Genetics and Molecular Biology

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator, DR1558

Deepti Appukuttan, Harinder Singh, Sun-Ha Park, Jong-Hyun Jung, Sunwook Jeong, Ho Seong Seo, Yong Jun Choi, Sangyong Lim
V. Müller, Editor
DOI: 10.1128/AEM.03371-15

ABSTRACT

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H2O2). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H2O2, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.

INTRODUCTION

Microorganisms are employed for the production of a wide variety of compounds, including biofuels, pharmaceuticals, enzymes, and other valuable products (1–3). The robustness of a strain is critical for the efficient functioning of industrial processes (4). Hence, many efforts, including metabolic engineering (reviewed in reference 5), have been directed toward developing physiological functionalities such as resistance to various stresses, inhibitors, and high concentrations of metabolites/substrates/end products (4). Compared to engineering cells for the increased production of a desired end product, engineering biological robustness to increase stress tolerance is more complex. Such cellular properties are often intricately controlled through multiple genes with several genetic checkpoints (6). Therefore, several novel strategies have been developed to increase the stress tolerance of microorganisms. One strategy involves the engineering of native, artificial, or exogenous transcriptional regulators. Since stress tolerance is a complex mechanism governed through multilayered regulatory networks, the use of regulators facilitates the modulation of the overall cellular processes of microbes, which cannot be achieved through the overexpression of a single functional gene (7, 8). Some notable examples include the engineering of the main σ70 factors of Escherichia coli and Lactobacillus plantarum to construct strains with ethanol and inorganic acid tolerance, respectively (7). Another global transcriptional factor, cyclic AMP receptor protein (CRP), was also engineered to increase oxidative stress and isobutanol tolerance in E. coli (9, 10).

Deinococcus radiodurans is one of the most highly stress resistant species reported (11). It can withstand extremely high doses of ionizing radiation, long periods of desiccation, UV radiation, and oxidizing agents and has a more efficient DNA repair system than any known species in the bacterial kingdom (12). The overexpression of IrrE (also named PprI), a global regulator of D. radiodurans that is essential for extreme radioresistance (13), has been reported to confer radiotolerance on E. coli (14), enhance salt tolerance in E. coli and Brassica napus (15), and improve osmotic-stress tolerance in ethanologenic strains of E. coli, thereby increasing ethanol production (16). This global regulator has also been artificially engineered to improve tolerance to alcohols, such as ethanol and butanol (17), and inhibitors, such as furfural, hydroxyl methyl furfural, and vanillin, which are routinely observed in lignocellulosic hydrolysates used in biorefineries (18).

In bacteria, two-component signal transduction systems (TCSs) sense, respond to, and adapt to environmental changes. A typical TCS comprises a sensor histidine kinase (HK) and a response regulator (RR). The HK detects environmental signals and passes these cues to the RR (19). The RR subsequently induces changes in the cell either directly, through binding to the promoter regions of genes via DNA binding domains, or indirectly, through effector molecules, leading to a cellular output against the environmental stress (20). Overexpression of the endogenous RR EvgA conferred acid resistance on E. coli (21). TCSs can be used to generate synthetic signal transduction pathways, which enable an organism to respond to different signals and elicit responses that are not native to the organism (22). In a synthetic TCS generated in plants, a bacterial RR, PhoB, was activated through the plant HK in response to exogenous cytokinin (23).

D. radiodurans contains 23 HKs and 29 RRs (24). Among these proteins, the RR DR1558 was maximally induced (approximately 5.64 times) and was reported to exhibit a recA-like expression pattern in response to 15 kGy of gamma radiation (25). The unique expression of DR1558 indicates that it might play an important role in stress resistance. Hence, the aim of the present study was to exploit DR1558 to improve the stress tolerance mechanism of E. coli. The DR1558 gene was successfully cloned and expressed in E. coli, resulting in a multistress-tolerant strain. Thus, we demonstrated a simple potential strategy of using heterogeneous RR expression to increase the stress tolerance of a surrogate host.

MATERIALS AND METHODS

Growth conditions.Escherichia coli DH5α was cultivated in LB medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) at 37°C under standard conditions. A stationary-phase culture grown for 18 h with shaking was used as the stock culture. The stock culture was inoculated into fresh LB broth at a 1:100 dilution and was grown at 37°C for all growth-related experiments. Recombinant E. coli strains were supplemented with ampicillin (100 μg/ml), and the rpoS mutant strain was grown without kanamycin.

Construction of plasmid and strains.The 678-bp open reading frame (ORF) of DR1558 was PCR amplified from D. radiodurans genomic DNA by use of primers DR1558-F and DR1558-R (see Table S1 in the supplemental material) and was cloned into the ApaI-HindIII site of the pRadgro plasmid, which harbors a deinococcal groESL promoter (26). The nucleotide sequence of dr1558 inserted into pRadgro was confirmed through nucleotide sequencing. The recombinant plasmid was transformed into E. coli to generate the Ec-1558 strain. The E. coli strain carrying the empty vector pRadgro was designated Ec-pR. To construct the E. coli rpoS mutant strain, the one-step gene inactivation method (i.e., the λ-Red recombination system) was used (27). Briefly, the RED helper plasmid pKD46 was transformed into E. coli to generate E. coli-pKD46. A kanamycin cassette from pKD13 was amplified using primers RpoS-F and RpoS-R, containing approximately 50 nucleotide homology arms identical to the upstream and downstream sequences of the rpoS gene, respectively (see Table S1). The purified PCR products were transformed into E. coli-pKD46 by electroporation and were subsequently screened on LB-kanamycin plates. The rpoS mutation was confirmed by PCR using the diagnostic primers Diag-RpoS-F and Diag-RpoS-R (see Table S1), followed by nucleotide sequencing.

