V. cholerae strains and their isogenic derivatives are listed in Supplementary Table S1. A V. cholerae clinical isolate from the 2010 Haiti cholera outbreak, IB5230, and the V. cholerae type strains O395, 569B, and N16961 were obtained from the culture collections of the International Vaccine Institute, Seoul, Korea (Mutreja et al., 2011; Kim et al., 2014). Isogenic derivatives of these V. cholerae strains carrying different toxT alleles were part of our laboratory collection or were newly constructed in this study (Lee et al., 2023; Baek et al., 2020; Kim et al., 2022).

Allele exchange at the 139th amino acid position: Allelic replacement of the chromosomal toxT gene with alternative toxT alleles in V. cholerae strains was conducted using established allelic-exchange procedures (Kim et al., 2014; Lee et al., 2023; Kim et al., 2017). Primers used for constructing isogenic derivatives of V. cholerae strains are listed in Supplementary Table S2. An 843-bp region of toxT—from 50 nucleotides upstream of the start codon to nucleotide 793—was amplified by PCR with primers toxT-XbaIF and toxT-SacIR using genomic DNA from MG116025 (toxT-SF) and N16961 (toxT-SY) as templates. Each PCR product was cloned into the suicide plasmid pCVD442 (Miller and Mekalanos, 1988), yielding pCVD-toxT-SF and pCVD-toxT-SY. A single-nucleotide polymorphism (SNP) at position 416—A416 in N16961 and T416 in MG116025—is located centrally within these fragments. These constructs were introduced into the respective V. cholerae strains, and allelic replacement was performed using standard pCVD442-mediated allelic-exchange procedures to swap toxT-SF and toxT-SY alleles between strains. Through this procedure, toxT-SF in MG116025 was replaced with toxT-SY, and the native toxT-SY alleles in O395 and N16961 were substituted with toxT-SF (Kim et al., 2014; Kim et al., 2017).

The classical biotype strain 569B contains a distinct toxT allele (toxT-AY). Conjugal delivery of pCVD-toxT-SF into 569B was used to introduce the toxT-AF allele, following the same pCVD442-mediated allelic-exchange procedure described above. Allele replacement and resolution of the pCVD442 vector were verified by Sanger sequencing.

Allele exchange at the 65th amino acid position: A series of allele-exchange plasmids was used to generate isogenic V. cholerae derivatives carrying SNPs at amino acid positions 65 and 139 of toxT. Four 1,308-bp DNA fragments—corresponding to the region from nucleotide 709 of tcpF to nucleotide 793 of toxT—were amplified by PCR using the primer pair TcpF-XbaIF and toxT-SacIR. Templates for amplification included genomic DNA from strains N16961, YJB003 (N16961-toxT-SF), 569B, and EJK007 (569B-toxT-AF) (Baek et al., 2020).

The PCR products were subcloned into the suicide vector pCVD442 to generate pCVD-toxT-Out-SY, pCVD-toxT-Out-SF, pCVD-toxT-Out-AY, and pCVD-toxT-Out-AF. Each construct was then introduced into the corresponding V. cholerae strains by conjugation, and allelic exchange was carried out to replace the native toxT allele with the corresponding alternative allele. Recombinants that had resolved the pCVD442 vector were selected on sucrose-containing agar plates, and the intended toxT alleles were confirmed by Sanger sequencing.

A 635-bp DNA fragment spanning from nucleotide 709 of tcpF to nucleotide 120 of toxT was amplified by PCR using the primers TcpF-KpnIF and TcpF-BamHIR (Lee et al., 2023). This fragment amplified from the genomic DNA of V. cholerae N16961 was inserted into the KpnI and BamHI sites of pUC18 to generate pUC-tcpF. A second 660-bp fragment, spanning nucleotide 616 of toxT to nucleotide 435 of tcpJ, was amplified using primers TcpJ-BamHIF and TcpJ-PstIR and cloned into the BamHI and PstI sites of pUC-tcpF, producing pUC-tcpF-tcpJ. Using primers TcpF-XbaIF and TcpH-SacIR, a 1,301-bp fragment was then amplified from pUC-tcpF-tcpJ and subcloned into the suicide vector pCVD442 to construct pCVD-del-toxT. This construct was conjugally transferred into V. cholerae strains to obtain toxT-deleted (ΔtoxT) isogenic derivatives through allelic exchange. After resolution of the pCVD442 vector, deletion of the targeted internal toxT region was verified by Sanger sequencing. The resulting ΔtoxT isogenic derivatives lacked 165 internal amino acids of ToxT corresponding to residues 41–205.

