Enhanced Multi-Stress Tolerance in Escherichia coli via the Heterologous Expression of Zymomonas mobilis recA: Implications for Industrial Strain Engineering

Abstract

This study investigated the role of the Zymomonas mobilis recA gene in conferring stress resistance when expressed in Escherichia coli. The recA gene was cloned and expressed in E. coli BL21(DE3), producing a 39 kDa polypeptide. The results of comparative analyses demonstrated that the recombinant strain significantly enhanced survival rates under various stress conditions. In oxidative stress tests, the recombinant E. coli pET-22b(+)-recA exhibited superior survival at 3 mM and 5 mM H₂O₂ concentrations. Heat stress experiments at 50 °C and 55 °C revealed increased survival for the recombinant strain. Under ethanol stress, particularly at 20% (v/v), E. coli pET-22b(+)-recA displayed higher viability than controls. UV-C exposure tests further highlighted the protective effect of recA expression, with the recombinant strain maintaining viability after 60 min of exposure, while control strains showed no survival. These results indicate that the Z. mobilis recA gene product enhances resistance to oxidative, heat, ethanol, and UV-C stresses when expressed in E. coli. This study elucidates the broad stress-protective functions of the RecA protein across bacterial species and suggests potential applications in developing stress-tolerant bacterial strains for biotechnological purposes.

1. Introduction

Bacteria persistently face variable and stressful environments, necessitating coordinated responses from global regulatory systems for survival and adaptation. One such system is the DNA damage response, known as the SOS response, which is a global regulatory network found in most bacteria. This response, which was first identified and has been extensively researched in Escherichia coli, focuses on repairing DNA damage [1]. It is a complex system that involves multiple genes and proteins working in concert to maintain genomic integrity under various stress conditions.

The recA gene, a highly conserved gene found in all free-living bacteria, is considered an important gene of the SOS response. It is one of the most slowly evolving genes involved in DNA metabolism, with an average sequence conservation of approximately 60–70% across the entire bacterial domain of life [2]. This high level of conservation underscores the critical importance of the RecA protein in bacterial survival and evolution. RecA plays a crucial role not only in DNA repair but also in recombination and in triggering the SOS response. Its multifaceted functions make it an interesting subject for study in various bacterial species, including Zymomonas mobilis, a Gram-negative bacterium known for its efficient ethanol production capabilities.

In E. coli, which serves as a model organism for understanding bacterial genetics and physiology, the SOS response is an inducible DNA repair pathway regulated primarily by two key proteins: LexA, a repressor, and RecA, an inducer. When DNA damage occurs, single-stranded DNA (ssDNA) accumulates as DNA polymerase stalls at the damaged site while helicase continues to unwind the DNA. RecA is induced (activated RecA) upon binding to the ssDNA, creating a nucleoprotein filament, which then promotes the self-cleavage of LexA, leading to the de-repression of over 50 SOS genes in E. coli [1]. This cascade of events allows the bacterial cell to rapidly respond to DNA damage and initiate repair processes. The SOS response in bacteria was initially discovered through observations of sensitivity to UV light. However, subsequent research has revealed that it is intrinsically linked to the natural response against many DNA-damaging agents, both endogenous and exogenous. Endogenous damage occurs naturally within the cell, often due to normal metabolic processes. Conversely, exogenous DNA damage occurs when exposed to extreme environmental, physical, or chemical agents that harm the DNA. Examples of exogenous damaging agents are UV and ionizing radiation, alkylating agents, and crosslinking agents [1,3]. Recent studies have expanded our understanding of the SOS response beyond its role in DNA repair. For instance, research has shown that the SOS response can influence bacterial evolution by promoting mutagenesis and horizontal gene transfer [4]. Furthermore, the SOS response has been implicated in the development of antibiotic resistance, making it a target of interest for novel therapeutic strategies [5].

While much of the knowledge about the SOS response and RecA comes from studies in E. coli, there is growing interest in understanding these systems in other bacterial species, particularly those with industrial or medical relevance. Z. mobilis, known for its unique metabolism and potential in bioethanol production, presents an interesting subject for such studies. This bacterium has shown remarkable tolerance to ethanol and other stresses, such as heat, osmosis, oxidative, and acetic acid, but the molecular mechanisms underlying this tolerance are not fully understood [6,7].

