Comparison of Copper-Tolerance Genes between Different Groups of Acidovorax citrulli

Acidovorax citrulli populations exhibit genetic and phenotypic variations, particularly in terms of copper tolerance. Group I strains of A. citrulli generally exhibit higher copper tolerance compared to group II strains. This study aims to identify genes involved in copper tolerance to better understand the differences in copper tolerance between group I and group II strains. Representative strains pslb65 (group I) and pslbtw14 (group II) were selected for comparison. Deletion mutants of putative copper-tolerance genes and their corresponding complementary strains were constructed. The copper tolerance of each strain was evaluated using the minimum inhibitory concentration method. The results showed that the copA, copZ, cueR, and cueO genes played major roles in copper tolerance in A. citrulli, while cusC-like, cusA-like, and cusB-like genes had minor effects. The different expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 under copper stress indicated that they had different mechanisms for coping with copper stress. Overall, this study provides insights into the mechanisms of copper tolerance in A. citrulli and highlights the importance of specific genes in copper tolerance.


Introduction
Bacterial fruit blotch (BFB) is a seed-borne bacterial disease caused by Acidovorax citrulli, which poses a serious threat to cucurbit crops [1][2][3].Under hot and humid conditions, BFB spreads rapidly, leading to fruit rot in later stages of the disease and causing serious losses to the watermelon and melon industries [4,5].Acidovorax citrulli populations can be divided into two genetically distinct groups, group I and II [6,7].Current methods for controlling BFB in the field rely heavily on copper-containing bactericides [8,9].However, only 2% of group I strains of A. citrulli are sensitive to copper sulfate at a concentration of 500 µg/mL (3.13 mM), while all tested group II strains are sensitive [10].The presence of copper-tolerant strains in A. citrulli has made prevention and control more challenging, resulting in an increase in group I strains [10].Therefore, it is crucial to study the copper-tolerance mechanism of A. citrulli and understand the disparity in copper tolerance between group I and II strains.This research can provide valuable insights for BFB prevention and treatment.
Copper is essential for bacterial metabolism, but excessive amounts can be harmful [11].Bacteria must tightly regulate copper homeostasis in the intracellular environment to maintain metabolism and vitality [12].Escherichia coli has multiple mechanisms for copper tolerance.In E. coli, copper ions are bound by the copper-chaperone protein CopZ upon entering the cytoplasm, which directs them to transcription regulators and the copperexporting P-type ATPase CopA.CopA actively pumps excess copper ions out of the cytoplasm and exports them to the Cus system.The periplasmic copper chaperone CusF assists in removing copper ions from the cell [13].In addition, copper ions in the periplasm can bind to multicopper oxidase CueO, which converts Cu + to the less toxic Cu 2+ [13], thus protecting periplasmic enzymes from copper-induced damage.Both the copper efflux P-type ATPase encoded by the copA gene and multicopper oxidase cueO in E. coli are regulated by CueR [13].
However, due to the differences in strains and methods for assessing growth on copper-amended media used in previous studies, direct comparisons cannot be made regarding the effects of these genes on copper tolerance in A. citrulli strains.Therefore, this study aims to address this gap by generating mutant and complementary strains based on known copper-tolerance genes from representative group I and group II strains.The roles of these genes in copper tolerance were assessed through measuring the minimum inhibitory concentration (MIC) of copper on solid medium, and the expression levels of copper-tolerance-related genes in the representative strains were compared.This study will shed light on the disparity in copper sensitivity among A. citrulli strains, and contribute to our understanding of the mechanisms underlying copper tolerance.Ultimately, the findings of this research will aid in the development of disease prevention and control strategies for BFB.

Strains, Plasmids, and Antibiotics
The strains and plasmids used in this study are listed in Table S1.The media and antibiotic concentrations used in the experiments were prepared as follows.KMB [19] solid medium was composed of the following ingredients per liter: Tryptone 20 g, K 2 HPO 4 1.5 g, MgSO 4 1.5 g, agar 15.0 g, and 1 L H 2 O.The antibiotic concentrations used were ampicillin (Amp) at 100 µg/mL, kanamycin (Kan) at 50 µg/mL, and chloramphenicol (Cm) at 25 µg/mL.

