(NbaA) and Its Mutants Alters the Sensitivity of Escherichia coli to

Article pubs.acs.org/jpr

Overexpression of Reactive Cysteine-Containing 2-Nitrobenzoate Nitroreductase (NbaA) and Its Mutants Alters the Sensitivity of Escherichia coli to Reactive Oxygen Species by Reprogramming a Regulatory Network of Disulfide-Bonded Proteins Yong-Hak Kim*,† and Myeong-Hee Yu*,‡ †

Department of Microbiology, Catholic University of Daegu School of Medicine, Daegu705-718, Republic of Korea Functional Proteomics Center, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul 136-791, Republic of Korea

‡

S Supporting Information *

ABSTRACT: The effects of redox-sensitive proteins on Escherichia coli were investigated by overexpressing Pseudomonas 2-nitrobenzoate nitroreductase (NbaA) and its mutants. Overexpression of wild-type and mutant NbaA proteins significantly altered the sensitivity of E. coli to antibiotics and reactive oxygen species regardless of the enzyme activity for reduction of 2-nitrobenzoic acid. The overexpressed proteins rendered cells 100−10000-fold more sensitive to superoxide anion (O2•−)-generating paraquat and 10−100-fold more resistant to H2O2. A significant increase in intracellular levels of O2•−, but not H2O2, was observed during expression of wild-type and truncated (Δ65−74, Δ193−216, and Δ65− 74Δ193−216) NbaA. From two-dimensional nonreducing/reducing sodium dodecyl sulfate− polyacrylamide gel electrophoresis and mass spectrometry analyses, 29 abundant proteins in the cytoplasm were identified to form interchain disulfide bonds, when cells were exposed to polymyxin B. Of them, down-regulation and modifications of SodB, KatE, and KatG were strongly associated with elevated cellular O2•− levels. Western blotting showed up-regulation of cell death signal sensor, CpxA, and down-regulation of cytoplasmic superoxide dismutase, SodB, with ∼2-fold up-regulation of heterodimeric integration host factor, Ihf. Activity gel assays revealed significant reduction of glyceraldehyde-3-phosphate dehydrogenase with constant levels of 6-phosphogluconate dehydrogenase. These changes would support a high level of NADPH to reduce H2O2-induced disulfide bonds by forced expression of thioredoxin A via thioredoxin reductase. Thus, overexpression of wild-type and truncated NbaA partially compensates for the lack of KatE and KatG to degrade H2O2, thereby enhancing disulfide bond formation in the cytoplasm, and modifies a regulatory network of disulfide-bonded proteins to increase intracellular O2•− levels. KEYWORDS: disulfide bond, reactive oxygen species sensitivity, antimicrobial susceptibility, superoxide anion, hydrogen peroxide, superoxide dismutase, catalase-peroxidase, regulatory pathway, cell death signal

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INTRODUCTION Proteomic studies on disulfide-bonded proteins, as a result of posttranslational modification, are not successfully employed in defining new redox pathways, which can lead to changes in gene expression.1 It is mainly due to the low abundance of redox signal transduction or regulatory proteins in cells.1 Moreover, cytoplasmic proteins do not contain structural disulfide bonds, because most cysteine residues have a pKa value of >8.0 and thus remain protonated at a physiological pH around 7.4.2 The reducing environment of the cytoplasm is tightly protected by redundant redox enzymes such as thioredoxins and glutaredoxins.3 Reactive cysteines in redoxsensitive proteins are likely to form disulfide bonds within the cytoplasm of unstressed as well as stressed cells primarily on the exposure to the oxidizing environment and chemicals.4 Because there exist a number of abundant proteins that interact with reactive cysteine-containing proteins, it is necessary to © 2012 American Chemical Society

interpret regulatory networks of disulfide-bonded proteome in conjunction with biochemical and genetic information. Reactive cysteine is vulnerable to oxidation and can cross-link through disulfide bonds to other cysteines in close proximity even under mild conditions.5 Cysteine is a rarely used amino acid that accounts for about 2% in eukaryotic proteins and about 1% in prokaryotic proteins.6 Some cysteines are highly conserved in the catalytic sites of thioredoxin-like redox proteins,7 peroxiredoxins, and flavoprotein reductases.8 They are subject to several forms of oxidative posttranslational modifications, including sulfenation (SOH), sulfination (SO2H), nitrosylation (SNO), disulfide formation, and glutathionylation, which play a catalytic or regulatory role in the cellular response to oxidative stress. In addition, oxidation of noncatalytic cysteines in a vast number of proteins can play a Received: December 29, 2011 Published: May 7, 2012 3219

dx.doi.org/10.1021/pr300221b | J. Proteome Res. 2012, 11, 3219−3230

Journal of Proteome Research

Article

obtained from Promega (Madison, WI). A fast protein liquid chromatography system, columns, and resins were supplied by GE Healthcare Life Sciences (Uppsala, Sweden). Protein mini gel kits and assay reagents were supplied by Bio-Rad (Hercules, CA) and Pierce (Thermo Fisher Scientific Inc., Rockford, IL). All other solvents and reagents used were of analytical grade and were obtained from USB Corporation (Cleveland, OH), J. T. Baker (Phillipsburg, NJ), and Merck (Darmstadt, Germany).

