Cu²⁺ regulated sulfonamides resistance gene (sul) via reactive oxygen species induced ArcA in a pathogenic Vibrio splendidus

Abstract

Purpose

To detect the sulfonamide resistance of Vibrio splendidus strain Vs, and characterize the factors that regulate the expression of sulfonamide resistance gene sul_Vs.

Methods

Minimal inhibitory concentration was measured using growth inhibition method, gene expression was measured using real-time reverse transcriptase PCR, reactive oxygen species (ROS) production under Cu²⁺ or Cu²⁺/NAC conditions was measured using an excitation wavelength of 485 nm, and an emission wavelength of 525 nm, and the binding of sul_Vs promoter and the transcriptional factor ArcA was measured using electrophoretic mobility shift assay (EMSA).

Result

sul_Vs gene was cloned and its expression was upregulated to 6.98- and 3.57-fold in the presence of sulfadiazine and sulfamethoxazole, respectively. Moreover, Cu²⁺ could also upregulate the expression of sul_Vs to 14.27-fold, the production of ROS to 4.37-fold, and the expressions of antioxidant-related genes and a transcriptional regulator arcA gene. After addition of N-Acetyl-L-cysteine, a ROS inhibitor, the production of ROS decreased to 53.1%, and the expression of arcA gene was also downregulated to 26%. EMSA showed that the purified recombinant ArcA could directly bind to the promoter region of sul_Vs with specificity.

Conclusion

V. splendidus strain Vs showed sulfonamide resistance due to sul_Vs, and Cu²⁺ could increase the level of ROS followed by ArcA activation as a transcriptional factor to increase the expression of sul_Vs.

Introduction

Vibrio splendidus is an opportunistic pathogen that ubiquitously presents in the marine environment. It can infect many hosts including fish, shellfish, and sea cucumber, leading to bacterial diseases (Helen et al. 2009; Liu et al. 2013; Zhang et al. 2016). Synthetic sulfonamides have been widely used to treat bacterial infections for their wide antibacterial spectrum and effective antibacterial action in humans and animals, since it was introduced into clinical practice from 1935. Sulfonamides function via competing with the natural substrate pamino-benzoic acid for dihydropteroate synthase, resulting reduced production of dihydrofolic acid and thus leading to death of bacteria (Stokstad and Jukes 1987). Now, levels of sulfonamides in the environment have reached a moderate pollution risk for their excessive and frequent use, and they have imposed persistent risks to the living organisms (Richardson et al. 2005; Hernando et al. 2006). Environmental bacteria can easily acquire antibiotic resistances after being exposed to residual antibiotics. Beside antibiotics, heavy metals were also widely used to treat bacterial diseases, leading to synergistic co-contamination of antibiotics and heavy metals (Mu et al. 2014; Wu et al. 2015). While abundance of antibiotic resistance genes (ARGs) was closely linked to the levels of residual antibiotics that exerted a selection pressure on microorganisms (McKinney et al. 2010), some reports showed that expression of ARGs showed no or weak correlation with antibiotic levels, but it was strongly correlated with levels of heavy metals (Baker-Austin et al. 2006).

Bacteria could produce reactive oxygen species (ROS), such as O²⁻, hydrogen peroxide (H₂O₂), hydroxyl radicals ([·OH]), nitric oxide (NO), and singlet oxygen ([·O₂]), which are important signaling molecules with strong oxidizing properties (Daniel et al. 2009). These chemicals play important roles in various signal transductions in vivo. When the cells are under heavy metals stress, the redox balance will be broken, and ROS will be generated in large quantities, and thus leading to oxidative stress (Sies 1997; James and Christopcher 2010). As a consequence, living organisms have developed enzymatic and non-enzymatic antioxidant defense systems to balance the excessive ROS. Among different kinds of ROS, O²⁻ was one of the most important species generated from NADPH by a typical environmental sensitive membrane protein (Han et al. 2012; Radermacher et al. 2012). Transcriptional regulator ArcA plays an important role in maintaining the ratio of NADH to NAD⁺ to avoid excessive production of ROS (Loui et al. 2009).

In the present study, the effects of Cu²⁺ on the expression of sul_Vs gene, ROS production, and expression of arcA gene were determined in V. splendidus. The relationship between ROS production and expressions of arcA and sul_Vs was examined. Based on the data obtained, a regulation pathway of Cu²⁺ on expression of sul_Vs was preliminarily proposed.