Four 852-bp DNA fragments were PCR-amplified, each containing the 828-bp toxT open reading frame (ORF) lacking the stop codon, a GCC codon inserted between amino acids 1 and 2 to maintain the reading frame, an 18-bp sequence encoding a C-terminal (His)₆-tag, and a TAA termination codon. PCR amplification was performed using primers toxT-NcoIF and toxT-SalIHisR with genomic DNA from V. cholerae O395 (toxT-SY) and its allele-exchange derivatives YJB001 (toxT-SF), EJK008 (toxT-AY), and EJK009 (toxT-AF). Each PCR product was digested with NcoI and SalI and ligated into the corresponding sites of the expression vector pBAD24 (Guzman et al., 1995), generating the constructs pBAD-toxT-SY-His, pBAD-toxT-SF-His, pBAD-toxT-AY-His, and pBAD-toxT-AF-His.

The resulting plasmids were transformed into E. coli DH5α and V. cholerae DHL010 (N16961-ΔtoxT). Expression of His-tagged ToxT from the P_BAD promoter was induced by supplementing cultures with L-arabinose to the bacterial culture at a final concentration of 0.2%. Production of the His-tagged ToxT protein was confirmed by Western blot analysis using an anti-His-tag (Cell Signaling, Danvers, MA, USA).

The tcpJ sequence is identical in both classical and El Tor V. cholerae O1 strains. A 762-bp tcpJ fragment was PCR-amplified from O395 genomic DNA using primers TcpJ-SalIF and TcpJ-SalIR. The PCR product was digested with SalI and ligated into the SalI site of the plasmids pBAD-toxT-SY-His, pBAD-toxT-SF-His, pBAD-toxT-AY-His, and pBAD-toxT-AF-His, generating pBAD-toxT-SY-His-tcpJ, pBAD-toxT-SF-His-tcpJ, pBAD-toxT-AY-His-tcpJ, and pBAD-toxT-AF-His-tcpJ, respectively. The orientation of the inserted tcpJ fragment was confirmed by DNA sequencing (Supplementary Figure S1).

Using the recombinant plasmids as templates, four 1,620-bp DNA fragments spanning from the initiation codon of His-tagged toxT to the termination codon of tcpJ were PCR-amplified with primers pBAD-toxT-XbaIF and pBAD-tcpJ-SacIR. The amplified products were digested with XbaI and SacI and inserted into the corresponding sites of the suicide vector pCVD442, generating pCVD442-toxT-SY-His-tcpJ, pCVD442-toxT-SF-His-tcpJ, pCVD442-toxT-AY-His-tcpJ, and pCVD442-toxT-AF-His-tcpJ.

These recombinant plasmids were conjugally transferred into the corresponding isogenic toxT allele derivatives of the O395, N16961, and IB5230 strains to construct isogenic derivatives harboring His-tagged toxT through the allelic exchange method. Excision of the suicide vector and replacement of toxT with toxT-His were screened on sucrose-containing agar plates, and the DNA sequence of the His-tagged toxT was confirmed by Sanger sequencing (Supplementary Figure S1).

An isogenic derivative of V. cholerae N16961, referred to as PM20, in which the ctxAB genes had been replaced by a kanamycin resistance cassette, served as the CTXΦ donor strain (Kim et al., 2017). PM20 was grown in LB medium supplemented with 20 ng/mL mitomycin C to induce CTXΦ production (Waldor and Mekalanos, 1996). Following induction, the culture was centrifuged, and the clarified supernatant containing CTXΦ particles was collected. This phage-containing supernatant was then mixed with recipient O395 cells that had been prepared under agglutinating conditions (30°C in LB medium, pH 6.5). After a 30-min incubation to allow CTXΦ infection, the mixture was plated onto LB agar plates containing kanamycin (100 μg/mL) to select for O395 transductants that had acquired CTXΦ.