role in protein stability, protein folding and refolding, regulation of signal transduction, and substrate-binding affinity and activity of enzyme.9−13 However, little has been known about the cellular response to reactive oxygen species (ROS), when reactive cysteine-containing proteins are overexpressed. In the course of characterizing a catalytic enzyme, 2nitrobenzoate nitroreductase (NbaA; GenBank accession number, BAF56676), we observed that NbaA is able to form various disulfide-bonded proteins at physiological condition. It is an unique enzyme that transforms 2-nitrobenzoic acid (NBA) to 2-hydroxyaminobenzoic acid.14−16 In the polypeptide of NbaA, there are two variable regions (amino acids 65− 74 and 193−216) and four cysteines at positions 39, 103, 141, and 194. The deletion of variable regions (Δ65−74, Δ193− 216, and Δ65−74Δ193−216) or alanine substitution of every cysteine (C39A, C103A, C141A, C194A, and C141AC194A) on NbaA modify or inactivate the enzyme activity for reduction of NBA but cannot abolish the random disulfide bond formation. Interestingly, we found that wild-type and mutant NbaA proteins have a significant potential to alter the sensitivity of Escherichia coli to various antibiotics and ROS, indicating a regulatory role in the cellular response to changes in intracellular ROS. The aim of the present study was to investigate the underlying mechanism of disulfide bond formation and ROS generation in NbaA derivative-transformed cells, which altered the sensitivity of E. coli to superoxide anion (O2•−) and hydrogen peroxide (H2O2). Their ability to form interchain disulfide bonds in cytoplasmic proteins was determined by Western blotting with anti-NbaA antibody. In this study, we used two-dimensional nonreducing/reducing sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) (called diagonal electrophoresis) to separate proteins that form interchain disulfide bonds under oxidizing conditions induced by polymyxin B treatment. To validate the proteomic findings, we analyzed intracellular concentrations of O2•− and H2O2, cell energy and redox balances, and the levels of several regulatory proteins. Our results show that the overexpression of reactive cysteine-containing proteins partially compensates for the lack of KatE and KatG to degrade H2O2thereby increasing the extent of disulfide bonds formed in the cytoplasmand modifies a regulatory network of disulfidebonded proteins to increase intracellular O2•− levels. These changes may account for oxidative stress-induced changes in the cellular sensitivity to various antibiotics and ROS.

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Strains and Culture Conditions

The complete DNA sequence of NbaA was amplified by polymerase chain reaction (PCR) and cloned into the pSD80 plasmid as previously described.16 The truncated (Δ65−74, Δ193−216, and Δ65−74Δ193−216) and site-specific mutants (C39A, C103A, C141A, C194A, and C141AC194A) were constructed using the PCR primers shown in Table S1 in the Supporting Information. The resulting plasmids were transformed into E. coli strain DH5α. As experimental controls, empty pSD80 plasmid was introduced into DH5α, and the pET28a(Kanr)-TrxA plasmid was cotransformed into strains containing NbaA derivative plasmids.17 Cells were routinely grown in LB media in the presence of 100 mg/L ampicillin or 40 mg/L kanamycin at 37 °C with aeration (180 rpm). When cells reached an optical density (OD600) of ∼0.5, the recombinant proteins were induced with 0.2 mM IPTG for 1 h. Protein Purification and Measurement of Enzyme Activity

Cells overexpressing wild-type and truncated NbaA were harvested at 4 °C, 3000g for 15 min, using a Sorval centrifuge and rotor. Washed cells were suspended in 3 volumes of buffer A containing 10 mM DTT, 1 mM EDTA, and 50 mM Tris/ HCl (pH 7.4) and disrupted by three passages through a prechilled French Pressure cell (max capacity, 3.5 mL). After centrifugation at 12000g for 30 min, the supernatant was applied to a DEAE Sepharose column (1.6 cm × 20 cm) equilibrated with buffer A at a flow rate of 2 mL min−1, and bound proteins were eluted by a 40 min linear gradient to 0.6 M NaCl + buffer A with collection of 2 mL fractions. Aliquots of each fraction were mixed into 50 mM sodium phosphate buffer (pH 7.4) containing 1 mM 2-NBA, 1 mM NADPH, 10 μM FMN, and 0.1 mM MnCl2 to determine the rate of NADPH oxidation at 340 nm (ε340nm = 6.21 mM−1 cm−1) using a Shimazu UV-1800 spectrophotometer (Shimazu Co., Kyoto, Japan) at room temperature. Fractions showing more than halfmaximum activity were combined and treated with 1 M ammonium sulfate prior to loading on a Phenyl Sepharose column (1.6 cm × 10 cm) equilibrated with 1 M ammonium sulfate in buffer A at 1 mL min−1. Bound proteins were eluted in a 40 min linear gradient to buffer A with collection of 1 mL fractions. Fractions containing more than half-maximum activity were combined as above and concentrated to approximately 500 μL using Centriplus YM-30 Centrifugal Filter Devices (Millipore Co., Bedford, MA) before application to a Superdex 200 column (1.6 cm × 60 cm) equilibrated with buffer A at a flow rate of 0.25 mL min−1. The protein concentration was determined using a Pierce Coomassie Plus Protein Assay kit and bovine serum albumin standard.

EXPERIMENTAL SECTION

Materials

The following reagents were purchased from Sigma (St. Louis, MO): 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), CHAPS, o-dianisidine, dithiothreitol (DTT), ethylenediaminetetraacetic acid disodium salt (EDTA), N-ethylmaleimide (NEM), glyceraldehyde-3-phosphate (G3P), horseradish peroxidase (HRP), iodoacetamide (IAA), isopropyl β-D1-thiogalactopyranoside (IPTG), NBA, 6-phosphogluconate (6PG), β-nicotinamide adenine dinucleotide (NAD), βnicotinamide adenine dinucleotide phosphate (NADP), βnicotinamide adenine dinucleotide 2′-phosphate, reduced form (NADPH), nitroblue tetrazolium (NBT), paraquat, phenazine methosulfate (PMS), riboflavin 5′-monophosphate (FMN), sodium salt dehydrate, thiazolyl blue tetrazolium bromide (MTT), and antibiotics. Sequencing-grade porcine trypsin was

Antimicrobial Susceptibility Tests and Plate Dilution Assays

Antimicrobial susceptibility tests for the determination of minimum inhibitory concentrations (MICs) of aminoglycosides (gentamicin, kanamycin, and streptomycin) and polymyxins (colistin and polymyxin B) were performed with exponentially 3220