Selection of Copper-Tolerant Representative Strains
Based on the copper sensitivity test conducted by Zhao [10], two strains (pslb9 and pslb65) from group I and four strains (pslbtw14, pslbtw32, pslbtw38, and Aac5) from group II were selected for further screening of representative strains.Copper sulfate (CuSO 4 •5H 2 O) concentrations ranging from 0 to 7.2 mM were used for the screening process.The strains were cultured in KMB liquid medium at 28 • C and 220 r/min for 12 h.Bacterial suspensions with an OD 600 of 0.3 (equivalent to 3 × 10 8 CFU/mL) were mixed with KMB liquid medium containing copper in a ratio of 1:100.The mixture was then added to 100-well polystyrene plates, and incubated at 28 • C with shaking in a Bioscreen C for monitoring growth (Bioscreen C • PRO, Helsinki, Finland).The OD 600 values were measured every 2 h.The experiment was conducted three times.

Construction and Verification of Deletion Mutants and Complementary Strains for Putative
Copper-Tolerance Genes Primers were designed using Primer Premier 5.0 (Canada).The DNA from pslb65 and pslbtw14 served as templates.The upstream and downstream fragments of the Aave_0032 (cueR) gene were amplified using primer pairs 0032-1F/1R and 0032-2F/2R, respectively.The resulting fragments were then fused using Overlapping PCR [20,21].The fused fragment was ligated with pK18mobsacB [22,23], which had been digested with EcoRI and HindIII.The ligated construct was then transformed into DH5α.The suicide-recombinant vectors were verified using primers M13F/M13R and sequencing.The mutants were constructed using the homologous recombination method with three parental strains and screened with sucrose [24][25][26].The mutants were validated using A. citrulli-specific primers WFB1/WFB2 [27] and target-gene-specific primers 0032-F/R.The validated mutants were sub-cultured and stored.The mutants for the other 11 genes were obtained using the same procedure.
To complement the mutants with impaired copper sensitivity, pslb65 was used as a template.Primer pair H0032F/H0032R was used to amplify Aave_0032.The amplified fragment was then ligated with pBBR1MCS-2, which had been digested with BamHI/HindIII.After confirming the correct complementary vector, it was introduced into the mutants ∆65-0032 and ∆14-0032 through three-parental hybridization.Verification was performed using target-gene-specific primers and Kan primers.Similar methods were used to obtain complementary strains for other genes.The primers used are listed in Table S2.

Determination of Copper MIC
The tested strains were cultured in KMB liquid medium at 28 • C and 220 r/min for 12 h.The bacterial suspensions were adjusted to an OD 600 of 0.3 and diluted to a concentration of 3 × 10 4 CFU/mL.Then, 10 µL of the bacterial suspension was spotted on KMB solid medium containing various concentrations of CuSO 4 •5H 2 O (0, 0.1, 0.2, 0.3, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, 4.8, 5.2, 5.6, 6.0, and 6.4 mM) [28].The plates were incubated at 28 • C for 6 days, and the MIC value was determined as the lowest concentration of copper sulfate that showed no bacterial growth.Each strain was tested in triplicate, and the experiment was conducted three times.