Kanamycin-resistant colonies were isolated, and the presence of the replicative form of the phage genome, pCTX-1kan, was verified by Sanger sequencing of the intergenic region between the kanamycin resistance and rstR. CTX-1kan virions produced from V. cholerae O395:pCTX-1kan cultures were subsequently used to transduce the recipient strains O395 (toxT-SY), O395-H (toxT-SY-His), EJK009 (toxT-AF), and EJK009-H (toxT-AF-His). Recipient cells were prepared under agglutinating conditions or cultured in LB medium at 37°C. Transduction efficiency was calculated as the ratio of kanamycin-resistant transductants to the total number of recipient cells.

Approximately 3 × 10⁸ cells (for His-tagged ToxT) or 1 × 10⁷ cells (for TcpA) were subjected to SDS-PAGE using 12 and 15% polyacrylamide gels, respectively. The PageRuler Prestained Protein Ladder (Thermo Scientific, Cat. No. 26616) was used as a molecular weight marker to estimate the apparent sizes of His-tagged ToxT and TcpA proteins. Following electrophoresis, proteins were transferred onto nitrocellulose membranes. Western blot analysis was then performed using anti-His antibody (Cell Signaling Technology, Danvers, MA, USA) and anti-TcpA antibody [a generous gift from Dr. W. F. Wade, Dartmouth University, Hanover, NH, USA (Taylor et al., 2004)]. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling Technology, 7074S) was used as the secondary antibody.

A real-time qRT-PCR assay was performed as previously described, with some modifications (Abuaita and Withey, 2011). Total RNA was extracted from approximately 2 × 10⁹ cells at each time point using the Nextractor-48 N® nucleic acid extractor system (Genolution, Seoul, Korea), followed by treatment with RNase-free DNase 1. cDNA was synthesized with 1 μg of RNA using the Thermo Scientific RevertAid First Strand cDNA synthesis kit (ThermoFisher). The concentrations of RNA and cDNA were determined using NanoDrop 2000 (ThermoFisher). Real-time PCR was carried out using 100 ng of synthesized cDNA and specific primers (Supplementary Table S2) with a CFX96RT-PCR system and iQ™ SYBR® Green Supermix (BioRad). The PCR conditions were 95°C for 2 min, followed by 35 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Each run concluded with a melting-curve ranging from 55°C to 95°C to validate specific amplification. Expression values were normalized to the housekeeping gene gyrA as described previously (Abuaita and Withey, 2011; Ray et al., 2011; Raskin et al., 2020). The relative expression levels of toxT, tcpA, and ctxAB were calculated using the 2^−ΔΔCT method (Raskin et al., 2020). The expression levels of toxT, tcpA, and ctxAB at each time point were individually normalized to the expression levels of the corresponding genes in the 4-h culture of O395-H (O395-toxT-SY-His). All quantitative data are presented as the mean ± standard deviation (SD) from three independent biological replicates.

For statistical evaluation, each condition was compared with this reference condition. At each time point, statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test, with the 4-h O395-H condition used as the control group. Only conditions that showed statistically significant decreases relative to the reference condition are indicated with asterisks in the figures; conditions that were not significantly different from or higher than the reference are not annotated. All statistical analyses were performed using GraphPad Prism software (version 9.2.0) and SPSS Statistics (version 29.0). A p value of < 0.05 was considered statistically significant.

All four native toxT alleles exhibited transcriptional activator functions in the classical biotype strain O395 (Lee et al., 2023). However, only certain alleles (e.g., toxT-AY and toxT-AF in N16961, and toxT-SY and toxT-SF in IB5230) detectable activity in other strain backgrounds (Lee et al., 2023). To confirm that all four toxT alleles maintain their transcriptional activator functions in El Tor biotype strains, we analyzed virulence gene expression by exogenously expressing toxT alleles in a toxT-deleted N16961 strain.