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grown cells at an optical density (OD600) of ∼0.2. Cells were diluted to a final OD600 of 0.001 (∼106 cells/mL) in Hinton− Mueller broth. One hundred microliters of cell suspension was placed into 96-multiwell plates including 11 consecutive 2-fold dilutions of an antibiotic and 1 control well in a row. Microtiter plates, sealed with sterile adhesive tapes, were incubated for 24 or 72 h on a rotary shaker (200 rpm) at 37 °C, and the MIC was determined at the lowest antimicrobial concentration that inhibited bacterial growth. To analyze antimicrobial susceptibility profiles of cells containing wild-type and truncated NbaA proteins, disk diffusion assays were performed using filter paper disks (diameter, 6 mm) impregnated with the following doses of antibiotics (μg per disk): cephalexin (30), chloramphenicol (5), colistin (10), erythromycin A (80), gentamicin (10), nalidixic acid (30), polymyxin B (10), rifampicin (5), streptomycin (10), and tetracycline (5). IPTG-induced cells were diluted to an appropriate cell density (OD600, 0.2; ∼108 cells mL−1) with sterile phosphate-based saline buffer (8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 1 L of H2O; pH 7.4) and evenly spread onto Hinton− Mueller agar plates (Becton, Dickinson Co., Sparks, MD; diameter, 90 mm) containing 20 mL of media and 0.2 mM IPTG. The surface water was dried for 2 h under a sterile bench before the application of antibiotic disks. Plates were incubated in a water-jacket incubator at 37 °C for 24 h to measure the diameters (mm) of clear inhibition zones around disks. For plate dilution assays against O2•−-generating paraquat and H2O2, 10 μL drops of serial 10-fold dilutions of IPTG-induced cells in the range of 101−105 colony-forming units were inoculated in parallel onto 0.2 mM IPTG-containing LB plates with or without the addition of 250 μM paraquat or 5 mM H2O2. Colony growth was observed after 24 h of incubation at 37 °C.

spots of disulfide-bonded proteins were shown below a diagonal line in the second dimension. Mass Spectrometric Analysis

Disulfide-bonded protein spots were excised from 2D gels and digested with trypsin for mass spectrometric analysis.18 Dried peptide extracts were dissolved in 0.4% acetic acid and analyzed with a nanoflow LC-LTQ Linear Ion Trap mass spectrometer (Thermo Fisher Scientific Inc.) installed with an Agilent Series 1200 nanoflow liquid chromatography system (Agilent, Santa Clara, CA) and a capillary column (75 μm inner diameter × 360 μm outer diameter × 15 cm length), which was packed inhouse with Magic C18AQ particles (5 μm, 200 Å pore size; Michrom Bioresources, Inc., Auburn, CA). The chromatographic conditions comprised a 45 min linear gradient from 5 to 40% acetonitrile (ACN) in 0.1% formic acid (FA), followed by a 5 min column-wash step in 80% ACN/0.1% FA and a 10 min column re-equilibration with 5% ACN/0.1% FA at a flow rate of 0.35 μL min−1. The full mass scan was performed between m/z 300 and 2000 and was followed by five datadependent MS-MS scans with the following options: isolation width, 1.5 m/z; normalized collision energy, 25%; dynamic exclusion duration, 30 s; and rejection of charge state 1 enabled in the Data Dependent Settings. The mass data were analyzed by two search methods using SEQUEST Cluster 3.1 (Thermo Finnigan LLC, San Jose, CA) and X!Tandem at the Global Proteomics Machine Organization (www.thegpm.org/). The SEQUEST search was carried out with potential modification of cysteine with IAA or NEM; precursor ion mass tolerance, ±1.5 Da; fragment mass error, ±0.5 Da; peptide probability, >95%; and protein probability, >99%. The X!Tandem search was performed with similar options, except for refinements of common potential modifications, such as oxidation (M, W), deamidation (N, Q), phosphorylation (S, T, Y), and acetylation (K). Probability assessments of peptide assignments and protein identifications were made using Scaffold version 3.0 (Proteome Software Inc., Portland, OR). Because two or more unique peptides were significantly detected by performing either SEQUEST or X!Tandem database searches, the reliability of protein identification was accepted. E. coli K-12 databases for the gene regulatory machinery (www.ecocyc.org), STRING 9.0 functional protein association networks (http:// string-db.org/), and KEGG pathways (www.genome.jp/kegg/ pathway.html) were analyzed using the identified proteins and their interactions with other effector proteins.

Cell Lysis and Protein Extraction

Cells were harvested and suspended in 3 volumes of lysis buffer [4% CHAPS, 1 mM EDTA-Na2, and a Complete Protease Inhibitor Cocktail tablet (Roche, Mannheim, Germany) in 50 mM Tris-HCl buffer (pH 8)]. To trap thiol groups, samples were treated overnight with 100 mM of NEM at 4 °C in the darkness. Cells were disrupted by 5 cycles consisting of 1 s ultrasonic vibration and 30 s of cooling on ice. Cell debris was removed by centrifugation for 15 min at 22000g. The protein concentration was determined using the Pierce Coomassie Plus Protein Assay Reagent and a bovine serum albumin standard (Thermo Fisher Scientific Inc.).

Activity Gel Assays

Diagonal Electrophoresis

The superoxide dismutases (SODs), SodA, SodB, and SodC, were separated by native polyacrylamide gels, in which enzyme activities were assayed at room temperature by the NBT method.19 The in-gel activity of glyceraldehyde-3-phosphate dehydrogenase (GapA) was measured by the formation of formazan dye with 1 mM G3P, 1 mM NAD, 10 μM PMS, and 20 μM MTT in 25 mM potassium phosphate buffer (pH 7.8). The activity of 6-phosphogluconate dehydrogenase (Gnd) was detected by the addition of 1 mM 6PG, 1 mM NADP, 10 μM PMS, and 20 μM MTT to the same buffer. Bifunctional hydroperoxidase I (catalase-peroxidase, KatG) was detected by the polymerization of o-dianisidine (final concentration, 0.1 mM) in the presence of 10 mM H2O2. After incubation of the gels for 1 h with 15 strokes per min in darkness, the dye reactions were stopped by washing the gels with water. The band intensity was calculated by determining the local average volume from the scan image with Molecular Dynamics