Analysis of Gene Expression Related to Copper Tolerance
The single colony of pslbtw14 and pslb65 was cultured in KMB liquid medium with different concentrations of Cu 2+ at 28 • C and 220 r/min for 12 h.For pslbtw14, the Cu 2+ concentrations were 0.8 mM and 2.0 mM, while for pslb65, the concentrations were 2 mM and 4 mM, with a control group receiving no copper treatment.The internal reference gene used was the rpoB gene.Genes expression levels related to copper metabolism were determined using the relative quantitative method (2 −∆∆CT ) [29], with the expression levels of each gene in wildtype strains pslbtw14 and pslb65 without copper treatment set as 1 for comparison.
∆65-0032 and ∆14-0032 were cultured in KMB liquid medium containing 1.6 mM Cu 2+ for 12 h, with pslb65 and pslbtw14 treated with 1.6 mM Cu 2+ serving as controls, respectively.The internal reference gene used was the rpoB gene.The gene expression levels in pslb65 and pslbtw14 treated with 1.6 mM copper were set as 1 for comparison, and the expression levels of related genes in ∆65-0032 and ∆14-0032 were calculated accordingly.
Total RNA was extracted from the cultures using a bacterial total RNA extraction kit (Yeasen, Shanghai, China).RNA reverse transcription was performed using the FastKing gDNA Dispelling RT Supermix kit (TIANGEN).The primer sequences for quantitative real-time PCR (qRT-PCR) are listed in Table 1.

Data Analysis
The experimental data were recorded and analyzed using Excel 2016 (Microsoft, Redmond, WA, USA).Statistical analysis and graphs were plotted using GraphPad Prism 9 (GraphPad, San Diego, CA, USA).Two-way analysis of variance (ANOVA) in GraphPad Prism 9 was used to determine the significance of gene expression levels related to copper metabolism following different copper treatments (with a confidence interval of 95%).Dunnett's multiple comparisons test was applied for further analysis.

Identification of Copper-Tolerant A. citrulli Strains
The copper tolerance of six selected A. citrulli wildtype strains was determined by measuring their copper MIC values (Figure 1).Group I strains had copper MIC values of 6.4 mM, while group II strains had copper MIC values of 2.8 mM.This indicated that group I strains were less sensitive to copper than group II strains.Additionally, the growth curves of the selected strains were measured in vitro at different copper concentrations.Clear differences were observed between group I and II strains at copper concentrations of 6.4 mM and 4 mM (Figure 2).At 6.4 mM, the growth rates of the two group I strains were similar within the 0~60 h timeframe.However, after 60 h, the growth of strain pslb65 surpassed that of strain pslb9 (Figure 2b).At a copper concentration of 4 mM, strain pslbtw14 exhibited higher growth than the other group II strains after 48 h (Figure 2a).Therefore, pslb65 and pslbtw14 were selected as representative strains for subsequent experiments. of 6.4 mM and 4 mM (Figure 2).At 6.4 mM, the growth rates of the two group I strains were similar within the 0~60 h timeframe.However, after 60 h, the growth of strain pslb65 surpassed that of strain pslb9 (Figure 2b).At a copper concentration of 4 mM, strain pslbtw14 exhibited higher growth than the other group II strains after 48 h (Figure 2a).Therefore, pslb65 and pslbtw14 were selected as representative strains for subsequent experiments.The growth curves of the tested strains were measured at 4 mM Cu 2+ (a) and 6.4 mM Cu 2+ (b).Strains pslb65 and pslb9 belong to group I, while pslbtw14, pslbtw38, pslbtw32, and Aac5 belong to group II.Bacterial suspensions with an OD600 of 0.3 (equivalent to 3 × 10 8 CFU/mL) were mixed with KMB liquid medium containing copper in a ratio of 1:100.The mixture was incubated at 28 °C with shaking in a Bioscreen C. The OD600 values were measured every 2 h.

Verification of Deletion Mutants and Complementary Strains for Putative Copper-Tolerance Genes
Deletion mutants for copper-tolerance genes were constructed using A. citrulli group I strain pslb65 and group II strain pslbtw14 as templates.The correctness of the deletion mutants was confirmed using target-gene-specific primers and A. citrulli-species-specific primers (Figures S1 and S2).Complementary strains were constructed using DNA from pslb65 as the template and verified using target-gene-specific primers and Kan primers (Figures S3 and S4). of 6.4 mM and 4 mM (Figure 2).At 6.4 mM, the growth rates of the two group I strains were similar within the 0~60 h timeframe.However, after 60 h, the growth of strain pslb65 surpassed that of strain pslb9 (Figure 2b).At a copper concentration of 4 mM, strain pslbtw14 exhibited higher growth than the other group II strains after 48 h (Figure 2a).Therefore, pslb65 and pslbtw14 were selected as representative strains for subsequent experiments.The growth curves of the tested strains were measured at 4 mM Cu 2+ (a) and 6.4 mM Cu 2+ (b).Strains pslb65 and pslb9 belong to group I, while pslbtw14, pslbtw38, pslbtw32, and Aac5 belong to group II.Bacterial suspensions with an OD600 of 0.3 (equivalent to 3 × 10 8 CFU/mL) were mixed with KMB liquid medium containing copper in a ratio of 1:100.The mixture was incubated at 28 °C with shaking in a Bioscreen C. The OD600 values were measured every 2 h.

