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Comparative Study
. 2017 Jan 6;292(1):121-133.
doi: 10.1074/jbc.M116.757500. Epub 2016 Nov 28.

Lineage-specific SoxR-mediated Regulation of an Endoribonuclease Protects Non-enteric Bacteria from Redox-active Compounds

Jisun Kim  1 , Chulwoo Park  1 , James A Imlay  2 , Woojun Park  3
Affiliations
Comparative Study

Lineage-specific SoxR-mediated Regulation of an Endoribonuclease Protects Non-enteric Bacteria from Redox-active Compounds

Jisun Kim et al. J Biol Chem. .

Abstract

Bacteria use redox-sensitive transcription factors to coordinate responses to redox stress. The [2Fe-2S] cluster-containing transcription factor SoxR is particularly tuned to protect cells against redox-active compounds (RACs). In enteric bacteria, SoxR is paired with a second transcription factor, SoxS, that activates downstream effectors. However, SoxS is absent in non-enteric bacteria, raising questions as to how SoxR functions. Here, we first show that SoxR of Acinetobacter oleivorans displayed similar activation profiles in response to RACs as did its homolog from Escherichia coli but controlled a different set of target genes, including sinE, which encodes an endoribonuclease. Expression, gel mobility shift, and mutational analyses indicated that sinE is a direct target of SoxR. Redox potentials and permeability of RACs determined optimal sinE induction. Bioinformatics suggested that only a few γ- and β-proteobacteria might have SoxR-regulated sinE Purified SinE, in the presence of Mg2+ ions, degrades rRNAs, thus inhibiting protein synthesis. Similarly, pretreatment of cells with RACs demonstrated a role for SinE in promoting persistence in the presence of antibiotics that inhibit protein synthesis. Our data improve our understanding of the physiology of soil microorganisms by suggesting that both non-enteric SoxR and its target SinE play protective roles in the presence of RACs and antibiotics.

Keywords: Acinetobacter; Escherichia coli (E. coli); SoxR; antibiotics; bacteria; biofilm; endoribonuclease; oxidative stress; reactive oxygen species (ROS); transcriptional regulation.

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Figures

FIGURE 1.
FIGURE 1.
Functional identification of SoxR. Shown is complementation of an E. coli soxR mutant with AoSoxR (A and B), PpSoxR (C), and EcSoxR (D). A, strain EH46 (pCR-AoSoxR) was grown anaerobically in LB or LB/nitrate medium. Cells in the exponential phase of growth were treated with 50 μm PMS for 2 h. The level of soxS′::lacZ reporter expression was measured by β-galactosidase activity using o-nitrophenyl-β-d-galactopyranoside as a substrate. B, C, and D, strains were grown aerobically in LB medium. The same concentration (0.1 mm) of PQ, MDs, PMS, PL, H2O2, and CHP and 0.01 mm PYO were used to treat exponentially growing cells. After incubation for 1 h, the expression of soxS′::lacZ was quantified by measuring β-galactosidase activity. All data show the average of three replicates, and error bars indicate S.D.
FIGURE 2.
FIGURE 2.
SoxR protection against high concentrations of PMS and PL. Cells were inoculated into nutrient medium, and the cultures were incubated at 30 °C. When the cultures reached the exponential phase, low concentrations of RACs (10 μm) were added. After 30 min of incubation, a higher concentration (50 μm) of the same compounds was added to culture. Cell growth was measured at A600. The graphs show the average of three replicates, and the error bars indicate S.D.
FIGURE 3.
FIGURE 3.
Gene expression analysis obtained from RNA-seq and qRT-PCR and binding of SoxR to the promoter region of target genes. A, comparison of gene expression patterns obtained from the RNA-seq analysis of exponentially growing wild type cells and PQ- or PMS-treated cells. The y axis shows RPKM values of each sample. B, relative expression of SoxR-controlled genes in the wild type and soxR mutant by qRT-PCR. The cells were grown to the exponential phase (A600 ∼0.4) and treated with paraquat (1 mm) for 15 min. All data represent the average of three replicates, and the error bars indicate S.D. C, binding of SoxR to the promoter region of target genes. Lane 1, free DNA (no protein); lane 2, purified SoxR, 3 nm; lane 3, purified SoxR, 7 nm; lane 4, purified SoxR, 13 nm; lane 5, purified SoxR, 25 nm; lane 6, purified SoxR, 40 nm; lane 7, purified SoxR, 70 nm; lane 8, purified SoxR, 70 nm with non-probing DNA. The nonspecific competitor poly(dI-dC) was added to all binding reactions. Protein-DNA complexes (B) and free DNA probes (F) are indicated with filled and open arrowheads, respectively.
FIGURE 4.
FIGURE 4.
Ranges of RAC concentrations for AoSoxR activation. Exponentially growing cells (A600 ∼0.4) were treated with various concentrations of RACs for 15 min. To evaluate SoxR activation, its direct target gene (sinE) was selected and subjected to gene expression analysis. The solvents and reduction potentials of the RACs are indicated in parentheses.
FIGURE 5.
FIGURE 5.
Function of SinE as a ribonuclease and inhibition of protein synthesis. A, in vitro RNase activity of SinE. Total RNA (1.5 μg) isolated from A. oleivorans DR1 was incubated with SinE (8–40 μm) and MgCl2 (2.5–5 mm) for 1 h at 30 °C. Reaction mixtures were loaded onto denaturing agarose gels and then stained with ethidium bromide to visualize 23S and 16S rRNA. EDTA (50 mm) was used to inhibit RNase activity. To compare residual 16S rRNA after reaction with SinE, semiquantitative real-time PCR was conducted. B, comparison of protein synthesis between the wild type and the soxR and sinE mutant under PMS treatment. The cells were grown to exponential phase (A600 ∼0.2) in 10-fold diluted nutrient medium and treated with PMS (10 μm). To measure protein synthesis, cells were labeled with 5 μCi/ml [35S]methionine. At different time intervals, each sample was mixed with liquid mixture and measured in a scintillation counter. Radioactivity was normalized by the A600 and expressed as a relative ratio, taking the radioactivity of 0 min samples to be 1. All data show the average of three replicates, and the error bars indicate S.D.
FIGURE 6.
FIGURE 6.
Comparison of RAC and antibiotic sensitivity in the wild type and soxR and sinE mutants. A, PMS sensitivity assay. Cells were grown overnight in nutrient medium and subsequently diluted 100-fold. The cells reached the mid-exponential phase (A600 ∼0.6), and serially diluted cells were spotted on nutrient agar with or without PMS. B, survival of cells after treatment with PMS or PL. Exponential phase cells were harvested, and ∼107 cells/ml were inoculated into fresh PBS (5 ml) containing 100 μm PMS or PL. At each time point, the cells were harvested and washed in PBS. The number of viable cells was determined by counting the cfu. C, effect of induced SinE on antibiotic resistance. PMS (25 μm) was added to cells in the exponential phase for 30 min. Cells were washed twice to remove PMS and inoculated into fresh nutrient broth with antibiotics (gentamicin, 5 μg/ml; tetracycline, 5 μg/ml; chloramphenicol, 100 μg/ml; ampicillin, 1000 μg/ml; rifampicin, 10 μg/ml). After 3 h of incubation, the number of viable cells was determined by counting the cfu. All data show the average of three replicates, and the error bars indicate S.D.
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