To analyze disulfide-bonded proteins, diagonal electrophoresis was performed under nonreducing and reducing conditions.6 NEM-treated protein samples (10 μg of protein per lane) were first electrophoresed by nonreducing SDS-PAGE. The firstdimensional gel was reduced by a 15 min treatment with 0.5% (w/v) DTT in equilibration buffer containing 6 M urea, 75 mM Tris-HCl (pH 8.8), 29.3% glycerol, 2% SDS, and a few particles of bromophenol blue. Subsequently, the gel was briefly washed with SDS-PAGE running buffer containing 25 mM Tris, 192 mM glycine, and 0.1% SDS before alkylation of the reduced cysteine residues with 4.5% (w/v) IAA in equilibration buffer for 15 min at room temperature. Each protein-containing lane was cut alongside the direction of migration, horizontally placed on top of the second-dimensional gel, and fixed with molten 0.5% agarose in SDS-PAGE running buffer. After gel electrophoresis, protein spots were visualized by silver staining, and 3221



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ImageQuant software (version 5.2; GE Healthcare Life Sciences). Detection and Determination of ROS

The production of O2•− was measured by the NBT reduction method.20 Harvested cells were lysed with 0.1 M NaOH and 0.1% SDS buffer containing 10 mM EDTA-Na2, and a blue pellet was obtained by centrifugation at 22000g for 15 min. The pellet was suspended in 1 mL of pyridine while being heated at 80 °C for 2 h to extract blue NBT formazan, which was measured at 540 nm (ε540nm = 0.72 mM cm−1). The concentration of H2O2 in the whole cell lysate was determined by adding 100 U of HRP and 1 mM ABTS, which produced a purple compound with maximum absorbance at 560 nm (ε560nm = 13.3 mM−1 cm−1).21 Determination of Cellular Adenine Nucleotide Levels

Cellular levels of ATP, ADP, AMP, NADH, NADPH, NAD, and NADP were determined by spiking whole cell lysate with 0.5 mM 3-acetylpyridine adenine dinucleotide as an internal standard, followed by high-performance liquid chromatography with UV detection at 260 nm.17

Figure 1. (A) Growth curves of E. coli strains containing NbaA derivatives before and after the addition of IPTG. (B) Western blot analysis of wild-type and truncated NbaA proteins using a rabbit polyclonal anti-NbaA antibody (upper panels) and Coomassie staining of total proteins in nonreduced SDS-PAGE denaturing gel (lower panels). Total proteins of 1-h IPTG-induced cells were extracted after 1 h incubation in the presence or absence of 10 mg/L polymyxin B (PMB). A trxA gene overexpression vector was cotransformed in E. coli strains (lanes 11−15) to examine the ability of TrxA to reduce disulfide bonds. (C) Patterns in the formation of interchain disulfide bonds of purified NbaA and the truncated mutant proteins. (D) Western blot analysis of wild-type NbaA and site-specific mutants (C39A, C103A, C141A, C194A, and C141AC194) with or without polymyxin B treatment for 1 h after 1 h of IPTG induction. As a standard, 20 pmol of purified NbaA was used in nonreducing conditions [NbaA(ox)] and reducing condition [NbaA(red)].

Western Blot Analysis

Protein expression levels and the formation of interchain disulfide bonds of wild-type and mutant NbaA proteins were examined by Western blotting using a rabbit polyclonal antiNbaA antibody. The levels of CpxA, SodA, SodB, SodC, IhfA, IhfB, and H-NS proteins were determined by using rabbit polyclonal antipeptide antibodies, as shown in Table S1 in the Supporting Information. Mouse monoclonal antibodies against E. coli CRP and two σ factors (σD and σS) were obtained from Neoclone (Madison, WI). To normalize protein levels, a housekeeping protein, 30S ribosomal subunit S1 (RpsA), was included in Western blotting experiments and detected with a mouse monoclonal anti-RpsA antibody. Standard enhanced chemiluminescence reagents and films (GE Healthcare Life Sciences, Piscataway, NJ) were used for Western blot detection with HRP-conjugated antirabbit and antimouse IgG antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

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type NbaA and the Δ65−74 mutant, since the former proteins were able to form more cross-links than the latter proteins. When NbaA derivatives were coexpressed with homologous TrxA, the extent of disulfide bonds formedeven with polymyxin B treatmentwas reduced to below normal levels (lanes 12−15). This showed that the forced expression of TrxA in E. coli cells was effective in reducing the extent of protein disulfide bonds in the cytoplasm. Using purified proteins, it was shown that NbaA and the Δ65−74 mutant were able to form multiple disulfide-bonded proteins generated by involvement of at least two cysteines, whereas the C-terminal truncation mutants, Δ193−216 and Δ65−74Δ193−216, formed mainly dimer size proteins, as shown in Figure 1C. Site-specific mutation showed that alanine substitution of every cysteine at positions 39, 103, 141, and 194 (C39A, C103A, C141A, C194A, and C141AC194A) did not abolish the formation of interchain disulfide bonds in the cells (Figure 1D). All strains containing wild-type NbaA and each of the site-specific mutants exhibited similar growth rates during IPTG induction. The band patterns of wild-type and mutant NbaA proteins in polymyxin B-treated cells indicated the interchain disulfide bond formations in cytoplasmic proteins. When the enzyme activities of wild-type and mutant NbaA proteins were determined with 2-NBA, the Δ193−216 mutant exhibited significantly reduced activity for 2-NBA, whereas the Δ65−74 mutant was similar to that of wild-type NbaA (Table 1). However, the deletion of both regions (Δ65−74Δ193−

RESULTS

Overexpression of NbaA Derivatives Affects the Growth of E. coli

To investigate the in vivo behavior and effects of wild-type and mutant NbaA proteins, NbaA derivative plasmids were transformed into E. coli strain DH5α. The use of the IPTGinducible pSD80 plasmid system enabled us to strictly control the expression of NbaA derivatives. Before treatment with IPTG, all strains harboring empty pSD80 or NbaA derivative plasmid displayed similar growth rates (Figure 1A). When the growth of cells overexpressing wild-type and truncated NbaA, the specific growth rates decreased in following order: wild-type NbaA > Δ65−74 > Δ193−216 > Δ65−74Δ193−216. The decline in specific growth rate was positively related to a reduction in protein expression level (Figure 1B, lanes 2−5). When the reducing environment of the cytoplasm was disrupted by treatment with 10 mg/L polymyxin B for 1 h, about 80% of the C-terminal truncation mutants (Δ193−216 and Δ65−74Δ193−216) formed random disulfide bonds in cytoplasmic proteins, whereas approximately 20% of wild-type NbaA and the Δ65−74 mutant participated in the formation of random disulfide bonds (lanes 7−10). The C-terminal truncation mutants appeared to be more reactive than wild3222