This study investigates the overexpression of the recA gene from Z. mobilis in E. coli BL21(DE3). This approach allows us to isolate and study the Z. mobilis recA gene in a well-characterized host system. The goal is to explore whether this overexpression leads to increased tolerance to various stresses, including oxidative, heat, ethanol, and UV-C stresses, the common stresses that occur during growth and ethanol fermentation. By expressing the Z. mobilis recA gene in E. coli, we can assess its specific effects on stress tolerance, potentially separate from the other factors in the Z. mobilis cellular environment. Understanding the role of recA in stress tolerance could have significant implications for industrial applications, particularly in improving ethanol production on an industrial scale. If the Z. mobilis RecA protein confers enhanced stress tolerance when expressed in E. coli, it could potentially be used to engineer more robust strains for industrial fermentation processes. Moreover, this study could provide insights into the evolution of stress response systems in different bacterial species and how they may be adapted for various environmental niches.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

This study utilized Z. mobilis TISTR548, obtained from the Thailand Institution of Science and Technological Research, as the source of the recA gene. It was cultured and maintained in a yeast extract peptone glucose (YPG) medium composed of 0.3% yeast extract, 0.5% peptone, and 3% glucose. For the gene cloning and expression, two E. coli strains were employed: E. coli DH5α served as the host for gene cloning, while E. coli BL21(DE3) was used for gene expression. Both E. coli strains were cultured and maintained in Luria–Bertani (LB) medium, which contained 1% tryptone, 1% sodium chloride, and 0.5% yeast extract. When required, ampicillin was added to the culture medium at a final concentration of 100 μg/mL. For their long-term preservation, all bacterial strains were stored in 15% glycerol at −80 °C.

2.2. Genomic DNA Isolation and PCR Amplification of Z. mobilis recA Gene

Genomic DNA from Z. mobilis TISTR548 was extracted using a slightly modified version of the standard phenol/chloroform method, as described by Sambrook et al. [8]. The extracted DNA was either used immediately for subsequent experiments or stored at −20 °C. The full-length recA gene of Z. mobilis (GenBank accession no. AF54827) was amplified using specific primers: Rec-NdeI-F (5′-GGG AAT TCC ATA TGA TGG CTC CGC AAA ATA AGG TTA-3′) and Rec-XhoI-R (5′-CCG CTC GAG ATC TTC AGG GAT AGA TGG ATC CAG-3′). The restriction sites of NdeI and XhoI were highlighted. The polymerase chain reaction (PCR) amplification was performed in a total volume of 25 µL, containing 1 µL ExTaq (Takara, Shiga, Japan), 2.5 µL 10× buffer, 2.5 µL dNTPs (2.5 mmol), 1 µL each of forward and reverse primers (10 μM), 1 µL DNA template (10 ng), and 16 µL nuclease-free water. The PCR protocol consisted of an initial denaturation at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 50 s, annealing at a temperature optimized for the primers used, and extension at 72 °C for 2 min. The amplified PCR products were analyzed by 1% agarose gel electrophoresis, and the target DNA bands were excised and purified using a GF-1 AmbiClean (Gel and PCR) purification kit (Vivantis, Selangor Darul Ehsan, Malaysia). The purified products were then ligated into the pGEM-T Easy vector (Promega, Fitchburg, WI, USA) using T4 DNA ligase according to the manufacturer’s protocol.

The recombinant plasmid containing the Z. mobilis recA gene was transformed into E. coli DH5α using the heat shock method [9]. Transformants were screened using blue–white selection on LB agar plates containing 100 µg/mL ampicillin, 0.1 M isopropyl β-D-1-thiogalactopyranoside (IPTG), and 20 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). The selected transformants were subjected to plasmid extraction using a GF1 plasmid DNA extraction kit (Vivantis, Selangor Darul Ehsan, Malaysia), and nucleotide sequences of the recA gene in the recombinant plasmid DNA were confirmed with DNA sequencing using the Sanger sequencing method (Macrogen, Seoul, Republic of Korea).

2.3. Construction of Expression Vector and Gene Transformation

The recombinant plasmid (pGEM-T Easy vector containing the recA gene) was digested with NdeI and XhoI restriction enzymes. The resulting recA fragment was separated with 1% agarose gel electrophoresis and purified using the GF-1 AmbiClean (Gel and PCR) purification kit (Vivantis, Selangor Darul Ehsan, Malaysia). The purified DNA fragment was then subcloned into the expression vector pET-22b(+), which had been previously digested with NdeI and XhoI. The resulting expression construct designated pET-22b(+)-recA was transformed into competent E. coli BL21(DE3) cells. Transformants were screened on LB agar plates supplemented with 100 μg/mL ampicillin. The presence of the recA inserted into selected colonies was confirmed with colony PCR. Following verification with nucleotide sequencing, the confirmed recombinant strains were used for subsequent experiments.