Survival studies.All the stress survival studies were performed with exponential-phase cultures. The Ec-1558 and Ec-pR strains were either exposed to different doses (100, 200, and 300 Gy) of radiation using a 60Co-gamma irradiator (Advanced Radiation Technology Institute, Republic of Korea) or treated with different concentrations of hydrogen peroxide (H2O2) at 37°C for 1 h. Acid stress survival studies were performed as described previously (28). Fifty microliters of the culture was transferred to 2 ml of LB acidified to pH 2.5 and was incubated at 37°C for different time intervals. For heat stress survival, the cultures were shifted from 37°C to 54°C for different time intervals. For osmotic-stress survival, the cultures were pelleted, resuspended in LB containing 2.5 M NaCl, and incubated at 37°C for different time intervals. Following the stress treatment, the cultures were serially diluted in 0.85% NaCl, spotted onto LB-ampicillin plates, and incubated at 37°C for 18 h prior to the enumeration of colonies. The survival fraction was calculated by dividing the number of colonies of samples by the number of colonies of controls.

Determination of ROS generation.The generation of reactive oxygen species (ROS) in E. coli strains was measured using H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) dye, a useful indicator for intracellular ROS, as described previously (9, 29), with certain modifications. Exponential-phase cells were washed and resuspended in phosphate-buffered saline (PBS). Two sets of 1-ml aliquots, corresponding to an optical density at 600 nm (OD600) of 0.1, were obtained for each of the E. coli strains. One set of aliquots was exposed to 10 mM H2O2 at 37°C for 30 min. The second set of aliquots served as unexposed controls. Subsequently, the samples were mixed with equal volumes of PBS containing 10 μM H2DCFDA. The culture-dye mixture was incubated at 37°C for 1 h in the dark. The fluorescence intensity was measured (excitation wavelength, 495 nm; emission wavelength, 527 nm) using an Infinite 200 spectrophotometer (Tecan, Switzerland). The emission values were normalized to the protein concentration.

qRT-PCR.Total RNA was isolated from exponentially growing cultures of strains Ec-1558 and Ec-pR, both from nonstressed and from stressed (5 mM H2O2 for 10 min) cells, by use of the RiboEx reagent (GeneAll, South Korea), treated with DNase, and purified by using the RNeasy minikit (Qiagen, Germany) according to the manufacturer's instructions. Purified RNA was resuspended in DEPC (diethyl pyrocarbonate)-treated sterile distilled water. The RNA quality and concentration were estimated after measurement of the optical densities of the solution at 260 and 280 nm using a GeneQuant pro instrument (Amersham Pharmacia Biotech, United Kingdom). One microgram of RNA from each of the samples was used for cDNA synthesis with random hexamers using the PrimeScript first-strand cDNA synthesis kit (TaKaRa Bio Inc., Japan). Quantitative real-time PCR (qRT-PCR) amplification was performed by using SYBR Premix Ex Taq (TaKaRa) and the Eco Real-Time PCR system (Illumina Inc., USA) according to the manufacturer's instructions. The housekeeping gene hcaT was used as an internal control (30). The primers used in qRT-PCR are listed in Table S1 in the supplemental material. All reactions were repeated in triplicate, and relative expression was calculated using the relative quantification method.

Preparation of E. coli cell extracts and zymogram studies.Crude E. coli cell extracts were prepared as described previously (31). Briefly, exponential-phase cells (OD600, ≈0.8) were washed and resuspended in PBS. The cells were lysed by sonication using a Vibra-Cell VCX 500/700 ultrasonic processor (Sonics, USA) at 4°C with a pulse of 1 s on and 1 s off until the lysate was clear. The cell lysates were subjected to centrifugation at 12,000 × g for 20 min at 4°C. The protein concentration was determined in the clarified supernatant by use of the Bradford colorimetric assay with bovine serum albumin (BSA) as the standard. For zymogram studies to detect in-gel catalase activity, 50 μg of protein of the cell lysate from E. coli strains was resolved on an 8% native PAGE gel, and staining was performed as described previously (31).

Western blot analysis.Exponential-phase cultures were harvested, resuspended in lysis buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 4 mM 2-mercaptoethaol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10% glycerol), and lysed using a VP-15S homogenizer (TAITEC, Japan). The cell extracts were collected, and the protein concentrations were determined by using a standard Bradford colorimetric assay with BSA as the standard. Subsequently, Western blot analyses were performed with monoclonal antibodies to RpoS (Abcam, United Kingdom) and DnaK (Enzo Life Sciences Inc., USA). Briefly, equivalent amounts of cell extract (10 μg) were resolved on an 8% Bis-Tris gel with morpholinepropanesulfonic acid (MOPS) buffer and were subsequently blotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with the primary antibody (1:5,000) and subsequently probed with a peroxidase-conjugated goat anti-mouse antibody (1:5,000) (Sigma-Aldrich, USA). The secondary antibody was detected by using the Pierce ECL Western blotting substrate according to the manufacturer's instructions (Thermo Scientific, USA).

EMSAs.The ORF of dr1558 was PCR amplified using primers listed in Table S1 in the supplemental material, cloned into the NcoI-XhoI site of pET28a, and transformed into E. coli BL-21(DE3). The resulting strain was cultured in LB containing kanamycin (50 μg/ml) at 30°C to an OD600 of 0.55. Protein expression was induced with 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) for 3 h. The DR15586×His protein was purified as described previously (32). The binding of DR1558 to the rpoS promoter DNA fragment was examined using the LightShift Chemiluminescent EMSA (electrophoretic mobility shift assay) kit (Thermo Scientific). Different regions of the rpoS promoter were PCR amplified using the biotinylated and nonbiotinylated primer sets rpoS_I/II/III_F and rpoS_I/II/III_R (see Table S1). In the biotinylated PCR, only the forward primer of each set was labeled with biotin. The PCR products were purified using the QIAquick gel extraction kit (Qiagen). The biotin-labeled rpoS promoter fragments (20 fmol) were incubated with 2 μg of purified DR1558 in EMSA buffer at room temperature for 20 min. Unlabeled rpoS promoter fragments, acting as competitor DNA, were added to one of the reaction mixtures in the set, and 200 ng of poly(dI-dC) (Sigma), acting as nonspecific competitor DNA, was added to the EMSA mixture for the second rpoS promoter fragment (PF-II). The EMSA reaction, blotting, and developing were performed according to the manufacturer's instructions.