An isogenic derivative of the N16961 strain, DHL010 (N16961-ΔtoxT) was individually transformed with recombinant pBAD plasmids, each carrying a different His-tagged toxT allele (Lee et al., 2023). The expression of His-tagged ToxT and TcpA was assessed in the presence of 0.2% arabinose or 0.5% glucose. The addition of glucose effectively suppressed pBAD-driven basal expression of His-tagged ToxT. Since the O395 and IB5230 strains lose viability in the presence of 0.5% glucose due to impaired neutral fermentation, this experiment was performed exclusively in N16961 (Lee et al., 2023). All four toxT alleles were successfully induced by the P_BAD promoter, leading to the expression of both His-tagged ToxT and TcpA in the presence of 0.2% arabinose. In contrast, neither ToxT nor TcpA was detected in the presence of 0.5% glucose (Supplementary Figure S2).

These results indicate that, once expressed at comparable levels in N16961, the toxT alleles exhibit no significant functional differences. This observation aligns with previous studies showing that all four toxT alleles in the O395 strain can activate virulence gene expression (Lee et al., 2023; Lee et al., 2024).

The introduction of a His-tag at the C-terminus of toxT alters the amino acid sequence of both ToxT and the adjacent TcpJ protein (Supplementary Figure S1), requiring an evaluation of the functional integrity of His-tagged ToxT (ToxT-His) relative to native ToxT. To assess the potential impact of these modifications, two complementary approaches were employed. First, the growth dynamics of isogenic derivatives expressing ToxT-His were compared to those of the parental strain expressing authentic ToxT under identical culture conditions. Second, the assembly and functionality of the TCP pilus—regulated by ToxT—were evaluated by measuring CTX phage transduction efficiency. Together, these analyses provide a comprehensive assessment of the functional equivalence of ToxT-His and native ToxT, enabling an evaluation of the effects of expression of His-tagging on ToxT and its downstream regulatory roles.

To assess whether integration of a His-tag at the C-terminus of the chromosomal toxT gene affects bacterial growth, growth curves were compared across four isogenic derivatives of each V. cholerae strain (Supplementary Figure S3). These included the parental strain containing the authentic toxT allele (toxT-SY), its derivative harboring the His-tagged toxT (toxT-SY-His), an isogenic derivative with an alternative toxT allele (toxT-AF), and its counterpart with the His-tagged alternative toxT allele (toxT-AF-His).

Bacteria were cultured in LB medium at pH 6.5 and 30°C, and their growth was monitored. After a brief lag phase, exponential growth was observed for up to 6 h of cultivation, with cell densities reaching approximately 5 × 10⁹ CFU/mL at the stationary phase. The growth curves of the isogenic derivatives of each strain were nearly identical, indicating that replacing the authentic toxT with either a His-tagged toxT or an alternative toxT (including His-tagged alternative toxT) did not significantly affect the growth rate (Supplementary Figure S3).

The functional assembly of the TCP pilus, mediated by TcpA expressed under the control of His-tagged ToxT, was evaluated by measuring CTXΦ transduction efficiency (Kim et al., 2014; Kim et al., 2017). Transduction experiments were conducted using the CTX Φ-1Km (in which ctxAB was replaced by a kanamycin-resistance cassette), produced from the replicative form of the CTX phage. Four isogenic derivatives of the V. cholerae strain O395 were used as recipients: O395 (toxT-SY), O395-H (His-tagged toxT-SY), EJK009 (toxT-AF), and EJK009-H (His-tagged toxT-AF). Transduction efficiency was determined as the ratio of transductants to the total number of recipient cells (Waldor and Mekalanos, 1996; Kim et al., 2017; Yu et al., 2018).

The observed CTXΦ transduction efficiencies were approximately 12.9% for O395, 12.8% for O395-H, 26.6% for EJK009, and 27.0% for EJK009-H (Supplementary Table S3). Consistent with previous reports that the toxT-AF allele enhances tcpA expression relative to toxT-SY, EJK009 (toxT-AF) and EJK009-H exhibited higher transduction efficiencies compared to O395 (toxT-SY) and O395-H (Lee et al., 2023; Kim et al., 2022). In contrast, when recipient cells were prepared in LB medium at 37°C, transduction efficiency was reduced by more than 10⁷-fold, indicating that functional TCP pili were not assembled under these conditions (Waldor and Mekalanos, 1996).