2.4. Protein Expression and SDS-PAGE Analysis

The recombinant E. coli BL21(DE3) strain harboring the Z. mobilis recA gene was cultured in LB broth supplemented with 100 μg/mL ampicillin. The culture was incubated at 37 °C with shaking at 200 rpm. When the bacterial growth reached the mid-exponential phase (after approximately 9 h of incubation), protein expression was induced by adding IPTG to a final concentration of 1.0 mM. The culture was then incubated for an additional 10 h to allow protein production. As controls, E. coli BL21(DE3) wild-type and transformant strains carrying an empty pET-22b(+) expression vector were cultured under identical conditions, both with and without IPTG induction.

Following incubation, bacterial cells were harvested using centrifugation at 5000 rpm for 10 min at 4 °C. The resulting cell pellets were resuspended in 1× phosphate-buffered saline (PBS, pH 7.4). To extract total cellular proteins, the resuspended cells were lyzed with sonication using a Bioruptor (Cosmo Bio, Tokyo, Japan). Sonication was performed for 10 min using a 50% pulse duty cycle with an output power setting of 5 [10]. The cell lysates were then centrifuged at 12,000 rpm for 20 min at 4 °C to remove cell debris, and the supernatants containing soluble proteins were collected.

The protein concentration of the cell-free extracts was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) as the standard. For the protein analysis, approximately 30 μg of total protein from each sample was mixed with SDS-PAGE sample buffer, denatured by heating at 95 °C for 5 min, and subjected to SDS-PAGE using a 12% acrylamide gel. Electrophoresis was conducted at 50 V for approximately 90 min. After electrophoresis, the proteins separated on the gel were visualized by staining with Coomassie Brilliant Blue R250 for 1 h, followed by destaining in a solution of 10% ethanol and 10% acetic acid until protein bands were clearly visible. The molecular weight of the expressed RecA protein was estimated by means of comparison with a standard protein marker run alongside the samples.

2.5. Stress Treatments of E. coli Recombinant Strains Harboring Z. mobilis recA Gene

The E. coli wild-type, a strain carrying an empty pET-22b(+) vector, and a recombinant E. coli pET-22b(+)-recA were cultured in Luria–Bertani (LB) medium at 37 °C and 200 rpm for 9 h, allowing growth to reach the mid-exponential phase (OD600 ≈ 0.6–0.8) [11]. Subsequently, the cells were subjected to various stress treatments to evaluate their resilience under adverse conditions, including oxidative stress, heat stress, ethanol stress, and UV-C exposure.

For oxidative stress, hydrogen peroxide (H₂O₂) was added to the culture medium at concentrations of 3 and 5 mM, followed by incubation at 37 °C for 2 min [12,13]. Heat shock treatment was conducted by exposing the bacterial cells to temperatures of 50 °C and 55 °C for 10 min [14], mimicking the thermal stress that bacteria may encounter in various environments or during industrial processes [15]. Ethanol stress was induced by culturing the bacterial cells in LB medium supplemented with 15% and 20% (v/v) absolute ethanol, followed by incubation at 37 °C for 2 h [16]. This treatment represents the challenges faced by bacteria in fermentation processes or when exposed to antimicrobial agents [17]. UV stress was conducted by exposing the bacterial cells to UV-C light (254 nm) at a dose of 30 J/m² for 30 and 60 min [18]. The effect of these stress conditions on cell viability was assessed using the drop plate technique, which allows for the enumeration of viable cells with high sensitivity [19]. Ten microliters of stressed cell suspensions were serially diluted and spotted onto LB agar plates, then incubated at 37 °C for 24 h. The resulting colonies were enumerated, and the percentage cell survival was calculated using the following formula:

% Cell survival = (CFU of stressed sample/CFU of unstressed control) × 100

where CFU represents colony-forming units [20].

2.6. Statistical Analysis

All stress treatments were performed twice with three replications, and data are expressed as the mean values ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) was applied for post hoc multiple group comparison at a probability of 0.05 (p ≤ 0.05).