Microarray and data analysis.Microarray analysis was performed using RNA samples isolated from exponentially growing cultures (OD600, ≈0.8) of strains Ec-pR and Ec-1558. First, the E. coli cultures were treated with ice-cold 5% phenol in an ethanol solution at a 1:10 (vol/vol) ratio to the culture and were incubated on ice for 5 min, followed by centrifugation. RNA was isolated from the pellets as mentioned under “qRT-PCR” above. The quality and integrity of the total RNAs prepared were confirmed using an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., USA) and a GeneQuant pro instrument (Amersham Pharmacia Biotech). The extracted RNA was amplified and was labeled by using Agilent's Low Input Quick Amp WT (whole-transcript) Labeling kit (Agilent Technologies, Inc.) according to the manufacturer's instructions. Briefly, 100 ng of total RNA was mixed with the WT primer mix provided in the kit and was incubated at 65°C for 10 min. Following the addition of the cDNA master mix, the reaction mixture was incubated at 40°C for 2 h for reverse transcription and at 70°C for 10 min to stop the reaction. The transcription master mix was added to the reaction mixture, and the mixture was incubated at 40°C for 2 h to generate labeled cRNA probes. The amplified and labeled cRNA was purified on an RNase minicolumn (Qiagen) according to the manufacturer's instructions and was quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Inc., USA). After the fragmentation of Cy3-labeled (control sample) and Cy5-labeled (experimental sample) cRNA, the cRNA was hybridized to the Escherichia coli K-12 oligonucleotide 3×15K microarray, which includes 4,294 gene-specific probes (99.1% of all genes) (MYcroarray Inc., USA). Hybridizations were conducted at 57°C for 17 h in an Agilent hybridization oven according to the manufacturer's instructions (Agilent Technologies, Inc.). Scans were performed using an Agilent DNA microarray scanner (Agilent Technologies, Inc.) with Agilent Feature Extraction software (version 10.7; Agilent Technologies, Inc.). The signal intensities were quantified using GeneSpring GX software (version 7.3.1; Agilent Technologies, Inc.). The genes were filtered by removing the flag-out genes in each experiment. Gene expression was normalized through LOWESS (locally weighted scatterplot smoothing) regression for 3 data sets obtained from 3 biological replicates. The genes were considered differentially expressed when the logarithmic gene expression ratios showed a 2-fold difference. The significance of the data was determined using Student's t test. P values of <0.01 were considered significant.

Microarray data accession number.The microarray data obtained in this study are available under accession number GSE70688 in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/).

RESULTS

Expression of DR1558 in E. coli increases hydrogen peroxide tolerance.Among D. radiodurans RRs, DR1558 was maximally induced in response to gamma radiation (25). Hence, E. coli expressing DR1558 (strain Ec-1558) was tested for gamma radiation resistance in comparison with E. coli harboring the empty vector pRadgro (Ec-pR) by exposing exponential-phase cultures to different doses of 60Co gamma radiation (0 to 300 Gy). Ec-1558 cells displayed a >1-log cycle increase in radiation resistance over that of the control (Ec-pR) at 300 Gy of gamma radiation (Fig. 1A). Because radiation stress induces oxidative stress through the generation of reactive oxygen species (ROS) (33), the E. coli strains were also examined for oxidative-stress tolerance under hydrogen peroxide (H2O2) stress (0 to 20 mM) conditions for 1 h. Under H2O2 stress, Ec-1558 cells showed high stress tolerance, with almost no loss of viability, even at 20 mM H2O2, while Ec-pR cells showed a 3-log cycle reduction at 10 mM H2O2, and no viable cells were obtained at 20 mM H2O2 (Fig. 1B). Thus, DR1558 overexpression increased H2O2 tolerance in E. coli.

FIG 1
FIG 1

Survival of E. coli harboring the empty vector pRadgro (Ec-pR) and E. coli expressing DR1558 (Ec-1558) under different stresses. Strains Ec-pR and Ec-1558 were exposed to different doses of 60Co gamma radiation (A) or H2O2 (B) for 1 h. The cultures were serially diluted and were spotted onto LB-ampicillin plates. The survival fraction was measured by comparison to the controls under nonstress conditions.

Phosphorylation is a prerequisite for DR1558 function in E. coli.In TCSs, the signal is transferred from an HK to the cognate RR. The signal transfer occurs through a series of phosphorylation reactions, whereby a phosphoryl group from the HK is transferred to a highly conserved aspartate (Asp) residue present on the RR, activating this protein to induce changes in the cell. Because of the high degree of conservation among TCS components, the HKs of one species are able to phosphorylate nonnative response regulators (19). We examined whether DR1558 acts as an RR in E. coli and consequently confers increased oxidative-stress tolerance. To this end, the response receiver domains of DR1558, along with those of two other characterized deinococcal response regulators, DR2418 (34) and DRB0091 (35), were identified using the Pfam database (http://pfam.xfam.org/) and were aligned using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) (Fig. 2A). Wang et al. (34) identified D54 as the Asp residue that undergoes phosphorylation, constituting the critical residue for the transduction of response signals. Based on sequence similarities with DR2418 and DRB0091, the D66 residue of DR1558 was identified as the residue likely involved in phosphorylation. Accordingly, this Asp residue was mutated to asparagine (Asn) and was cloned into E. coli (Ec-1558D66N), and subsequently, H2O2 tolerance was examined. The mutated DR1558 did not confer H2O2 tolerance on the host cell (Fig. 2B), suggesting that D66 is an important residue in the transduction of the signal to combat H2O2 stress. This demonstrates that DR1558 acts as an RR and enhances H2O2 tolerance in E. coli.