Verification of Deletion Mutants and Complementary Strains for Putative Copper-Tolerance Genes
Deletion mutants for copper-tolerance genes were constructed using A. citrulli group I strain pslb65 and group II strain pslbtw14 as templates.The correctness of the deletion mutants was confirmed using target-gene-specific primers and A. citrulli-species-specific primers (Figures S1 and S2).Complementary strains were constructed using DNA from pslb65 as the template and verified using target-gene-specific primers and Kan primers (Figures S3 and S4).

Verification of Deletion Mutants and Complementary Strains for Putative Copper-Tolerance Genes
Deletion mutants for copper-tolerance genes were constructed using A. citrulli group I strain pslb65 and group II strain pslbtw14 as templates.The correctness of the deletion mutants was confirmed using target-gene-specific primers and A. citrulli-species-specific primers (Figures S1 and S2).Complementary strains were constructed using DNA from pslb65 as the template and verified using target-gene-specific primers and Kan primers (Figures S3 and S4).

Copper-Tolerance Phenotypes of Representative Strains and Mutants
The mutants of representative strains pslb65 (group I) and pslbtw14 (group II) with the same gene deletion exhibited different levels of sensitivity to copper.Strain pslb65 had a copper MIC value of 6.4 mM, and the deletion of certain genes associated with copper tolerance in pslb65 decreased its tolerance to Cu 2+ (Figure 3).For instance, ∆65-0034 (Aave_0034-gene-deletion mutant derived from pslb65, similar below) failed to grow at a copper concentration of 0.5 mM, ∆65-0033 failed to grow at 1.3 mM, and ∆65-0032 and ∆65-1810 failed to grow at 2.8 mM (Figure 3b).∆65-1811 did not grow on KMB plates with a Cu 2+ concentration of 5.6 mM, and ∆65-0387, ∆65-0388, and ∆65-0389 did not grow on KMB plates with a Cu 2+ concentration of 6 mM.In group I strain pslb65, deletion of the tolC (Aave_1811) gene had a greater impact on copper tolerance compared to cusC-like (Aave_0387), cusA-like (Aave_0388), and cusB-like (Aave_0389).Conversely, ∆65-0038, ∆65-0039, ∆65-2798, and ∆65-4663 exhibited similar copper-tolerance levels to pslb65 (Figure 3).The copper MIC value for pslbtw14 was 2.8 mM (Figure 3a).∆14-0034 did not grow on a KMB plate containing 0.3 mM copper, and ∆14-0033 lost its ability to grow at a copper concentration of 1.3 mM.At a copper concentration of 2.4 mM, ∆14-0032, ∆14-0387, ∆14-0388, ∆14-0389, and ∆14-1810 lost their ability to grow.However, the deletion of Aave_0038, Aave_0039, Aave_1811, Aave_2798, and Aave_4663 genes in pslbtw14 did not affect the copper-tolerance level of the strains.Overall, strain pslb65 exhibited greater tolerance to copper stress compared to pslbtw14.The deletion mutants of the same gene in strain pslb65 generally showed lower copper sensitivity compared to pslbtw14, except for Aave_0033.
The copper MIC values for ∆65-0033 and ∆14-0033 were both 1.3 mM.Furthermore, the deletion of the Aave_0034 gene resulted in the loss of basic copper tolerance in both pslb65 and pslbtw14.The gene organization of the copper-tolerant genes characterized in this study is shown in Figure 4.