3. Results and Discussion

3.1. Cloning and Expression of recA Gene in E. coli

The recA gene from Z. mobilis was successfully amplified with PCR using specific primers, as described in Section 2. Agarose gel electrophoresis analysis revealed a PCR product of approximately 1 kb in size. Following subcloning and sequencing of this DNA fragment, the ORF of the recA gene was determined to be 1065 bp long (accession no. PP437198), encoding a protein of 354 amino acid residues. The predicted molecular weight of the gene product was calculated to be 39 kDa. Homology analysis demonstrated that the nucleotide sequence of the recA gene from Z. mobilis TISTR548 shared 99% identity with the recA gene of Z. mobilis ATCC29191 (accession no. PP437198.2). This high degree of similarity suggests the conservation of the recA gene among different strains of Z. mobilis.

The Z. mobilis recA gene was subcloned into an expression vector for heterologous expression and subsequently transformed into E. coli BL21(DE3) cells. Recombinant strains were selected based on the antibiotic resistance conferred by the vector-encoded marker gene. Following confirmation through colony PCR, restriction digestion analysis of the recombinant plasmid, and nucleotide sequencing, a single verified strain designated E. coli pET-22b(+)-recA was chosen for the gene expression experiments

SDS-PAGE analysis was performed to assess protein expression. A distinct protein band of approximately 39 kDa was observed in the recombinant E. coli BL21(DE3) strain following induction with IPTG. This band was absent in other cell types, including uninduced samples and control strains. The presence of this band, corresponding to the predicted molecular weight of the RecA protein, provided strong evidence that the recA gene from Z. mobilis was successfully cloned and expressed in E. coli BL21(DE3) cells (Figure 1).

3.2. Effect of Stress Conditions on Growth of Recombinant E. coli pET-22b(+)-recA

To evaluate the role of the recA gene product in conferring oxidative stress, survival tests were conducted using hydrogen peroxide (H₂O₂) on three strains: wild-type E. coli BL21(DE3), E. coli BL21(DE3) harboring an empty pET-22b(+) vector, and E. coli BL21(DE3) expressing the recA gene from Z. mobilis (designated as E. coli pET-22b(+)-recA). All strains were exposed to 0 mM (control), 3 mM, and 5 mM H₂O₂ for 2 h, and their growth performance on the LB agar plate was compared (Figure 2). The results demonstrated that the recombinant strain E. coli pET-22b(+)-recA exhibited significantly higher survival rates than the other strains. Under the control treatment, all strains exhibited 100% survival. However, at 3 mM H₂O₂, E. coli pET-22b(+)-recA showed 75% survival, which statistically was significantly higher than both the wild-type and E. coli BL21(DE3) harboring an empty vector. When subjected to the more severe oxidative stress of 5 mM H₂O₂, E. coli pET-22b(+)-recA maintained a survival rate of 5.63%, while the wild-type and E. coli BL21(DE3) carrying an empty vector showed dramatically reduced survival rates of 0.30% and 0.29%, respectively.

These findings align with previous studies demonstrating the role of the recA gene in oxidative stress tolerance. Rodríguez-Rojas et al. [21] reported that bacterial survival under H₂O₂ stress is quantitatively driven by genes primarily under OxyR control, with RecA contributing to this survival mechanism. The protective role of RecA against oxidative damage has been well documented across various bacterial species, including E. coli [22], Salmonella enterica serovar Typhimurium [23], Lactococcus lactis [24], Neisseria gonorrhoeae [25], and Bacteroides fragilis [26]. In Acinetobacter baumannii, RecA has been shown to be crucial for bacterial survival following exposure to oxidative stress produced by reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI), likely through its role in the recombinational repair pathway [18]. Similarly, in N. gonorrhoeae, which lacks a classical SOS response, RecA and DNA recombinational repair enzymes confer resistance to oxidative damage [25].

The consistent observation of a recA-mediated protection against oxidative stress across diverse bacterial species underscored its conserved and critical role in maintaining genomic integrity under adverse conditions. These findings further corroborated the importance of recA in enhancing bacterial survival under oxidative stress, particularly in the context of its heterologous expression in E. coli.

While no statistically significant difference in cell viability was observed under a control condition (37 °C), the recombinant E. coli pET-22b(+)-recA exhibited a statistically higher survival rate under heat stress at 50 °C and 55 °C than the other tested strains (Figure 3). This observed increase in survival suggests a potential role of recA overexpression in heat stress tolerance.