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Table 1. Kinetic Parameters of Purified and Crude Enzymes of Wild-Type and Mutant NbaA for NBA Reductiona proteins

enzymes

purified

NbaA Δ65−74 Δ193−216 Δ65−74Δ193−216 NbaA C39A C103A C141A C194A C141AC194A

crude

vmax 555 521 62 ND 252 221 252 149 184 61

K0.5

± 13 ± 17 ±3 ± ± ± ± ± ±

267 206 300 ND 602 231 137 417 149 265

46 9 15 29 11 1

vmax/K0.5

±1 ± 10 ± 26 ± ± ± ± ± ±

h

2.08 2.53 0.21 ND 0.42 0.96 1.84 0.36 1.23 0.23

133 12 16 94 22 4

3.78 3.60 5.95 ND 1.75 2.87 2.81 2.19 5.85 6.54

± 0.04 ± 0.66 ± 1.54 ± ± ± ± ± ±

0.22 0.42 0.82 0.57 2.56 0.51

a Apparent kinetic parameters: vmax, apparent maximum velocity (μM min−1); K0.5, half-saturation constant (μM); and h, Hill coefficient estimated from the Hill equation, v/vmax = Sh/(K0.5h + Sh), fit to the sigmoid curve of the NADHP oxidation rate (v) by concentration of NBA (S), and results are expressed as means ± SDs. ND, not detected. Enzyme reactions with various concentrations of NBA were carried out at 25 °C with 0.1 μM monomer of wild-type or mutant NbaA protein in 50 mM sodium phosphate buffer (pH 7.4) containing 1 mM NADPH, 0.1 mM MnCl2, and 10 μM FMN. Molar concentrations of the crude enzymes were determined by Western blotting using a rabbit polyclonal anti-NbaA antibody and purified NbaA standard as shown in Figure 1D.

Table 2. MICs of Aminoglycosides and Polymyxins antibiotics

pSD80

NbaA

Δ65−74

Δ193−216

Δ65−74 Δ193−216

C39A

C103A

C141A

C194A

C141A C194A

gentamicin kanamycin streptomycin colistin polymyxin B

1 2 4 0.5 4

32 256 >128 0.25 0.5

32 256 >128 128 512 >128 0.25 0.5

32 256 >128 512 >128 128 0.25 0.5

32 256 >128 2-fold) as compared with those of the pSD80 control (Figure 3F). Assuming that the spot density of a disulfide-bonded protein is proportional to the total protein concentration in the cell, the expression levels of LacI, which represses lacZ gene expression by binding to the Lac promoter in pSD80, were markedly increased by IPTG induction. The LacI repressor spot was not detected for the pSD80 control, which represents only a tiny fraction of the proteins in normal cells. The expression of the lacI gene located just upstream of the lac promoteris positively regulated by the binding of catabolite activator protein (CAP).28 Because CAP is only able to bind to DNA when cAMP is bound to CAP, low cellular levels of cAMP inhibit the binding of CAP to DNA, thereby preventing the expression of the lacI gene or lac operon by RNA polymerase. Thus, upregulation of LacI during expression of NbaA derivatives may be related to elevated cAMP levels under stressful conditions, caused by the overproduction of reactive cysteine-containing proteins, that is, Δ193−216 and Δ65−74Δ193−216. This indicates the negative control of reactive cysteine-containing proteins expressed from a Lac promoter-controlled expression system. LacZ, encoded by the pSD80 plasmid, was significantly down-regulated in accordance with LacI up-regulation. Notably, the overexpression of NbaA derivatives produced down-regulation of a monofunctional catalase, KatE. Moreover, a bifunctional catalase-peroxidase, KatG, was able to form an interchain disulfide bond under oxidative conditions. Because these two enzymes are required for the detoxification of H2O2 in aerobically growing cells, their down-regulation and oxidative modification could lead to a local concentration of H2O2, resulting in the formation of protein disulfide bonds in the cytoplasm. Under such conditions, several abundant proteins, including glyceraldehyde-3-phosphate dehydrogenase (GapA), aconitases (AcnA and AcnB), 50S ribosomal proteins (RplB and RplF), elongation factor TufA (a duplicate of TufB), and tryptophanase TnaA, may form interchain disulfide bonds, leading to posttranslational down-regulation and modifications of various biological functions and activities. A carbamidomethyl cysteine (C298) residue in a tryptic peptide, TLC298VVQEGFPTYGGLEGGAMER, of TnaA (UniProt ID: P0A853) was identified from the spot 12 by tandem mass spectrometry. The cysteine 298 is in the active site of tryptophan indole-lyase.29 The spot intensity of disulfidebonded TnaA in NbaA derivative-containing cells was 1.2−1.3fold higher than the control value, suggesting that a larger portion of TnaA was inactivated by the disulfide bond formation in the NbaA derivative-containing cells. TnaA catalyzes indole production, which serves an intercommuni-

cable signal for the transcription of multiple antimicrobial resistance systems in E. coli population.30 It is possible that the disulfide bond formation of TnaA leading to enzyme inactivation confers a certain degree of sensitivity to various antibiotics and O2•−-generating paraquat. Analyses of Protein Association Networks and Metabolic Pathways

Scientific databases, namely, the E. coli K-12 MG1655 database (http://ecocyc.org/), STRING 9.0 protein association networks (http://string-db.org/), and KEGG metabolic pathways (http://www.genome.jp/kegg/), were analyzed to assess the biological relevance of the identified proteins for the sensitivity of cells to ROS. As shown in Figure 4, a total of 27 proteins,