Copper-Tolerance Phenotypes of Representative Strains and Mutants
The mutants of representative strains pslb65 (group I) and pslbtw14 (group II) with the same gene deletion exhibited different levels of sensitivity to copper.Strain pslb65 had a copper MIC value of 6.4 mM, and the deletion of certain genes associated with copper tolerance in pslb65 decreased its tolerance to Cu 2+ (Figure 3).For instance, Δ65-0034 (Aave_0034-gene-deletion mutant derived from pslb65, similar below) failed to grow at a copper concentration of 0.5 mM, Δ65-0033 failed to grow at 1.3 mM, and Δ65-0032 and Δ65-1810 failed to grow at 2.8 mM (Figure 3b).Δ65-1811 did not grow on KMB plates with a Cu 2+ concentration of 5.6 mM, and Δ65-0387, Δ65-0388, and Δ65-0389 did not grow on KMB plates with a Cu 2+ concentration of 6 mM.In group I strain pslb65, deletion of the tolC (Aave_1811) gene had a greater impact on copper tolerance compared to cusC-like (Aave_0387), cusA-like (Aave_0388), and cusB-like (Aave_0389).Conversely, Δ65-0038, Δ65-0039, Δ65-2798, and Δ65-4663 exhibited similar copper-tolerance levels to pslb65 (Figure 3).The copper MIC value for pslbtw14 was 2.8 mM (Figure 3a).Δ14-0034 did not grow on a KMB plate containing 0.3 mM copper, and Δ14-0033 lost its ability to grow at a copper concentration of 1.3 mM.At a copper concentration of 2.4 mM, Δ14-0032, Δ14-0387, Δ14-0388, Δ14-0389, and Δ14-1810 lost their ability to grow.However, the deletion of Aave_0038, Aave_0039, Aave_1811, Aave_2798, and Aave_4663 genes in pslbtw14 did not affect the copper-tolerance level of the strains.Overall, strain pslb65 exhibited greater tolerance to copper stress compared to pslbtw14.The deletion mutants of the same gene in strain pslb65 generally showed lower copper sensitivity compared to pslbtw14, except for Aave_0033.The copper MIC values for Δ65-0033 and Δ14-0033 were both 1.3 mM.Furthermore, the deletion of the Aave_0034 gene resulted in the loss of basic copper tolerance in both pslb65 and pslbtw14.The gene organization of the coppertolerant genes characterized in this study is shown in Figure 4.A schematic representation of the organization of copper-tolerance genes in the Acidovorax citrulli genome AAC00-1 (Genbank accession NC_008752.1).Genes with a major impact on copper tolerance are shown in blue; genes with a minor influence are shown in green.Genes that may not be directly associated with copper tolerance are shown in gray.The tolC (Aave_1811) gene shown in red exhibited a reduction in copper tolerance only when deleted in pslb65.The Cue system in E. coli is composed of CopA, CueO, and CueR, with copA and cueO being regulated by CueR.The Cus system is composed of CusA, CusB, CusC, and others.CopA primarily exports copper ions from the cytoplasm, while Cus system exports copper ions from the periplasm to maintain copper balance in the cell.CueO may oxidize toxic Cu + ions in the periplasm to the less toxic Cu 2+ ions.The mutant strain MIC results in A. citrulli suggest a similar copper-tolerance mechanism to that of E. coli.