These results suggest that functional TCP was assembled, with no substantial differences in transduction efficiency between isogenic derivatives expressing His-tagged ToxT and those expressing native ToxT. Moreover, the findings indicate that the addition of the His-tag did not noticeably affect TcpA production, TCP assembly, or the overall regulation of virulence genes. Thus, His-tagged ToxT appears to retain its regulatory function with minimal impact under the tested conditions (Kim et al., 2017).

All four native toxT alleles (toxT-SY, toxT-SF, toxT-AY, and toxT-AF) have been shown to activate virulence gene expression under virulence-inducing conditions in the classical biotype strain O395 (Lee et al., 2023; Choi et al., 2023). To assess the functionality of native and His-tagged toxT variants, we compared the expression of toxT at the mRNA level by RT-PCR and at the protein level using Western blotting (anti-His-tag) in isogenic derivatives expressing the toxT-AF allele, with (EJK009-H) or without (EJK009) a His-tag, across different growth phases (Figure 1). These isogenic derivatives were cultured under two conditions: agglutinating conditions (30°C in LB medium, pH adjusted to 6.5) and virulence-repressing conditions (LB medium at 37°C, pH adjusted to 8.5).

As a baseline, expression levels in O395-H (His-tagged toxT-SY) were used for normalization, since in the parental O395 strain TcpA first became detectable by Western blot after 4 h under agglutinating conditions. At this reference point, transcript levels of toxT, tcpA, and ctxAB in O395-H were set to 1 (100%), and the relative expression of these genes was compared across strains carrying different toxT alleles.

Under virulence-inducing culture conditions, EJK009 (toxT-AF) and EJK009-H (His-tagged toxT-AF) displayed very similar timing and magnitude of virulence gene expression, with only minor variation (Figures 1A,B). toxT transcripts were detectable as early as 2–3 h post-inoculation in both strains, with expression peaking at approximately 6–8 h. Consistent with transcript levels, ToxT protein was detectable by Western blot analysis as early as 3 h post-cultivation. Under virulence-inducing conditions, ToxT protein levels peaked between 3 and 6 h, gradually decreasing thereafter, with nearly undetectable levels after 14 h (Figure 1B). Under virulence-repressing conditions, only minimal levels of toxT mRNA were detected, and no detectable ToxT protein was observed (Figure 1C).

Taken together, these results indicate that His-tagged toxT constructs closely approximate the expression dynamics of native toxT, supporting their use as reliable surrogates for studying virulence gene regulation in V. cholerae (Lee et al., 2023). As described below, the expression patterns of tcpA were comparable between His-tagged and native toxT alleles across different strain backgrounds (summarized in Supplementary Table S4).

Based on the observation that His-tagged toxT-AF exhibited expression levels comparable to those of native toxT-AF, we next investigated whether both native ToxT-AF and His-tagged ToxT-AF similarly activated the expression of downstream target genes, tcpA and ctxAB. The expression of these target genes appeared to follow a slight delay relative to toxT expression.

Under virulence-inducing culture conditions, tcpA and ctxAB mRNA became detectable approximately 3–4 h after cultivation (Figures 1D,E,G,H), with TcpA protein also first detected at this time. In the His-tagged variant, a modest delay was observed in the peak expression of tcpA and ctxAB. TcpA protein levels were maintained up to 24 h in both isogenic derivatives (Figures 1D,E). In contrast, under virulence-repressing conditions, no expression of tcpA or ctxAB was detected (Figures 1F,I).

These results suggest that the addition of a His-tag does not alter the capacity of ToxT-AF to activate transcription of virulence genes under the tested conditions.