Figure 4. Network and pathway analyses of disulfide-bonded proteins. String 8.3 network analysis (http://string-db.org/) of identified proteins was performed with interactive proteins (probability >0.99). A strong confidence of the predicted protein−protein interaction is shown as follows: thick line for >0.9 and thin line for >0.7. Proteins grouped by function are shown in eight pathways: I, translation; II, transcription; III, superoxide radical degradation; IV, defense against damage from ROS generation; V, tricarboxylic acid/glyoxylate cycle; VI, synthesis and repair of iron−sulfur clusters; VII, glycolysis; and VIII, fatty acid biosynthesis.

excluding LacZ and LacI, strongly interact with each other through translation (RplB, RplF, and TufA/B), transcription (RpoB), superoxide radical degradation (KatE, KatG, and SodB), defense against damage from ROS generation (AhpC, TrxB), fatty acid biosynthesis (FabB, FabI), glycolysis (GapA, GatY), the TCA cycle (AcnA, AcnB, and SucA), and synthesis and repair of iron−sulfur clusters (IscS). Among them, SodB, KatE, and KatG are essential for superoxide radical degradation processes under aerobic conditions. They are further linked to antioxidant systems, including AhpC and TrxA in combination with thioredoxin reductase (TrxB), which are involved in the reduction of H2O2. TrxB is essential for NADPH-dependent reduction of TrxA, which catalyzes thiol-disulfide exchange with disulfide-bonded proteins. Activity Gel Assays for Superoxide Radical Degradation and Glycolytic Pathways

To understand the biochemical basis of oxidative stress caused by the overexpression of NbaA derivatives, the enzyme activities of SODs and KatG were determined by activity gel assays. In native gels, SodA, SodB, and SodC were clearly distinguished by the use of two inhibitors, KCN and H2O2, which inhibit SodB and SodC, respectively, but not SodA (results not shown). The SodB activity was markedly reduced during the expression of NbaA derivatives (Figure 5A). Because SodB is required for cytoplasmic O2•− degradation, its lowered activity could elevate intracellular O2•− levels. In addition, the 3225



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pro-antioxidant NADPH may serve as the ultimate electron source for antioxidant systems, for example, the Trx system, to protect cells from ROS-induced oxidative damage. However, the total adenine dinucleotide concentrations in the four strains (178 ± 43.8 nmol per mg of protein) were significantly lowered (p = 0.033; Student's two-way t test) as compared with the control value (260 ± 18.5 nmol per mg of protein). In addition, NbaA derivative-containing strains had lower concentrations of total adenine mononucleotides (36.0 ± 11.7 nmol [ATP] + [ADP] + [AMP] per mg of protein) than the control strains (59.2 ± 5.36 nmol per mg of protein), even though the ATP ratios did not vary significantly. The total adenine nucleotides comprised a pool of energy molecules, which was directly related to the specific growth rates of cells.

Figure 5. Activity gel assays for (A) SODs (SodA, SodB, and SodC; inverted images), (B) bifunctional catalase-peroxidase (KatG), (C) glyceraldehyde-3-phosphate dehydrogenase (GapA), and 6-phosphogluconate dehydrogenase (Gnd) present in 10 μg of protein loaded in each lane. Relative band intensity compared to the pSD80 control strains expressed as 1 unit is shown below each band. (D) Western blot analyses of CpxA, SodA, SodB, SodC, RpsA, σD, σS, H-NS, CRP, IhfA, and IhfB. After normalization of the protein bands in the columns by using the relative levels of a housekeeping protein, RpsA (S1), relative band intensities to the pSD80 control strain expressed as 1 unit are shown in each row.

Western Blot Analyses of Oxidative Stress Regulatory Proteins

Western blot analyses showed that overexpression of NbaA derivatives caused down-regulation of SodB with simultaneous up-regulation of a cell death signal sensor, CpxA (Figure 5D). Endogenous ROS generation may have been triggered by activation of the two-component Cpx system. Simultaneous changes in the levels of CpxA and SodB could ultimately result in the increase in cellular O2•− levels. It is possible that transcription of the sodB gene was repressed by a 2-fold upregulation of the integration host factor (Ihf), consisting of heterodimeric IhfA and IhfB, during the 1 h period of NbaA derivative expression. In contrast, there was no significant relationship between levels of SodB and other repressors, including cAMP receptor protein (CRP) and histone-like nucleoid structuring protein (H-NS), which are known to repress transcription of the sodB gene by interaction with the iron-responsive global regulator, Fur, under stationary or starvation conditions.32,33 In particular, when the C-terminal truncation mutants (Δ193−216 and Δ65−74Δ193−216) were induced, the σD levels were lowered without significant changes in the levels of σS. Although the molecular mechanism of the reduction of σD is unclear, lowering the ratio of σD to σS could have inhibitory effects on the specific growth rates of E. coli cells.

degradation of H2O2 by KatG activity was also decreased during the expression of NbaA derivatives (Figure 5B). Reduced activities of these enzymes for ROS degradation produce an increase in intracellular O2•− levels. However, the intracellular levels of H2O2 did not significantly change in cells containing wild-type and truncated NbaA proteins, supporting the idea that they could compensate for the lack of catalase-peroxidase activity to degrade H2O2 during the process of disulfide bond formation. Overexpression of NbaA derivatives resulted in reduced activity of GapA with constant activity of 6-phosphogluconate dehydrogenase (Gnd) (Figure 5C, upper and lower panels). GapA plays a pivotal role in glycolysis and gluconeogenesis to the extent that lowering GapA activity not only reduces the rate of NADH production and G3P conversion to glycerate-3phosphate, but it also activates the Entner−Doudoroff pathway, which joins the pentose phosphate pathway via Gnd to produce ribulose-5-phosphate and NADPH. These changes result in an increase in the levels of NADPH, which serves as the ultimate electron source for the defense against H2O2-induced oxidative damage.31 As shown in Table 4, the potential activities of 4 NbaA derivative-containing strains for NADPH production were calculated to be 100- to several 1000-fold higher than those of the control strains if the ratio of adenine dinucleotides (AD) was assumed to express an equilibrium state as follows: [NAD][NADPH]/[NADP][NADH]. The increased levels of

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DISCUSSION In the present study, we demonstrated that the overexpression of wild-type and mutant NbaA proteins alters cellular sensitivity to various antibiotics and ROS by reprogramming a regulatory network of disulfide proteins in E. coli cells, thereby resulting in

Table 4. Cellular Levels of Adenine Nucleotides (nmol mg Protein−1) after 1 h of IPTG Treatmenta

a Results from four independent measurements are shown as the mean ± SD. Statistically significant difference from Student's two-way t tests between strains expressing control (pSD80 and pSD80+TrxA) and NbaA derivatives: ***, p < 0.01. bATP ratio = [ATP]/{[ATP] + [ADP] + [AMP]}. cAD (adenine dinucleotide) ratio = {[NAD] × [NADPH]}/{[NADP] × [NADH]}. dBelow the limit of quantification.