FIG 2
FIG 2

(A) Multiple-sequence alignment of the response receiver domains of three deinococcal RRs, DR2418, DRB0091, and DR1558, using ClustalW. Asterisks indicate positions that have a single fully conserved residue. Colons indicate conservation between groups with strongly similar properties, scoring >0.5 in the Gonnet PAM 250 matrix, and periods indicate conservation between groups with weakly similar properties, scoring ≤0.5 in the Gonnet PAM 250 matrix. The conserved aspartate residues that are critical for phosphorylation and subsequent activation of the response regulator are boxed. (B) Survival of E. coli harboring the empty vector pRadgro (Ec-pR), E. coli expressing DR1558 (Ec-1558), and E. coli expressing mutated DR1558 (Ec-1558D66N). These strains were exposed to different doses of H2O2 for 1 h, followed by serial dilution and spotting onto LB-ampicillin plates.

ROS levels decrease in Ec-1558 cells.Oxidative stress results from uncontrolled ROS in the cell. ROS damage almost all vital components of the cell, from nucleic acids and protein to lipids and the cell membrane. Thus, to measure the amount of cellular ROS generated in Ec-pR and Ec-1558 cells, we compared the ROS levels in resting (0 mM H2O2) cells and cells treated with 10 mM H2O2 by use of H2DCFDA, a dye widely used for the measurement of cellular ROS. As expected, ROS levels in H2O2-treated Ec-1558 cells were much lower (approximately 14 times) than those in the corresponding Ec-pR cells (Fig. 3). Moreover, ROS levels in resting Ec-1558 cells were approximately 40% lower than those in control cells (Fig. 3). This result explains the increased oxidative-stress tolerance observed in Ec-1558 cells: ROS levels inside the cells remain relatively low, even under H2O2 stress.

FIG 3
FIG 3

Intracellular ROS assay. The Ec-pR and Ec-1558 strains were either left untreated or exposed to 5 mM H2O2, and the relative ROS levels were measured using H2DCFDA dye. Intracellular ROS levels are expressed as arbitrary fluorescence units.

Expression profiles of important oxidative-stress-responsive genes.Having established that Ec-1558 cells have lower levels of ROS, we examined the expression profiles of important oxidative-stress-responsive genes, such as katG and katE (encoding catalases), dps (DNA-binding protein from starved cells), ahpC (subunit C of alkyl hydroperoxide reductase), and sodA (superoxide dismutase) (36, 37), using quantitative real-time PCR (qRT-PCR). Upon H2O2 treatment, Ec-1558 cells exhibited the induction of all genes tested, among which katE was not induced in Ec-pR (Fig. 4A). The katE gene encodes a major catalase in stationary-phase cultures and is not induced in the exponential phase (38). The expression of katG and katE was significantly more activated in Ec-1558 than in Ec-pR (Fig. 4A), suggesting that the low ROS level in Ec-1558 is attributable to the induction of catalases under H2O2 stress. However, we observed inherently higher levels of katE and dps in Ec-1558 cells than in Ec-pR cells, even without H2O2 treatment (Fig. 4A). To determine whether the higher katE transcript levels are translated into higher KatE protein levels, we examined the catalase activities of resting Ec-pR and Ec-1558 cells by use of cell lysate-based zymogram studies. As shown in Fig. 4B, the band corresponding to catalase activity (labeled with a number sign) was precisely cut from Coomassie blue-stained gels and was analyzed by nanoscale liquid chromatography-tandem mass spectrometry (nLC–MS-MS) using a dual-cell linear ion trap Orbitrap mass spectrometer (LTQ Velos; Thermo Scientific). The proteins were identified as KatE catalase after the MS-MS spectra were searched against a protein database of E. coli sequences using SEQUEST. The results clearly showed the enhanced KatE catalase activity of Ec-1558 cells relative to that of Ec-pR control cells (Fig. 4B).

FIG 4
FIG 4

(A) Transcriptional changes in oxidative-stress-responsive genes in strains Ec-pR and Ec-1558. Total RNA was isolated from Ec-pR and Ec-1558 cells that were either left untreated or treated with 5 mM H2O2 for 15 min. The relative expression values were determined by defining the mRNA level of each gene from untreated Ec-pR cells as 1. Error bars indicate standard errors from three replicates. Asterisks indicate statistical significance (P, <0.01 by the t test). (B) Zymogram showing the in-gel catalase activities of Ec-pR (lane 1) and Ec-1558 (lane 2). Fifty micrograms of cell-free lysates from each strain was resolved on nondenaturing gels, and bands were stained for the detection of catalase activity. The activity band (#) was identified as KatE by nLC–MS-MS.

Increased levels of RpoS in Ec-1558 cells.There are four important regulators of the oxidative-stress response in E. coli: OxyR, RpoS, SoxS, and Fur (37). We examined the expression levels of these regulators using qRT-PCR. The oxyR transcript levels were similar in Ec-pR and Ec-1558 cells, and the expression levels of soxS and fur were lower in Ec-1558 than in Ec-pR cells (Fig. 5A). However, the rpoS mRNA levels were 3 times higher in Ec-1558 cells than in Ec-pR cells (Fig. 5A), suggesting that RpoS is involved in the increased H2O2 resistance of Ec-1558. To confirm that the higher rpoS transcript levels are translated into higher protein levels, Western blot analyses were performed to detect the levels of RpoS protein in both E. coli strains. As shown in Fig. 5B, RpoS protein levels were higher in Ec-1558 cells than in Ec-pR cells. Considering that katE expression and dps expression are activated through RpoS (39), it is likely that the inherently higher levels of RpoS in Ec-1558 prepare the cells for stress, thereby leading to higher oxidative-stress tolerance.