Copper-Tolerant Phenotype of the Complementary Strains
The copA (Aave_0034), copZ (Aave_0033), cueR (Aave_0032), and cueO (Aave_1810) genes showed 100% sequence identity between pslb65 and pslbtw14.However, the cusAlike (Aave_0388) gene had seven single nucleotide polymorphisms (SNPs), the cusB-like (Aave_0389) gene had three SNPs, the cusC-like (Aave_0387) gene had six SNPs, and the tolC (Aave_1811) gene had two SNPs.Subsequent analysis of the amino acid sequences revealed that these SNPs in the cusA-like, cusB-like, cusC-like, and tolC homologous genes resulted in differences in the amino acid sequences between A. citrulli strain pslb65 and pslbtw14.To investigate whether these genetic differences contributed to the distinct copper sensitivities of the two groups, this study used pslb65 as a template to construct complementary strains of mutant strains, and measured copper MIC of these strains.
The copper-tolerance levels of the complementary strains Δ65-0032p65-0032 (Δ65-0032 complemented with Aave_0032 gene of pslb65, similar below), Δ65-0034p65-0034, Δ65-1811p65-1811, and Δ65-0389p65-0389 were restored to the wildtype level (Figure 5b).However, Δ65-0387p65-0387 and Δ65-0388p65-0388 did not recover their copper sensitivity.The copper tolerance of Δ65-0033p65-0033 was partially restored but did not reach the wildtype level.Surprisingly, the copper-tolerance level of Δ65-1810p65-1810 was even higher than that of the wildtype strain.Comparing the complementary strains Δ14-0033p65-0033, Δ14-1810p65-1810, Δ14-1811p65-1811, Δ14-0387p65-0387, Δ14-0388p65-0388, and Δ14-0389p65-0389 with the wildtype strain pslbtw14, their copper-tolerance levels were consistent (Figure 5a).Δ14-0034p65-0034 and Δ14-0032p65-0032 exhibited higher copper-tolerance levels compared to pslbtw14.(Figure 5a).A schematic representation of the organization of copper-tolerance genes in the Acidovorax citrulli genome AAC00-1 (Genbank accession NC_008752.1).Genes with a major impact on copper tolerance are shown in blue; genes with a minor influence are shown in green.Genes that may not be directly associated with copper tolerance are shown in gray.The tolC (Aave_1811) gene shown in red exhibited a reduction in copper tolerance only when deleted in pslb65.The Cue system in E. coli is composed of CopA, CueO, and CueR, with copA and cueO being regulated by CueR.The Cus system is composed of CusA, CusB, CusC, and others.CopA primarily exports copper ions from the cytoplasm, while Cus system exports copper ions from the periplasm to maintain copper balance in the cell.CueO may oxidize toxic Cu + ions in the periplasm to the less toxic Cu 2+ ions.The mutant strain MIC results in A. citrulli suggest a similar copper-tolerance mechanism to that of E. coli.

Copper-Tolerant Phenotype of the Complementary Strains
The copA (Aave_0034), copZ (Aave_0033), cueR (Aave_0032), and cueO (Aave_1810) genes showed 100% sequence identity between pslb65 and pslbtw14.However, the cusAlike (Aave_0388) gene had seven single nucleotide polymorphisms (SNPs), the cusB-like (Aave_0389) gene had three SNPs, the cusC-like (Aave_0387) gene had six SNPs, and the tolC (Aave_1811) gene had two SNPs.Subsequent analysis of the amino acid sequences revealed that these SNPs in the cusA-like, cusB-like, cusC-like, and tolC homologous genes resulted in differences in the amino acid sequences between A. citrulli strain pslb65 and pslbtw14.To investigate whether these genetic differences contributed to the distinct copper sensitivities of the two groups, this study used pslb65 as a template to construct complementary strains of mutant strains, and measured copper MIC of these strains.