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high sensitivity to O2•− and high resistance to H2O2. They trigger endogenous O2•− generation through the activation of the Cpx system, as seen with bactericidal drugs,22,23 and downregulate SodB levels by producing a ∼2-fold increase in levels of the heterodimeric Ihf protein, consisting of IhfA and IhfB. These changes result in increased intracellular O2•− levels, which confer high sensitivity to O2•−-generating paraquat but not to H2O2. In contrast, wild-type and mutant NbaA proteins provide protection from H2O2-mediated cell killing by reversible oxidative modifications, resulting in enhanced disulfide bond formation in cytoplasmic proteins, as schematically illustrated in Figure 6.

appear to exert similar effects as hypoxic treatment, which simultaneously lowers cellular levels of SodB, KatE, and KatG.45,46 Therefore, they effectively potentiate O2•−-generating drugs by elevating O2•− levels in cells. Overexpression of NbaA derivatives caused the up-regulation of CpxA, which triggers endogenous ROS generation as a common cell death mechanism of bactericidal drugs.22 The Cpx system functions in a stress response pathway to control several dozen genes encoding cell envelope proteins involved in protein folding (e.g., DsbA and PpiA) and degradation (e.g., DegP) in response to cell envelope damage caused by mistranslation of membrane proteins under stressful conditions.22,23,47 However, it was not known how bactericidal antibiotics stimulate the production of ROS. The present study suggests that NbaA derivatives cause not only the up-regulation of CpxA but also the down-regulation of SodB, KatE, and KatG. Expression of the sodB gene was repressed by the 2-fold upregulation of IhfA and IhfB, which comprise a global transcriptional regulator, Ihf, and is consistent with a previous report.48 These changes may lead to an ultimate increase in intracellular O2•− levels. During physiological stress, two σ factors, σD and σS, compete for binding to the RNA polymerase core, in both the exponential and the stationary phase.49,50 Levels of the exponential sigma factor, σD, usually remain constant, while those of an alternative subunit of RNA polymerase, σS, vary between 5- and 10-fold during entry into the stationary phase. The decrease in the σD/σS ratio negatively regulates σDdependent housekeeping genes, such as TCA cycle genes, which leads to slow cellular growth and low oxygen consumption.51 However, the rpoS transcript encoding σS is rapidly degraded under stressful conditions, such as antibiotic treatment in the exponential phase.52,53 In NbaA derivativecontaining cells, the levels of σD were found to decrease unusually, while the levels of σS remained nearly constant. Although it is not yet clear how the σD levels decreased, the attenuated σD/σS ratio will lead to reductions in adenine (di)nucleotide pools, which are directly related to the specific growth rate of cells. The wild-type and mutant NbaA proteins appear to reduce H2O2 and then form interchain disulfide bonds. The cystine disulfide is likely to be reduced to the thiol by the involvement of TrxA in combination with TrxB.4,54 This ability to reduce H2O2 to a subtoxic level renders high resistance to H2O2, even though cells contain lower levels of KatE and KatG.55 Furthermore, cells expressing NbaA derivatives lower the GapA activity, while the Gnd activity remains constant. These changes will turn the glycolysis to the pentose phosphate pathway via Gnd to support high NADPH levels.56 NADPH is the ultimate electron source for most antioxidant systems in prokaryotic and eukaryotic cells and provides protection from H2O2-induced oxidative damage.2,31 TrxB transfers electrons from NADPH to TrxA, which catalyzes thiol-disulfide exchange with disulfide−bonded proteins as alternatives to catalases and peroxidases for H2O2 degradation. However, the antioxidant system may be overwhelmed or inactivated during severe oxidative stress, leaving cytosolic cysteine residues susceptible to oxidation.4 The oxidative damage to TrxB will lead to irreversible oxidative modifications of cytoplasmic proteins, as seen in Figure 4. It was previously reported that the activity level of Trx corresponded to a decrease in oxidative modifications of proteins and lipids in plant mitochondria.57 The impairment of Trx function by a reduction in its own

Figure 6. Proposed model for a regulatory network of disulfidebonded proteins that respond to the oxidative stress induced by overexpression of reactive cysteine-containing NbaA and its mutant proteins. The down-regulation (↓) of the exponential growth factor, σD, lowers the σD/σS ratio, which relates to inhibition of specific growth rate of cells and depletion of NADH. Together with upregulation (↑) of the cell death signal sensor CpxA, repression of the sodB gene by up-regulation of the host integration factor (Ihf) consisting of IhfA and IhfB accounts for the increase in intracellular O2•− levels. Instead of low levels of KatE and KatG, NbaA and mutant proteins play a role in scavenging H2O2 and then form interchain disulfide bonds that are reduced by thioredoxin (TrxA) together with NADPH-dependent thioredoxin reductase (TrxB).