Given that the expression levels of His-tagged toxT-AF—and consequently tcpA and ctxAB—were comparable to those of native toxT-AF in the O395 strain, we reasoned that the virulence gene expression pattern in isogenic derivatives carrying the His-tagged toxT allele could serve as a proxy for those carrying the corresponding native toxT allele. On this basis, we proceeded to investigate virulence gene expression in isogenic derivatives harboring His-tagged toxT alleles.

toxT-SY: The O395 strain harbors the toxT-SY allele. Under virulence-inducing culture conditions, toxT mRNA in O395-H (His-tagged toxT-SY) was detectable within 2 h of cultivation, reaching its highest levels at 5 h (Figure 2A). His-tagged ToxT protein was first observed at 3 h, with protein levels peaking between 3 and 4 h (Figure 2A). Both toxT mRNA and ToxT protein levels began to decline after reaching their peaks, with toxT mRNA decreasing by 8 h and ToxT protein levels dropping by 12 h. Under virulence-repressing conditions, toxT mRNA levels were minimal, and ToxT protein production was negligible (Figure 2B).

The expression of tcpA and ctxAB mRNA was first detected between 3 and 4 h after cultivation, with peak expression occurring at approximately 5–6 h, followed by a gradual decrease (Figures 2C,E). TcpA protein levels became detectable starting at 4 h, and remained detectable up to 24 h post-cultivation (Figure 2C). No expression of tcpA and ctxAB was detected under virulence-repressing conditions (Figures 2D,F).

toxT-SF: The expression of His-tagged toxT-SF in YJB001-H under virulence-inducing conditions was first detectable within 1 h of culture, peaking at 2 h before gradually decreasing (Figure 2G). His-tagged ToxT was detectable starting at 2 h, with peak expression occurring between 2 and 4 h (Figure 2G). In contrast, under virulence-repressing conditions, toxT mRNA levels were minimal, and His-tagged ToxT was negligible (Figure 2H).

The mRNAs of tcpA and ctxAB were first detected at 3 h post-cultivation, reaching peak levels at 8 h, followed by a decline (Figures 2I,K). TcpA protein levels were first detectable at 4 h and TcpA protein levels remained substantial for up to 24 h (Figure 2I). Under virulence-repressing conditions, no expression of tcpA or ctxAB was observed (Figures 2J,L).

toxT-AY: The expression of His-tagged toxT-AY mRNA in EJK008-H (His-tagged toxT-AY) was first detected at 2 h post-cultivation under agglutinating conditions, with mRNA levels reaching a peak at 5 h before progressively declining (Supplementary Figure S4A). His-tagged ToxT protein was similarly detected starting at 2 h (Supplementary Figure S4A). Under virulence-repressing conditions, both toxT-AY mRNA and His-tagged ToxT protein were present at negligible levels (Supplementary Figure S4B).

Expression of the downstream targets tcpA and ctxAB was observed beginning at 2 h post-cultivation, peaking at 4 h before gradually declining (Supplementary Figures S4C,E). TcpA protein was first detected at 3 h and remained at substantial levels up to 24 h (Supplementary Figure S4C). Under virulence-repressing conditions, the expression of tcpA and ctxAB was negligible (Supplementary Figures S4D,F).

We investigated two V. cholerae El Tor biotype strains: the Wave 1 prototype strain N16961 and the Wave 3 atypical El Tor strain IB5230 (Mutreja et al., 2011; Chin et al., 2011). El Tor biotype strains typically express virulence genes under AKI conditions but not when cultured in LB medium (Lee et al., 2023; Kim et al., 2022; Lee et al., 2024). In previous work, we showed that introducing alternative toxT alleles (toxT-AY and toxT-AF) into N16961 enabled the strain to express virulence genes in aerated LB medium, even without pH adjustment to 6.5 (Lee et al., 2023; Baek et al., 2020). In the present study, we monitored virulence gene expression in El Tor biotype strains cultured in LB medium without pH adjustment, to assess whether allele-specific or strain-specific regulation could drive virulence gene induction under these otherwise non-inducing conditions.