Materials and methods

Bacterial strains, culture conditions, and chemicals

The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown in Luria-Bertani Lysis Broth (LB) at 37 °C. V. splendidus strain Vs was isolated from the diseased Apostichopus japonicus (Zhang et al. 2016), and was grown in 2216E medium (5 g tryptone, 1 g yeast extract, and 0.01 g FePO₄ in 1 L aged seawater) at 28 °C. Ampicillin (Amp), kanamycin (Kan), or X-gal (5-bromo-4-chloro-3-indolyl-beta-Dgalactopyranoside) was separately supplemented at concentrations of 50, 50, or 40 μg/ml, when it was needed.

Table 1 Bacterial strains and plasmids used in this study

Cell density was measured at 600 nm using an UV–Vis (Molecular Device, USA). N-Acetyl-L-cysteine (NAC) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Electrophoretic mobility shift assay (EMSA) Gel-shift binding buffer (5×) was purchased from Beyotime (Shanghai, China). 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Sigma-Aldrich. Taq and Pfu DNA polymerases were purchased from Takara (China). Restriction endonucleases were purchased from New England Biolabs and used in accordance with the manufacturer’s specifications. Unless otherwise stated, all other chemicals used in this study were of the highest purity and were purchased from Sangon (Shanghai, China).

Plasmid construction

Plasmid extraction, DNA extraction from agarose gels, and the purification of PCR products were performed using the respective kits from Omega Bio-Tek (GA) according to the manufacturer’s instructions. The primers used in this study are listed in Table 2. DNA sequencing was carried out by Sangon (Shanghai, China). pET28a-arcA was constructed by ligating arcA, amplified with the forward primer arcAF3 and reverse primer arcAR3, into pET-28a, between the BamH I / Xho I sites. To construct pSC11-Psul_Vs, Psul_Vs was amplified by PCR using Pfu DNA polymerase with primers of P3F6 and P3R6. The PCR product was phosphorylated using T4 polynucleotide kinase, and then was inserted into the Swa I site of pSC11, a low-copy-number plasmid with a pSC100 replication origin and a promoterless lacZ gene as a reporter of heterologous promoter activity (Zhang et al. 2008).

Table 2 Primers used in this study

Expression and purification of the recombinant ArcA

E. coli BL21(DE3) harboring the plasmid pET28a-arcA was grown to mid-log phase, and expression of the recombinant ArcA was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 μM. After grown for an additional 6 h, the cells were harvested by centrifugation and the His-tagged ArcA was purified with nickel-nitrilotriacetic acid agarose under native conditions according to the recommendations of the manufacturer (QIAGEN, Valencia, CA) (Zhang and Sun 2007).

EMSA

EMSA was carried out according to the previous description by Wang et al. (2008). Fifty nanograms of Psul_Vs or negative control DNA amplified using the primers P3F6/P3R6 or P1F1/P1R1 was separately mixed with various amounts of purified recombinant ArcA, EMSA/Gel-shift binding buffer (5×) and the nuclease-free water were added to make up the volume of 10 μl, and incubated at 25 °C for 45 min. The samples were then electrophoresed in a non-denatured 8% polyacrylamide gel in an ice bath, and the gel was dyed with nucleic acid dye after electrophoresis for 1 h. Observation was conducted under a gel imaging system (Junyi, China).

ROS measurement

V. splendidus strain Vs was cultured in 2216E medium to approximately OD₆₀₀ of 0.2, and then it was departed into two parts. One part was added with 0.5 mM Cu²⁺, and the other part without any addition was used as a control. Cell precipitates were obtained from the culture at a time intervals of 15, 30, and 45 min by centrifugation at 6000g for 5 min. To quantify ROS production after sole NAC or the combined treatment of NAC and Cu²⁺, V. splendidus strain Vs was cultured in 2216E medium to approximately OD₆₀₀ of 0.2, and then it was departed into three parts. One millimolar NAC, or both 0.5 mM Cu²⁺ and 1 mM NAC was added into separate part and one part without addition was used as a control. The cell pellet was washed three times with PBS (1×), and then it was resuspended in PBS (1×) to an OD₆₀₀ of 0.1. DCFH-DA was used to treat samples, followed by the quantitation of intracellular ROS production. ROS was determined according to the protocol of Varioskan™, and fluorescence was detected using an excitation wavelength of 485 nm, and an emission wavelength of 525 nm (Thermo Fisher Scientific, MA).