FIG 5
FIG 5

(A) Transcriptional changes in important oxidative-stress regulators in strains Ec-pR and Ec-1558. The expression of the oxidative-stress regulator genes oxyR, rpoS, soxS, and fur in Ec-1558 relative to that in Ec-pR was determined. Error bars indicate standard errors from three replicates. (B) Detection of the RpoS protein in Ec-pR and Ec-1558 cells by Western blotting. Ten micrograms of a cell extract from each E. coli strain was subjected to Western blotting. The anti-DnaK antibody was used as a loading control.

The multistress tolerance of Ec-1558 cells is conferred through RpoS.The RpoS protein, alternative sigma factor σS, associates with RNA polymerase and, through the transcription of genes belonging to the rpoS regulon, activates a general stress response that protects the bacterial cell from harmful environmental conditions (40). To further examine this mechanism, Ec-1558 cells were exposed to different stress conditions, including a low pH, a high temperature, and high NaCl concentrations, and the survival of these cells was compared with that of Ec-pR cells. In all the stress tolerance studies conducted, Ec-1558 cells showed superior survival relative to Ec-pR cells (Fig. 6). To further confirm the importance of RpoS in conferring multistress tolerance on Ec-1558 cells, an E. coli rpoS mutant (ΔrpoS) was generated. The empty vector pRadgro and the recombinant plasmid, pRadgro containing DR1558, were introduced into the rpoS mutant strain to yield the ΔrpoS-pR and ΔrpoS-1558 strains, respectively. These strains were tested for survival under different stresses (Fig. 6). In general, the stress tolerance of rpoS mutant strains was greatly compromised relative to that of wild-type strains, and this result was expected, since RpoS acts as a general stress regulator. At 5 mM H2O2, the oxidative-stress tolerance of the ΔrpoS-pR strain was 2-log cycles lower than that of the corresponding wild-type strain, Ec-pR (Fig. 6A). As expected, the ΔrpoS-1558 strain did not exhibit a remarkable increase in H2O2 tolerance, such as that observed for Ec-1558. Only a marginal increase of approximately 1-log cycle for the ΔrpoS-1558 strain relative to the ΔrpoS-pR strain was observed (Fig. 6A). Similar results were obtained for acid (Fig. 6B) and heat (Fig. 6D) stresses, where DR1558 did not substantially increase the stress tolerance of E. coli in the rpoS mutant background. Although the ΔrpoS-1558 strain showed higher survival than Ec-pR under osmotic (NaCl) stress, the tolerance of this strain was lower than that of Ec-1558 (Fig. 6C). These results indicate that the enhanced stress tolerance conferred by DR1558 expression is mediated primarily through RpoS in E. coli.

FIG 6
FIG 6

Survival of the Ec-pR, Ec-1558, ΔrpoS-pR, and ΔrpoS-1558 strains under different stresses. E. coli harboring the empty vector pRadgro (Ec-pR), E. coli expressing DR1558 (Ec-1558), the E. coli rpoS mutant harboring the empty vector pRadgro (the ΔrpoS-pR strain), and the E. coli rpoS mutant expressing DR1558 (the ΔrpoS-1558 strain) were exposed to oxidative stress (H2O2 for 1 h) (A), acid stress (pH 2.5) (B), salt stress (2.5 M NaCl) (C), and heat stress (54°C) (D). After the stress treatments, the cultures were diluted and were spotted onto LB-ampicillin plates. The survival fraction was measured by comparison to controls under nonstress conditions.

DR1558 binds to the rpoS promoter.DR1558 belongs to the NarL/FixJ family of bacterial RRs, which has a helix-turn-helix (HTH) DNA-binding motif, indicating that this protein can function as a transcriptional regulator. To determine whether DR1558 binds to the rpoS promoter and directly regulates rpoS gene expression, an electrophoretic mobility shift assay (EMSA) was performed. The E. coli rpoS gene is situated downstream of the nlpD gene, and the major promoter of rpoS is embedded within the nlpD gene (rpoSp1 in Fig. 7), 565 nucleotides upstream of the start codon of rpoS (41). We generated two promoter fragments (PFs) by PCR, covering the regions from nucleotide −1035 to −550 and from nucleotide −600 to −50 upstream of the start codon of the rpoS gene. The presence of DR1558 shifted the fragment comprising nucleotides −600 to −50 (Fig. 7, PF-I) but not the fragment comprising nucleotides −1035 to −550 (data not shown). To identify the precise region where DR1558 binds, we divided the PF-I region into two fragments, one from nucleotide −600 to −300 and one from nucleotide −350 to +1. The EMSA results revealed that DR1558 binds to the 350-bp region upstream from the translation start codon of rpoS (data not shown). This region was further dissected into two parts, namely, PF-II and PF-III, containing bases −350 to −150 and bases −200 to +1 of the rpoS promoter region, respectively (Fig. 7). A dramatically shifted band corresponding to the DR1558-PFII complex was detected (Fig. 7, PF-II, lane 2), whereas no DNA shift was observed for PF-III (Fig. 7, PF-III, lane 2). Thus, the binding site of DR1558 is located in a 200-nucleotide region between bases −350 and −150 upstream of the rpoS gene. High-throughput transcriptional start site (TSS) mapping at a genomewide scale revealed that rpoS has an additional TSS (Fig. 7, rpoSp2) at bp −173 upstream of the rpoS start codon (42). This result suggests that rpoSp2 transcription is activated through the direct binding of DR1558, resulting in a high level of RpoS expression in Ec-1558 cells (Fig. 5).

FIG 7
FIG 7

EMSA to examine the binding of DR1558 to the rpoS promoter. A schematic representation of the rpoS gene and the upstream region is shown with the positions of the three rpoS promoter fragments (PFs), PF-I, PF-II, and PF-III. The different rpoS promoter fragments were mixed with purified DR1558 proteins. Lanes 1, biotin-labeled PF; lanes 2, biotin-labeled PF mixed with DR1558; lanes 3, biotin-labeled PF mixed with DR1558 and unlabeled PFs, which act as competitors.