Higher than normal levels of ROS are generated when cells are poorly regulated or exposed to a toxic chemical or environmental stress. The oxidative stress caused by the generation of ROS leads to oxidative modifications of cellular components, which are related to cell damage, senescence, and death.34−38 To combat oxidative stress, cells produce various defense systems, such as SODs, catalases-peroxidases, glutaredoxins, peroxiredoxins, and thioredoxins. These enzymes are necessary for maintaining the reducing environment of the cytoplasm. Thus, any defect in the antioxidant system or energy metabolism causes significant ROS elevation, leading to oxidative modifications of proteins in the cytoplasm.39,40 At physiological pH, SODs rapidly convert O2•− to H2O2, and catalase-peroxidases detoxify H2O2 to H2O and 1/2O2.41 H2O2 is at least as hazardous as O2•− and ultimately causes cell death by the iron-catalyzed decomposition of H2O2 to •OH radicals.42 Intracellular H2O2 levels are especially increased when a high level of SOD exists.43 Furthermore, H2O2-induced oxidative damage is particularly high when cells lack defense systems.3,36,40,44 The wild-type and mutant NbaA proteins 3227



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CONCLUSIONS Redox-sensitive NbaA and its mutant proteins have similar effects on E. coli, resulting in high sensitivity to O2•− and high resistance to H2O2. During overexpression of these proteins, the cellular levels of SodB, KatE, and KatG decreased, while that of CpxA increased. The expression of the sodB gene appears to be repressed by 2-fold up-regulation of a global transcriptional factor (Ihf), consisting of IhfA and IhfB. These changes may result in elevation of intracellular O2•− levels, which is consistent with the increased sensitivity of cells to O2•−-generating paraquat and various antibiotics. In contrast, intracellular H2O2 levels are kept nearly constant and are similar to normal concentrations in the cytoplasm. Despite the low activities of KatE and KatG, wild-type and mutant NbaA proteins are able to scavenge H2O2 and form interchain disulfide bonds nonspecifically in cytoplasmic proteins. Further studies will be needed to elicit the mechanism of disulfide bond formation in wild-type and truncated NbaA proteins involving thiol-disulfide exchange reactions via the Trx system with the cost of NADPH. To support the high levels of NADPH, glycolysis turns to the pentose-phosphate pathway by downregulation of GapA with a constant level of Gnd available. Our results show a new aspect of a regulatory network of disulfidebonded proteins in cells, which responds to oxidative stress. The proteomic approach used in this study provides an insight into the mechanistic relationships between the formation of disulfide-bonded proteins and the generation of ROS under oxidative stress conditions, induced by polymyxin B treatment. This relationship will help us to identify ways to potentiate bactericidal effects of O2•−-generating drugs in future.

activity will amplify oxidative stress-induced disulfide bond formation in the cytoplasm, which enables cells to respond rapidly to a changing environment by modulating the activity of its target proteins. Diagonal 2D gel-based mass spectrometry is a useful method for quantitative analysis of disulfide-bonded proteins from whole cell extracts treated with a thiol-blocking agent, for example, NEM. Using this technique, we identified 29 proteins, principally involved in the formation of protein disulfide bonds in E. coli. The smeared band patterns of wild-type and mutant NbaA proteins in nonreducing SDS-PAGE gels represent random (nonspecific) formation of the interchain disulfide bonds with abundant proteins in E. coli cells. The list of disulfide-bonded proteins in E. coli cells is strikingly similar to the finding of Cumming and colleagues who showed interchain disulfide formation of redox active proteins, such as peroxiredoxins, thioredoxin reductase, in the cytosolic fraction of a mammalian neuronal cell line treated with diamide or hydrogen peroxide.4 Brennan and colleagues also found the widespread of interchain disulfide bonds in metabolic and antioxidant enzymes, structural proteins, signaling molecules, and molecular chaperones during myocyte oxidative stress and suggested that these oxidative associations are likely to be fundamental in the cellular response to redox changes.58 Disulfide bond formation of GapA is widely found in prokaryotic and eukaryotic cells, which indicates that GapA plays an essential role not only in glucose metabolism but also in oxidative stress-induced cell death.59 In E. coli, protein disulfide bond formation is likely to lead to posttranslational down-regulation and modifications of growth-related proteins, such as elongation factor EF-Tu (TufA/B), ribosomal protein subunits (RplB and RplF), and RNA polymerase β subunit (RpoB). Ji et al. showed that antisense RNA inhibition of such growth-related genes rapidly induces cell cycle arrest in prokaryotic cells.60 It has been suggested that the translational fidelity of the ribosome is reduced by increasing tRNA misacylation and mistranslation under oxidative conditions.23−26 Any conformational changes in the RNA polymerase and ribosome subunits are likely to modify the fidelity of transcription and translation, thereby resulting in aberrant protein synthesis, which may trigger endogenous ROS generation through activation of the Cpx system via a common pathway of bactericidal drugs. The proteomic approach employed in this study provides further insights into the regulatory role of disulfide-bonded proteins that act as reactive oxygen sensors in the cell. Aconitases (AcnA and AcnB), which function as a protective buffer against the basal level of oxidative stress, regulate the TCA cycle in response to intracellular O2•− levels.61,62 The O2•−-sensitive iron−sulfur [Fe−S] cluster of aconitases may act as a reactive oxygen sensor.63 To repair the [Fe−S] cluster damaged by oxidation, cysteine desulfurase (IscS) is a key element for rapid reactivation of the oxidized cluster in the presence of iron and reducing agent.64,65 IscS catalyzes the transfer of sulfur from cysteine to the coregulated scaffold protein, IscU, via a disulfide linkage between the Cys63 of IscU and the Cys328 of IscS.66 If the sulfur transfer from IscS to IscU is inhibited by random disulfide cross-linking with other proteins, the failure of IscS may result in loss of the ability to add sulfur to tRNA, prevent repair of the [Fe−S] clusters, and generate a number of other sulfur- and selenium-dependent proteins, thus propagating the oxidative damage to other cell components that cause cell death.

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ASSOCIATED CONTENT

* Supporting Information S

Supplemental Tables S1−S3 and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-53-650-4338. Fax: +82-2825-5206. E-mail: ykim@cu. ac.kr (Y.-H.K.). Tel: +82-2-958-6911. Fax: +82-2-958-6919. Email: [email protected] (M.-H.Y.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. John B. Sutherland (Division of Microbiology, National Center for Toxicological Research, U.S. FDA) for critical review of the manuscript. This research was partly supported by the 21C R&D program at the Functional Proteomic Centeradministered by the Ministry of Education, Science, and Technologyand by the Marine and Extreme Genome Research Center programme of the Ministry of Land, Transport, and Maritime Affairs, Korea.

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