Sample collection for real-time reverse transcriptase PCR (RT-PCR)

To detect the effects of SDZ and SMZ on the expression of sul_Vs, V. splendidus strain Vs was cultured in 2216E medium to approximately OD₆₀₀ of 0.2, then cells were divided into three aliquots. One aliquot was supplemented with 64 μg/ml SDZ, the second aliquot was supplemented with 32 μg/ml SMZ, and the third aliquot was continuously cultured in medium without addition of SDZ or SMZ. All the three aliquots were cultured for another 10, 20, and 30 min.

To determine the effect of Cu²⁺ on the expression of arcA and other antioxidant-related genes, V. splendidus strain Vs was grown in 2216E medium to OD₆₀₀ of approximately 0.2, and then it was departed into two parts. One part was supplemented with 0.5 mM Cu²⁺, and the other part without any addition was used as a control. Then, each sample was cultured for another 30 and 45 min. To detect the effects of simultaneous presence of Cu²⁺ and NAC on the expression of arcA, V. splendidus strain Vs was grown in 2216E medium to OD₆₀₀ of approximately 0.2, then cells were divided into three aliquots. One aliquot was supplemented with 1 mM NAC, the second aliquot was simultaneously supplemented with 0.5 mM Cu²⁺ and 1 mM NAC, respectively, and the third aliquot without any addition was used as a control. Then, each sample was cultured for another 30 and 45 min.

After treated under the above conditions, all of the cells were quickly freezed in − 80 °C pre-incubated ethanol, and then centrifuged to collect the pellets. The cell pellets were then resuspended in RNAlater, and then centrifuged again to collect the pellets. Then, the cell pellets were used for RNA extraction.

Real-time RT-PCR

Total RNA was extracted using the bacterial RNA kit (Omega), followed by the treatment of RNase-free DNase (Promega, USA) for 1 h. The DNA-free RNAs were used for the synthesis of cDNA using the reverse transcriptase (Taraka, China). Real-time RT-PCR was carried out as described by Zhang et al. (2013b). Real-time RT-PCR was carried out in an ABI 7500 real-time detection system (Applied Biosystems) by using the SYBR ExScript RT-PCR kit (Takara, China). The primers for arcA and antioxidant-related genes were arcAdl F4/arcAdl R4, FtF1/R1, SODF1/R1, NoxF1/R1, and SoxRF1/R1. Each assay was performed in triplicate using 16S rRNA as a control. The primers used for 16S rRNA were 933F and 16SRTR1. The comparative threshold cycle method (2^−△△CT method) was used to analyze the relative mRNA levels of arcA and antioxidant-related genes.

Database search and data analysis

Searches for nucleotide and amino acid sequence similarities were conducted using the BLAST programs from the NCBI (National Center for Biotechnology Information). Isolated of V. splendidus strain Vs has been deposited into the China General Microbiological Culture Collection (CGMCC, Beijing, China) with accession numbers CGMCC No. 7.242. The homologous analysis of amino acid sequence was analyzed by MEGA 5.1 (Tamura et al. 2007) and CLUSTAL X (Larkin et al. 2007). Bacterial promoter prediction was performed using BPROM (http://www.softberry.com). Statistical analyses were performed by using the two-tailed Student t test. One-way ANOVA was performed to determine the significant differences between experimental and control groups at each specific time point, and the significance level was defined as *, P < 0.05, and **, P < 0.01.