Global transcriptional profiling of Ec-1558 cells.To obtain insight into the global gene expression profile of Ec-1558 cells, we performed DNA microarray analysis using RNA isolated from exponential-phase cultures of strains Ec-pR and Ec-1558. The microarray experiment was conducted in three biological replicates, and only those genes whose expression changed significantly, more than 2-fold (P < 0.01), were considered. A total of 972 genes were altered in Ec-1558 cells: 499 and 473 genes were up- and downregulated, respectively (see Tables S2 and S3 in the supplemental material).To validate the microarray data, qRT-PCR was performed on eight genes: six induced genes, hchA, hdeA, mscS, otsA, wrbA, and yccV, and two repressed genes, alsA and fadA. The expression ratios of the selected genes correlated well (R2 = 0.89) with those obtained from the microarray results (see Fig. S1 in the supplemental material). Based on Clusters of Orthologous Groups (COG) analysis, approximately 50% of the downregulated genes were involved in carbon and amino acid transport and energy metabolism processes (COG categories C, E, and G) (see Fig. S2 in the supplemental material).

The differential gene expression pattern observed in Ec-1558 is represented in Fig. 8. Ec-1558 cells showed reduced expression of the malEFGK genes (encoding an ABC-type maltose transporter system), the dppABCDF genes (dipeptide ABC transporter), and the atp operon (FoF1 ATP synthase complex). The microarray data also showed that most genes involved in glycolysis and the pentose phosphate pathway (PPP) were activated in Ec-1558, while genes encoding the tricarboxylic acid (TCA) cycle enzymes showed decreased expression (Fig. 8). These changes in central carbon metabolism could have implications for the σS-dependent oxidative resistance of Ec-1558 (Fig. 6A), since RpoS decreases the expression of genes required for energy metabolism, including enzymes of the TCA cycle and the ATP synthase operon, to reduce ROS production (43). In addition, NADPH plays an important role in the detoxification of ROS (44); thus, NADPH production through activation of the PPP could also contribute to the oxidative resistance of Ec-1558. Of the three cytochrome oxidases, only the appCB genes, encoding cytochrome bd-II oxidase, were activated, and their expression is also dependent on σS (45). Since the multistress resistance of Ec-1558 manifested only in the presence of rpoS (Fig. 6), these results support the hypothesis that this altered gene expression is mediated by RpoS in Ec-1558. Thus, we examined the number of σS-dependent genes that were altered in Ec-1558. Among the 499 upregulated genes, 132 were σS-dependent genes (see Table S2 in the supplemental material). Interestingly, more than half of the σS-dependent genes showed a >5-fold increase in expression (see Table S2). Taking our findings together, the overall increase in the expression of stress-responsive genes, mediated primarily through RpoS, confers tolerance to various abiotic stresses on Ec-1558 cells.

FIG 8
FIG 8

Schematic illustrating the differential expression of energy metabolism and transport system genes in strain Ec-1558. The number after each gene shows the expression ratio (fold change in the expression of the gene in Ec-1558 from its expression in Ec-pR). N.D., not determined.

Genes involved in the stress resistance of Ec-1558.Microarray analysis revealed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated (see Table S2 in the supplemental material). These genes were classified into groups on the basis of involvement in the stress response.

Oxidative stress.Oxidative-stress-responsive genes, such as katE and dps, were upregulated in microarrays (Table 1), a finding that was also confirmed by qRT-PCR (Fig. 4). In addition, iron storage proteins, or bacterioferritins, such as yciE (24.7-fold), yciF (16.6-fold), and bfr (12.7-fold), were highly induced in Ec-1558 (Table 1). Ferrous ions play an important role in oxidative stress. Free ferrous ions react with H2O2 to produce hydroxyl radicals (·OH) through Fenton's reaction (46). These hydroxyl radicals subsequently induce widespread damage to DNA, proteins, and even lipids (47). Thus, the vulnerability of a cell depends on the availability of free ferrous ions. Increasing the concentrations of bacterioferritins such as YciEF, Bfr, and Dps increases the sequestration of free intracellular iron, which could be one of the potential defense systems against hydrogen peroxide stress in Ec-1558. Moreover, certain other genes, such as ydeI, ygiW, and yhcN, have also been implicated in the oxidative-stress response. The exact functions of these genes are not known; however, deletion of these genes has been associated with decreased survival under oxidative-stress conditions (48). These genes were also found to be induced in Ec-1558 cells (Table 1; see also Table S2 in the supplemental material). Among the eight upregulated oxidative-stress-responsive genes mentioned above, seven (all except yhcN [Table 1]) were σS dependent, reinforcing the critical role of RpoS in DR1558-mediated stress tolerance.

View this table:
TABLE 1

Top induced genes implicated in the oxidative-stress response

Acid stress.The amino acid-dependent acid resistance system has been well studied in E. coli. The decarboxylation of amino acids, such as glutamate, arginine, and lysine, consumes intracellular protons, which helps maintain the pH homeostasis of the intracellular environment. The antiporter specific to each amino acid operates this system through the exchange of external substrates for internal products (49). Among the top 10 upregulated genes (see Table S2 in the supplemental material), 6 were located in the E. coli acid fitness island (AFI) (50). The AFI has direct implications for the glutamate-dependent acid resistance (GDAR) system of glutamate decarboxylases (GadA and GadB) and the glutamate antiporter GadC (50, 51). Recently, another GDAR system was discovered in E. coli, in which the glutaminase YbaS mediates acid resistance along with GadC (52). The microarray data showed that these genes were strongly upregulated in Ec-1558 (Fig. 9; see also Table S2). These AFI genes are controlled through RpoS (39), suggesting that the GDAR system likely confers acid tolerance on Ec-1558 cells.

