4 expression by Western blotting in the absence of environmental stress. The constitutive Orf4 expressions in strain WT710 and WT711 are clearly shown. OD₆₀₀, optical density at 600nm. (B) Orf4 overexpression increases cell viability under the condition of 50°C heat stress or 1.5 mM t-BOOH treatment. A cell culture was harvested every 30 min to measure cell viability by plating count. The diamond, square, asterisk, triangle, and circle symbols denote the survival rate of wild-type B. cereus, WT708, WT709 (orf4-null mutant harboring pRF304), WT710, and WT711, respectively. The means of three replications are displayed for cell viability at each recorded time point.