Results

V. splendidus showed resistance to sulfonamides via sul _Vs

The MICs of SDZ, SMZ, and TMP are listed in Table 3. The inhibitory effect of SDZ on growth of V. splendidus strain Vs was dose dependent. V. splendidus strain Vs showed obvious growth in medium supplemented with 512 μg/ml SDZ, but showed no obvious growth in medium supplemented with 1024 μg/ml SDZ. Thus, the MIC of SDZ to V. splendidus strain Vs was 1024 μg/ml under the test conditions. We have sequenced the whole genomic DNA of V. splendidus strain Vs (unpublished data); one dihydropteroate synthase gene was annotated and it showed 88.69% homolog to the nucleotide sequence of dihydropteroate synthase gene from the published chromosome of V. splendidus strain LGP32 (No. FM954972). Nucleotide sequence analysis showed that ORF was 831 bp and coded dihydropteroate synthase with an estimated molecular mass of 30.06 kDa and theoretical pI was 5.12. The homology analysis showed that the dihydropteroate synthase gene of V. splendidus showed best homology to sulfonamide resistance gene (Fig. 1a), and thus, it was named sul_Vs. Comparison with the known sequence data of sul_s indicated that the best matches for the sul_Vs were the suls from Bacillus amyloliquefaciens (CP000560.1), and Thermoanaerobacter kivui (AIS53244.1), which showed, 43.63% and 42.91% identity to the amino acid sequence of sul_Vs, respectively. The expression of sul_Vs significantly was upregulated to 6.98-fold and 3.57-fold in the presence of SDZ and SMZ (Fig. 1b).

Table 3 MICs of antibiotics to V. splendidus strain Vs

Fig. 1

Phylogenetic tree of suls from V. splendidus strain Vs and other bacteria constructed using MEGA 5.1 and CLUSTAL X (a). The one signed with dot indicated sul_Vs from V. splendidus strain Vs. Fold differences in mRNA level of sul_Vs in the presence of SDZ or SMZ (b). mRNA level of sul_Vs was normalized to that of 16S rRNA at each time point, and the mRNA level of sul_Vs in the cells treated with SDZ or SMZ was compared to that in the control cells at each time point. Data are the means for three independent experiments and are presented as the means ± SD. **, P < 0.01

Cu²⁺ induced ROS and expressions of sul _Vs and arcA genes

Expression of sul_Vs could be upregulated to 14.27-fold in the presence of Cu²⁺. Beside that, Cu²⁺ also induced the production of ROS and the expression of arcA gene. ROS increased 2.85-, 4.37-, and 2.54-fold at 15, 30, and 45 min, respectively, after the addition of Cu²⁺ (Fig. 2). The mRNA levels of antioxidant-related genes also correspondingly increased at 30 and 45 min, respectively, and the highest stimulatory effect was obtained in the cells collected at 45 min (Fig. 3a–d). Taken all these results together, it proved that Cu²⁺ could stimulate the production of ROS in V. splendidus. Meanwhile, the expression of arcA gene was also upregulated to 1.4- and 2.48-fold at 30 and 45 min, respectively (Fig. 3e), compared with that in the cells without contact of Cu²⁺.

Fig. 2

Fluorescence value of ROS in the presence of Cu²⁺. Samples were treated using DCFH-DA probe and the intracellular ROS production was qualified. The fluorescence value in Cu²⁺-treated cells was compared to that in the control cells at each time point. Data are the means for three independent experiments and are presented as the means ± SD. **, P < 0.01

Fig. 3

Fold difference in mRNA levels of antioxidant-related genes in the presence of Cu²⁺ (a is for ft, b is for nox, c is for sod, d is for soxR, and e is for arcA). The mRNA level of each gene was normalized to that of 16S rRNA, and the mRNA level of each gene in the Cu²⁺-treated cells was compared to that in the control cells at each time point. Data are the means for three independent experiments and are presented as the means ± SD. *, P < 0.05 and **, P < 0.01

ROS affected the expression of arcA gene

To see if ROS could regulate the expression of arcA gene, a ROS inhibitor, N-Acetyl-L-cysteine (NAC), was applied. The effect of NAC on growth of V. splendidus strain Vs was first determined. 0.01 mM, 0.1 mM, and 1 mM NAC exhibited approximately the same trend without affecting growth (Fig. 4a). Then, the concentrations of NAC used to determine its affection on ROS production were 0.01, 0.1, and 1 mM. Under the three tested levels, addition of NAC inhibited ROS production in V. splendidus strain Vs. The quantity of ROS decreased to 88.3%, 81.4%, and 53.1% at 15, 30, and 45 min, respectively, after the addition of NAC (Fig. 4b). One millimole NAC showed approximately 50% inhibitory effect on ROS production without affecting growth (Fig. 5), so 1 mM NAC was added into bacterial culture to test its effect on the expression of arcA gene. The expression of arcA gene decreased to 26% in the presence of 1 mM NAC compared to that without NAC treatment (Fig. 6). So, it suggested that ROS could regulate the expression of arcA gene.