FIG 9
FIG 9

Schematic illustrating the GDAR system operating in strain Ec-1558 and the chromosomal organization of the GDAR and AFI genes. GadC exchanges extracellular glutamine/glutamate and intracellular γ-aminobutyric acid (GABA), whereas GadA/GadB and YbaS convert glutamine/glutamate to GABA. GadX and GadE are transcriptional regulators of the gadB and ybaS genes. The number above each acid fitness island gene represents its expression ratio.

NaCl osmotic stress.The ompC and ompF genes, encoding outer membrane porins, are representative osmoregulated genes. The regulation of these genes is not restricted to salt stress; however, high osmolarity results in the activation of ompC and the repression of ompF (53). A high ratio of ompC to ompF expression (approximately 150-fold) indicates that Ec-1558 cells mimic the transcriptional repertoire of E. coli exposed to hyperosmotic conditions (Fig. 10). The activation of osmotically inducible (osm) genes in strain Ec-1558 is consistent with this assumption; these genes include osmB (activated 8.6-fold), osmC (8.3-fold), osmE (5.4-fold), osmF (4.1-fold), and osmY (17-fold) (see Table S2 in the supplemental material). In E. coli, the most rapid response to osmotic shock involves the stimulation of potassium (K+) uptake, mediated by the Trk and Kdp systems (54). Both K+ transport systems were activated in Ec-1558 along with the K+ efflux system (Kef) (Fig. 10). Glycine betaine, ectoine, proline, and trehalose are the most widely used compatible solutes against osmotic stress in bacteria (55). The ProP-mediated transport of compatible solutes is stimulated under conditions of high osmotic stress, reflecting increased proP expression (56). The expression of proP, which is regulated through RpoS (57), was upregulated in Ec-1558 (4.1-fold) (Fig. 10). Other σS-dependent osmoregulatory genes, such as otsA and otsB, which encode the enzymes for trehalose biosynthesis (58), were also highly expressed in Ec-1558 (Fig. 10). Trehalose complexes with the TreR repressor protein repress the catabolic treBC operon (58). The downregulation of treBC (trehalose degradation) expression and the upregulation of otsAB (trehalose synthesis) expression indirectly indicate the high intracellular concentration of trehalose (Fig. 10; see also Table S3 in the supplemental material). E. coli cells possess mechanosensitive channels (Msc) for the release of cytoplasmic solutes to avoid bursting (59). Both msc genes, mscS and mscL, were induced in Ec-1558. Furthermore, the expression of mscS (21.1-fold) was higher than that of mscL (2.6-fold) in Ec-1558 (Fig. 10). This finding is consistent with a previous report stating that σS has higher affinity for the mscS promoter than for the mscL promoter (59). Therefore, the NaCl resistance of Ec-1558 reflects the upregulation of σS-dependent (proP, ots, and msc) and σS-independent (trkG, kdpFABC, and kefA) genes, and the latter confers some resistance to NaCl stress even in the absence of RpoS (Fig. 6C, ΔrpoS-1558 strain).

FIG 10
FIG 10

Schematic illustrating the osmotic-stress tolerance mechanism functioning in strain Ec-1558. The number after each gene represents the expression ratio (fold change in the expression of the gene in Ec-1558 from its expression in Ec-pR). Abbreviations: B, glycine betaine; E, ectoine; P, proline; T, trehalose. Genes with P values of <0.05 are marked with asterisks. OM, outer membrane; IM, inner membrane.

Heat shock.In E. coli, heat shock results in the induction of heat shock proteins (HSPs) and molecular chaperones, such as DnaK, DnaJ, GroEL, and GroES, and ATP-dependent proteases, such as ClpP, DegP, FtsH, HslUV, and Lon, whose expression is controlled through the alternative sigma factor σ32, encoded by the rpoH gene (60). However, we did not observe activation of genes encoding these typical HSPs in Ec-1558 (see Table S2 in the supplemental material). The expression of the ibpA gene, which is dependent on σ32 (61), was decreased in Ec-1558 cells (Table 2). Instead, some σS-dependent genes, such as cbpAM (62), hchA (63), and yhbO (39), encoding chaperones and proteases, were upregulated (see Table S2). CbpA and HchA act as molecular chaperones, like DnaJ, which interacts with denatured proteins to prevent aggregation (64, 65). A yhbO mutant strain is highly sensitive not only to heat shock but also to oxidative, UV, and pH stresses (66). In addition, otsAB and dps, whose expression levels increased in strain Ec-1558 (Fig. 10; see also Table S2), also contribute to heat resistance in E. coli (67, 68). In conclusion, the increased expression of stress-responsive genes obtained from microarray data, under the direct or indirect control of RpoS, confirms the role of RpoS in the multistress tolerance of Ec-1558.

View this table:
TABLE 2

Top induced genes implicated in the heat stress response

DISCUSSION

TCSs facilitate the detection of stress signals in affected organisms, which mount a cellular response to combat the stress. Although it has been known that TCS is associated with stress resistance in various organisms (19, 20), there is limited information about the use of TCSs to increase the stress resistance of an organism. In E. coli, for example, overexpression of the native RR EvgA activates the expression of drug efflux pumps and acid resistance-related genes, thereby enhancing drug, acid, and even heat resistance (21, 69, 70). In addition, nonnative TCS components have been employed to generate new synthetic transduction pathways for the acquisition of functions not native to the organism (23). Because TCS components form the first line of contact between an environmental signal and cellular components, they represent good candidates for manipulating cell behaviors and responses to the environment.