The V. cholerae N16961 strain carries the authentic toxT-SY allele but does not exhibit virulence gene expression under LB culture conditions (Lee et al., 2023; Lee et al., 2024). An isogenic derivative in which toxT-SY was replaced with toxT-SF similarly failed to induce virulence gene expression (Lee et al., 2023). In contrast, previous studies have shown that substitution with either toxT-AY or toxT-AF enabled virulence gene expression in LB medium at 30°C (Lee et al., 2023; Choi et al., 2023; Lee et al., 2024).

In this study, we investigated virulence gene expression in isogenic derivatives of N16961 carrying four different His-tagged toxT alleles. The isogenic derivatives carrying His-tagged toxT-AF (DHL009-H) or His-tagged toxT-AY (DHL008-H) exhibited virulence gene expression during the early exponential phase at 30°C (Figures 3A,G). Once mRNA transcription was initiated, ToxT protein was detectable shortly after transcript initiation and remained detectable for approximately 12 h (Figures 3A,G). The expression patterns of the downstream targets tcpA and ctxAB followed a similar temporal profile (Figures 3C,E,I,K). Additionally, no expression of tcpA or ctxAB was detected at 37°C (Figures 3B,D,F,H,J,L).

By contrast, no virulence gene expression was observed in isogenic derivatives carrying His-tagged toxT-SY (N16961-H) or His-tagged toxT-SF (YJB003-H) alleles (Figures 4A–L), consistent with results obtained for the corresponding native alleles. Overall, tcpA and ctxAB expression patterns observed with His-tagged toxT alleles were in agreement with prior reports for native toxT alleles (Supplementary Table S4) (Lee et al., 2023; Lee et al., 2024).

The atypical El Tor strain V. cholerae IB5230 also carries the authentic toxT-SY allele. Unlike N16961, IB5230 expressed virulence genes with either the toxT-SY or toxT-SF allele. In contrast, replacement of toxT-SY allele with toxT-AY or toxT-AF allele ab prevented detectable virulence gene expression (Lee et al., 2023; Lee et al., 2024).

YJB020-H (His-tagged toxT-SF) expressed virulence genes at both 30°C and 37°C although ctxAB expression was restricted to 30°C. At 30°C, toxT mRNA became detectable starting 1 h after inoculation, reaching peak levels at approximately 4–5 h (Figure 5A). ToxT protein became detectable between 1 and 2 h, with substantial levels maintained for approximately 10 h. Expression of tcpA and ctxAB followed shortly after toxT expression (Figures 5C,E).

At 37°C, toxT mRNA was detectable as early as 1 h after inoculation, reaching its peak at approximately 3–4 h. ToxT protein also appeared between 1 and 2 h but declined more rapidly than under the 30°C condition (Figure 5B). The expression of tcpA and ctxAB appeared to be differentially regulated at 37°C. While tcpA mRNA and TcpA protein were detected (Figure 5D), ctxAB expression was not observed (Figure 5F). These findings were consistent with results from YJB020 carrying the native toxT-SF allele (Lee et al., 2023; Lee et al., 2024).

In the isogenic derivative of IB5230 harboring the His-tagged toxT-SY allele (IB5230-H), a temperature-dependent shift in toxT and virulence gene expression was observed. At 30°C, no expression of toxT, tcpA, or ctxAB was detected (Figures 5G,I,K). In contrast, when cultured at 37°C, both toxT and ctxAB were expressed; however, tcpA expression remained absent under these conditions (Figures 5H,J,L).

The isogenic derivative of IB5230 harboring the His-tagged toxT-AF allele (DHL021-H) did not exhibit toxT, tcpA, or ctxAB expression at either 30°C or 37°C (Figures 6A–F). Similarly, in DHL020-H, which harbors the His-tagged toxT-AY allele, expression of toxT, tcpA, and ctxAB remained negligible under both temperature conditions (Figures 6G–L).

In IB5230 derivatives carrying His-tagged toxT alleles, the tcpA and ctxAB expression patterns observed—whether expression was detected at 37°C or showed differential regulation—closely paralleled those obtained with the corresponding native toxT alleles (Lee et al., 2023). These results indicate that His-tagging did not alter allele-specific regulatory outcomes and further highlight a strain-specific mode of virulence gene regulation in IB5230.