Fig. 4

OD₆₀₀ (a) and ROS production (b) in the presence of different concentration of NAC. 0.01 mM, 0.1 mM, or 1 mM NAC were respectively supplemented into the culture medium. After cultured for 15, 30, 45 min, and 24 h, aliquots were taken for the measurement of absorbance at 600 nm (a), and remaining samples were used for ROS detection (b). The fluorescence value in NAC-treated cells was compared to that in the control cells at each time point. Data are the means for three independent experiments and are presented as the means ± SD. *, P < 0.05, and **, P < 0.01

Fig. 5

Fluorescence value of ROS in the presence of Cu²⁺, NAC or both Cu²⁺ and NAC. The fluorescence value in cells treated with Cu²⁺, NAC or both Cu²⁺ and NAC was compared to that in the control cells at each time point. Data are the means for three independent experiments and are presented as the means ± SD. **, P < 0.01

Fig. 6

Fold difference in mRNA level of arcA gene in the presence of Cu²⁺, NAC or both Cu²⁺ and NAC. The mRNA level of arcA gene was normalized to that of 16S rRNA, and the mRNA level of arcA gene in cells treated with Cu²⁺, NAC or both Cu²⁺ and NAC was compared to that in the control cells at each time point. Data are the means for three independent experiments and are presented as the means ± SD. *, P < 0.05 and **, P < 0.01

Cu²⁺ regulated the expression of arcA through ROS pathway

Since Cu²⁺ could simultaneously upregulate ROS and expression of arcA, we wondered whether Cu²⁺ regulated arcA expression through ROS or not. This was realized through the addition of NAC. When the NAC and Cu²⁺ were simultaneously added into the culture, ROS production decreased to 77.7%, 78%, and 50.6% at 15, 30, and 45 min, respectively, while ROS production was upregulated to 4.37-fold at 30 min when only Cu²⁺ was added (Fig. 5). Similarly, after NAC and Cu²⁺ were simultaneously added into culture for 30 or 45 min, the expression of arcA also decreased to 67% or 80%, respectively, while expression of arcA was upregulated to 1.4- and 2.3-fold in the presence of Cu²⁺ alone at these two time points (Fig. 6).

ArcA directly regulated expression of sul _vs gene

Prediction of the 1000 bp upstream of sul_vs gene showed that one promoter, named Psul_Vs, showed an ArcA binding site (Fig. 7a). Psul_Vs was cloned into promoterless plasmid pSC11 and it developed blue colonies on X-gal agar plates, which suggested that Psul_Vs was an active promoters. To further detect whether ArcA could directly bind to the promoter region of Psul_Vs, recombinant ArcA was firstly obtained. orf of arcA is 717 bp, and it codes a protein of 238 aa with an estimated molecular mass of 27.1 kDa, and a theoretical pI of 5.29. Comparison with the known ArcA amino acid sequence indicated that the best matches for the amino acid sequence of ArcA were those of Vibrio tasmaniensis (CAV17486.1) and Vibrio alginolyticus (WP_115385736.1), which showed, respectively, 100% and 99% identity to the sequence ArcA in V. splendidus strain Vs. Pure recombinant ArcA was purified from BL21(DE3)/pET28a-arcA induced by IPTG under native condition.

Fig. 7

The prediction of promoter regions of sul_Vs and binding sites of ArcA (a). The − 35 and − 10 regions of the promoter Psul_Vs have been identified. Bold and italic part represents the ArcA binding site, predicted using both BPROM and consistent sequence of 5′- nGTTAATTAn-3′(n is A or T) in E. coli (Lynch and Lin 1996). Recombinant ArcA was purified from BL21(DE3)/pET28a-arcA (b). Lane 1, protein Marker; lane 2, proteins from un-induced BL21(DE3)/pET28a-arcA; lane 3, proteins from induced BL21(DE3)/pET28a-arcA; lane 4, purified ArcA. Specific binding of ArcA to the promoter regions of sul_Vs determined by EMSA (c). Lane 1, ArcA and Psul_Vs; lane 2, ArcA; lane 3, Psul_Vs. A 303 bp DNA fragment from the upstream of promoter was used as a negative control (d). Lane 1, ArcA and negative DNA control; lane 2, negative DNA; lane 3, ArcA

Purified active recombinant ArcA, Psul_Vs DNA, and negative control DNA were obtained for EMSA. A distinctive protein band for the purified ArcA was detected (Fig. 7b). The EMSA result indicated that ArcA could bind to Psul_Vs with specificity, which predicted to possess the potential ArcA binding site, while it could not bind to the other DNA fragment (Fig. 7c, d). It indicated that ArcA was a transcriptional factor of sul_Vs. Taken together, these results demonstrated that ArcA could regulate the expression of sul_Vs gene, probably via direct interaction with the promoter region of sul_Vs.