In the present study, we demonstrated that overexpression of DR1558, an exogenous RR from the polyextremophilic organism D. radiodurans, conferred multistress tolerance on E. coli. Ec-1558 cells were rendered resistant to multiple stresses, such as H2O2, low-pH, thermal, and NaCl stresses (Fig. 6). Site-directed mutational studies on the conserved Asp residue of DR1558 showed that DR1558 employs the host phosphorylation system (Fig. 2). Although RRs are phosphorylated primarily through cognate HKs, they can be phosphorylated by other HKs or acetyl-phosphates in the absence of cognate HKs. Cross talk with other TCSs reflects the remarkable conservation shared in the response receiver domains of various RRs (71, 72). Such heterogeneous cross talk across different kingdoms, where plant TCS components function in bacteria (73) and vice versa (23), have been reported.

The multistress tolerance of Ec-1558 cells was highly compromised in the absence of RpoS (Fig. 6). Gene expression studies, where several stress-responsive genes whose expression is dependent on RpoS were upregulated (see Table S2 in the supplemental material), also corroborated the results of phenotypic stress response studies. Although we have classified σS-dependent genes into those related to four different types of stresses, including oxidative (Table 1), acid (Fig. 9), salt (Fig. 10), and heat (Table 2) stresses, σS-dependent genes provide cross-protection against several different stresses. This cross-protection in E. coli occurs as a result of rpoS induction and σS accumulation upon challenge with a specific environmental stress (40, 74). Collectively, the results indicate that the multistress tolerance phenotype conferred by DR1558 is likely to be routed through RpoS in Ec-1558 cells. Although RpoS functions primarily as a general stress regulator in the stationary phase, a role for this protein in the stress tolerance of exponential-phase cultures cannot be ruled out. In the present study, we detected increased rpoS transcript and protein levels in exponentially growing Ec-1558 cells (Fig. 5). There are reports of RpoS conferring stress tolerance on exponential-phase cultures (75). Moreover, increased rpoS levels have been detected under oxidative-stress conditions in the exponential phase (76).

σS accumulation plays a key role in engineering stress tolerance in E. coli. Recently, IrrE was hypothesized to play a role in increasing stress tolerance in E. coli through the promotion of σS accumulation in the cell via the upregulation of AppY, IraD, and IraM, which prevents the proteolysis of σS (77). Previously, Gaida et al. (78) engineered synthetic tolerance in E. coli through the overexpression of small RNAs (sRNAs) that facilitated σS accumulation in the cell. However, direct overexpression of the rpoS gene did not confer significant stress tolerance (74, 78). This result emphasizes the fact that σS levels are critically regulated in the cell, and a simple strategy, such as overexpression of the rpoS gene, cannot confer stress resistance. rpoS regulation in E. coli is highly complex and is modulated through different proteins, adaptors, and noncoding RNAs that affect the transcription, translation, and posttranslational stability of this gene (40, 74). In these two studies (77, 78), σS accumulation was facilitated through the increased posttranslational stability of this protein in the cell. In the present study, DR1558 increased rpoS transcript levels (Fig. 5A) through direct binding to the rpoS promoter (Fig. 7), resulting in σS accumulation (Fig. 5B). However, the possibility that other regulatory mechanisms, occurring at the levels of translation, protein activity, and degradation, led to the σS accumulation in Ec-1558 cells cannot be ruled out.

Here, the overexpressed DR1558 rewired the signal transduction pathway of E. coli, leading to a global change in gene expression through an increase in the levels of RpoS: approximately 22% of E. coli genes (972 genes) were significantly altered (see Tables S2 and S3 in the supplemental material). In E. coli, approximately 140 genes constitute the core RpoS regulon, but approximately 500 genes are under the direct or indirect control of RpoS, suggesting that σS-dependent transcription is associated with additional regulators (40). Many bacteria, including E. coli, use small signaling molecules, such as indole, autoinducer 2 (AI-2), and cyclic di-GMP (c-di-GMP), to sense extracellular environmental conditions and the intracellular physiological status (79, 80). A detailed examination revealed the differential expression of several genes associated with the synthesis or uptake of these signaling molecules. In E. coli, indole is produced from tryptophan through a tryptophanase enzyme (TnaA) or is imported through the TnaB transporter (80). Both of these genes were severely downregulated in Ec-1558 (tnaA expression and tnaB expression were repressed 0.002- and 0.014-fold, respectively) (see Table S3 in the supplemental material). In AI-2, the luxS gene, encoding AI-2 synthase, was activated, whereas the lsrB and lsrD genes, encoding AI-2 transporters, were repressed approximately 4- and 3-fold, respectively (see Tables S2 and S3). The AI-2 kinase, encoded by lsrK, was repressed approximately 2.6-fold (see Table S3). The diguanylate cyclase DosC and the phosphodiesterase DosP control the production and removal of c-di-GMP (81). The dosCP genes showed increased expression of approximately 11- and 4.5-fold, respectively, in Ec-1558 (see Table S2). These results imply that perturbations of small-molecule signaling pathways occur in Ec-1558 cells. Notably, the expression levels of tnaA and dosCP are dependent on RpoS (82, 83). Therefore, the DR1558-regulated genes in Ec-1558 not only constitute the RpoS stress regulon but also include genes affected by small signaling molecules.

Engineering robustness in industrial microbes is an essential prerequisite for the improvement of overall production efficiency. One of the strategies for achieving biological robustness is global transcription machinery engineering (gTME) through the introduction of exogenous global regulators (6). In this study, we employed an exogenous RR to introduce an alternate signal transduction pathway, which resulted in multistress tolerance. In particular, extremophiles living under harsh environmental conditions could serve as reservoirs of genes/regulators that could confer robust phenotypes on industrial strains. The unique TCSs of these bacteria could serve as a rich resource for modulating and introducing new signal transduction pathways into surrogate hosts, facilitating the acquisition of novel traits. Thus, we propose the use of exogenous RRs from extremophiles as a promising gTME approach with broad applications.

ACKNOWLEDGMENT

This research was supported by the Nuclear R&D Program of the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea.

FOOTNOTES

    • Received 20 October 2015.
    • Accepted 20 November 2015.
    • Accepted manuscript posted online 11 December 2015.

  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03371-15.

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