The heat shock response in bacteria primarily involves the expression of heat shock proteins (HSPs) encoded by several heat shock genes, such as grpE, dnaK, dnaJ, groEL, and groES. These HSPs function as intracellular chaperones, stabilizing and assembling partially unfolded proteins to prevent their aggregation under stress conditions. Under normal physiological growth conditions at ambient temperature, these mechanisms help maintain cellular homeostasis [27].

Our findings align with previous studies that have established connections between RecA and heat stress tolerance in various bacterial species. For instance, in Listeria monocytogenes, exposure to elevated temperatures (48 °C) leads to the upregulation of recA expression alongside other heat shock regulons [14]. Similarly, in Lc. lactis, disruption of the recA gene resulted in temperature sensitivity, highlighting RecA’s role in heat stress tolerance [28]. This connection was further demonstrated in A. baumannii, where recA mutant strains showed a 10-fold lower survival rate than wild-type strains after 40 min of exposure to 55 °C heat stress [18].

The enhanced survival of our recombinant E. coli pET-22b(+)-recA strain under heat stress conditions (Figure 3) is consistent with these previous findings. The protective effect observed in our study may be attributed to RecA’s known function in DNA repair, as heat stress can induce DNA damage, particularly double-strand breaks. The activation of the SOS response, mediated by RecA, triggers various DNA repair mechanisms that could contribute to increased bacterial survival under heat stress conditions. These results suggest potential applications for recA overexpression in enhancing bacterial resilience to thermal stress, though further investigation would be needed to fully understand the underlying mechanisms.

The role of recA in bacterial stress responses to ethanol was also examined after exposing the cells to 0% (control), 15%, and 20% (v/v) ethanol concentrations. As illustrated in Figure 4, no statistically significant difference in cell viability was observed under a control condition without ethanol stress among the tested strains. However, the recombinant E. coli pET-22b(+)-recA exhibited a significantly higher survival rate under severe conditions than the wild-type and the strain harboring an empty pET-22b(+) vector. Notably, almost no growth of the wild-type and strain harboring an empty plasmid was observed at 20% (v/v) ethanol stress. This marginal increase in cell viability suggests a potential protective role of recA in ethanol stress conditions.

The present findings align with the complex nature of bacterial ethanol tolerance mechanisms. As reported by Nicolaou, Gaida, and Papoutsakis [29], increasing ethanol concentration leads to the involvement of various genes in ethanol tolerance, not limited to the upregulation of the recA gene. This multifaceted response includes the activation of genes related to cell envelope maintenance, peptidoglycan biosynthesis, and unsaturated fatty acid biosynthesis. All of these processes contribute to the development of ethanol-tolerant bacteria. While RecA is primarily known for its role in DNA repair and the SOS response, its potential contribution to ethanol tolerance is poorly understood. However, several studies have suggested possible mechanisms. Ethanol can cause DNA damage, including strand breaks and base modifications. RecA functions in DNA repair and may help mitigate this damage, indirectly contributing to ethanol tolerance [30]. Additionally, RecA regulates the SOS response, which can be triggered by various stresses, including ethanol exposure. The activation of the SOS response may contribute to overall cellular resilience [31]. Moreover, RecA has been shown to have chaperone-like activities, which could potentially help stabilize proteins under ethanol stress conditions [32].

The overexpression of protein chaperones, such as GroESL, has been shown to enhance ethanol tolerance in E. coli [29,33] and Z. mobilis [34,35]. These findings have parallels with the role of heat shock proteins in heat stress tolerance, suggesting a potential overlap in stress response mechanisms. The slight increase in survival observed in the recombinant E. coli pET-22b(+)-recA strain under ethanol stress at 15% and 20% (v/v) may be due to the synergistic effects between RecA and other heat stress response elements.