Discussion

Although strains of Vibrio sp. had been reported to show resistance to different antibiotics (Okoh and Igbinosa 2010), our study was the first to report V. splendidus showed sulfonamide resistance to SDZ, SMZ, and SXT. The MIC of SDZ was 2.93-fold higher than the Performance Standards for Antimicrobial Susceptibility Testing (Jorgensen and Ferraro 2009). sul_Vs was homology to sul3, a sulfonamide resistance gene occurring in Salmonella sp. carrying class 1 integron with aadA and dfrA gene cassettes (Antunes et al. 2005). High antibiotic resistances have been frequently associated with bacterial pathogenesis (Beceiro et al. 2013); thus, the multiple antibiotic resistances may facilitate the infective ability of V. splendidus strain Vs. In natural environment, antibiotic resistance and heavy metals resistance are very frequently associated in the same organism, and furthermore, both resistances were due to the genes in the same plasmid, transposon, or integrin (Baker-Austin et al. 2006; Graham et al. 2011). Cu²⁺ has been reported to be closely related to ARGs (Berg et al. 2005). Cu²⁺ treatment has positive correlation to the erythromycin resistance genes in urban wastewater (Gao et al. 2015), carbapenem antibiotic (Caille et al. 2007), and tetracycline, erythromycin, and β-lactams (Graham et al. 2011). Our study was the first time to report that Cu²⁺ could significantly upregulate the expression of sul_Vs.

Although Cu²⁺ is an essential microelement that is necessary for a wide range of metabolic processes in living organisms, an accumulation of an over-dosage of Cu²⁺ can be toxic to bacteria with strain specificity (Ali et al. 2013). Similar to the effects of other heavy metals, excess of Cu²⁺ was also associated with ROS production (Luna et al. 2015). In our present study, Cu²⁺ stimulated the ROS production in V. splendidus, similar to that occurred in E. coli (Santo et al. 2011a) and Candida albicans (Santo et al. 2011b). Furthermore, we tested the response of several antioxidant enzymes, including SoxR, the redox regulator of the expression of SOD (Imlay 2008), NADH oxidase (Nox), one enzyme produce H₂O₂ which activates SOD and removes ROS (Gui et al. 2008), and Ferritin (Ft) that could alleviate the cytotoxicity caused by H₂O₂ and Fe²⁺ stresses, thus preventing the damage of [•OH] caused by Fenton reaction (Zhao et al. 2002; Imlay 2008). The upregulation of these antioxidant enzyme genes further suggested that Cu²⁺ could stimulate the generation of ROS and the bacterial cells have to initiate the antioxidant processes to balance ROS level.

ArcA is a transcriptional regulator that plays an important role in tolerating ROS (Salmon et al. 2003). We found that NAC could inhibit the production of ROS in V. splendidus, just like what happened in the other bacterial such as Listeria monocytegenes (Zhang et al. 2013a), and arcA gene decreased responding to ROS level like in E. coli (Salmon et al. 2003; Loui et al. 2009). In V. splendidus strain Vs, after NAC was added, ROS was inhibited, followed by reduced expression of arcA, which indicated that the expression of arcA was related to ROS. Moverover, NAC could inhibit the upregulatory effects of Cu²⁺, which indicated that Cu²⁺ could also regulate arcA through the ROS mediated pathway. And after that, ArcA acted as a transcriptional regulator of sul_Vs by directly and specifically binding to sul promoter region.

Taking all the results and references together, we proposed the following regulation pathway of Cu²⁺ on sul_Vs expression in V. splendidus strain Vs: Cu²⁺ stimulated ROS production and higher ROS upregulated the expression of arcA gene. ArcA directly bound to the promoter region of sul_Vs and upregulated the expression of sul_Vs gene. Our study provides a basis for studying the combination effects of environmental factors on bacterial resistance.