Many studies have also highlighted the importance of membrane composition in ethanol tolerance. For instance, Luo et al. [36] demonstrated that increasing the proportion of saturated fatty acids in the cell membrane enhanced ethanol tolerance in E. coli. While RecA is not directly involved in membrane composition, its role in maintaining genomic integrity could indirectly support the expression of genes involved in membrane modification under stress conditions. Our results, combined with existing studies, suggest a potential role for RecA in bacterial ethanol tolerance, albeit a subtle one. The marginal protective effect observed in recA-overexpressing strains opens avenues for further investigation into the mechanisms by which recA might contribute to ethanol stress resistance. Future studies should focus on elucidating the specific molecular pathways through which recA might confer ethanol tolerance, investigating potential interactions between RecA and other known ethanol tolerance factors, such as membrane modification enzymes or stress-responsive chaperones, exploring the effects of recA overexpression on gene expression profiles under ethanol stress conditions, and examining the potential synergistic effects of combining recA overexpression with other strategies for enhancing ethanol tolerance, such as membrane engineering or chaperone overexpression. These investigations could provide valuable insights for biotechnological applications, particularly in developing more robust strains for industrial processes involving ethanol production or exposure.

The role of the recA gene in DNA repair mechanisms, particularly in response to UV-C exposure, has been well documented in previous studies [1,3]. To further investigate this phenomenon, this study examined the effect of UV-C on cell viability in recombinant E. coli strains overexpressing the Z. mobilis recA gene. The results, summarized in Figure 5, demonstrated that E. coli pET-22b(+)-recA exhibited significantly higher survival rates under UV-C treatments than wild-type E. coli and strains carrying an empty pET-22b(+) vector.

Notably, after 60 min of UV-C exposure, approximately 3.3% of the recombinant E. coli pET-22b(+)-recA cells remained viable, whereas virtually no surviving cells were observed in the wild-type and empty vector control groups. This marked difference in survival rates underscored the protective function of RecA protein overexpression and aligns with previous research on the critical role of recA in DNA repair mechanisms.

These findings extend the work of Chatterjee and Walker [3], who elucidated the multifaceted roles of RecA in bacterial DNA repair processes, and Podlesek et al. [1], who reported on the importance of RecA in UV-induced DNA damage repair across various bacterial species. The present study quantifies the survival advantage conferred by recA overexpression in E. coli under prolonged UV-C exposure, providing a more precise understanding of its protective capabilities.

The substantial difference in survival rates between recA-overexpressing strains and controls highlights the potential of recA overexpression as a strategy for enhancing bacterial resistance to UV-induced DNA damage. These results complement the work of Buchmeier et al. [23], who demonstrated the protective effects of RecA against oxidative stress in Sal. enterica. Although their study focused on different bacterial species and stress conditions, it similarly emphasized the broad protective capabilities of the RecA protein. Furthermore, the observations of this study also align with the review by Kreuzer [37] on the complex interplay between RecA-mediated homologous recombination and other DNA repair pathways. The enhanced survival of the recA-overexpressing strain in this study provides additional evidence for the central role of RecA in coordinating effective DNA repair responses to severe genotoxic stress.

This study not only corroborates previous findings on the importance of RecA in UV-C resistance but also quantifies the survival advantage conferred by recA overexpression under extended UV-C exposure. These results contribute to our understanding of bacterial DNA repair mechanisms and may have implications for developing UV-resistant strains for various biotechnological applications.

4. Conclusions

Based on the experimental results presented, it can be concluded that the recA gene from Z. mobilis, when expressed in E. coli, confers an enhanced resistance to various stress conditions. The recombinant strain E. coli pET-22b(+)-recA demonstrated significantly improved survival rates under oxidative stress induced by H₂O₂, heat stress, ethanol stress, and UV-C exposure compared to wild-type E. coli and E. coli harboring an empty vector. The most pronounced protective effect was observed in response to oxidative stress, where the recombinant strain maintained substantially higher viability at both 3 mM and 5 mM H₂O₂ concentrations. Similarly, the recombinant strain exhibited a superior tolerance to severe ethanol stress, particularly at a 20% (v/v) concentration, where the wild-type and empty vector control strains showed almost no growth. The enhanced survival under UV-C exposure further corroborates the role of RecA in DNA repair mechanisms, with the recombinant strain maintaining viability even after prolonged UV-C treatment.

These findings suggest that the Z. mobilis recA gene product is crucial in conferring resistance to multiple stressors, likely through its involvement in stress response pathways and DNA repair mechanisms. The ability of the recombinant E. coli strain to better withstand these diverse stress conditions highlights the potential of the Z. mobilis recA gene as a valuable tool for enhancing bacterial stress tolerance. This research not only expands our understanding of the RecA protein’s functions across different bacterial species but also opens up possibilities for developing more robust bacterial strains for various biotechnological applications, particularly in environments characterized by oxidative, heat, ethanol, or UV stress.