Analysis of Copper-Tolerance-Related Genes Expression in Wildtype Strains
The expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 after copper treatment were measured, with a copper-free treatment as the control.Significant differences were observed in the expression levels of these genes under copper stress.At 0.8 and 2.0 mM Cu 2+ concentrations, cueR, copZ, copA, cusC-like, cusA-like, cusB-like, cueO, and tolC were significantly upregulated in pslbtw14 (Figure 6a).However, in pslb65, cusClike, cusA-like, cusB-like, and tolC were significantly downregulated at a low copper concentration (2.0 mM), but at a high copper concentration (4.0 mM), all genes except cusA-like and cusB-like were significantly upregulated (Figure 6b).
At a concentration of 2.0 mM Cu 2+ , the expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 differed, with the genes in pslbtw14 showing significant upregulation.Conversely, the expression levels of cusC-like, cusA-like, cusB-like, and tolC genes in pslb65 were significantly downregulated.and its complementary strains.The notation p65-0032 represents pBBR1MCS-2 carrying the Aave_0032 gene of pslb65, the complementary strain ∆14-0032p65-0032 represents the mutant strain ∆14-0032 carrying p65-0032, and the complementary strain ∆65-0032p65-0032 represents the mutant strain ∆65-0032 carrying p65-0032.Similar notations apply to the other strains.The strains were cultured in KMB liquid medium at 28 • C and 220 r/min for 12 h, and the bacterial suspensions were diluted to 3 × 10 4 CFU/mL.A 10 µL bacterial suspension was spotted on KMB solid medium mixed with varying concentrations of CuSO 4 •5H 2 O.The picture shows the colony growth after 6 days of culture.

Analysis of Copper-Tolerance-Related Genes Expression in Wildtype Strains
The expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 after copper treatment were measured, with a copper-free treatment as the control.Significant differences were observed in the expression levels of these genes under copper stress.At 0.8 and 2.0 mM Cu 2+ concentrations, cueR, copZ, copA, cusC-like, cusA-like, cusB-like, cueO, and tolC were significantly upregulated in pslbtw14 (Figure 6a).However, in pslb65, cusC-like, cusA-like, cusB-like, and tolC were significantly downregulated at a low copper concentration (2.0 mM), but at a high copper concentration (4.0 mM), all genes except cusA-like and cusB-like were significantly upregulated (Figure 6b).
At a concentration of 2.0 mM Cu 2+ , the expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 differed, with the genes in pslbtw14 showing significant upregulation.Conversely, the expression levels of cusC-like, cusA-like, cusB-like, and tolC genes in pslb65 were significantly downregulated.

Results of Analysis on the Expression of Copper-Tolerance-Related Genes in Mutant Strains
The copper-tolerance-related genes of strains pslb65, ∆65-0032, pslbtw14, and ∆14-0032 were analyzed using qRT-PCR after treatment with 1.6 mM copper-containing KMB liquid medium.Compared to pslbtw14, the expression levels of copA and cueO in ∆14-0032 were significantly downregulated, while the expression levels of copZ remained unchanged (Figure 7a).However, in the group I strain, the expression levels of copA, cueO and copZ in ∆65-0032 were significantly downregulated compared with pslb65 (Figure 7b), with a much greater decrease compared to ∆14-0032.

Results of Analysis on the Expression of Copper-tolerance-related Genes in Mutant Strains
The copper-tolerance-related genes of strains pslb65, Δ65-0032, pslbtw14, and Δ14-0032 were analyzed using qRT-PCR after treatment with 1.6 mM copper-containing KMB liquid medium.Compared to pslbtw14, the expression levels of copA and cueO in Δ14- 0032 were significantly downregulated, while the expression levels of copZ remained unchanged (Figure 7a).However, in the group I strain, the expression levels of copA, cueO and copZ in Δ65-0032 were significantly downregulated compared with pslb65 (Figure 7b), with a much greater decrease compared to Δ14-0032.

Results of Analysis on the Expression of Copper-tolerance-related Genes in Mutant Strains
The copper-tolerance-related genes of strains pslb65, Δ65-0032, pslbtw14, and Δ14-0032 were analyzed using qRT-PCR after treatment with 1.6 mM copper-containing KMB liquid medium.Compared to pslbtw14, the expression levels of copA and cueO in Δ14- 0032 were significantly downregulated, while the expression levels of copZ remained unchanged (Figure 7a).However, in the group I strain, the expression levels of copA, cueO and copZ in Δ65-0032 were significantly downregulated compared with pslb65 (Figure 7b), with a much greater decrease compared to Δ14-0032.

Discussion
In this study, we investigated the copper tolerance of different strains of A. citrulli and identified various copper-tolerance genes.The results showed that group I strains exhibited higher copper tolerance compared to group II strains, as evidenced by their higher copper MIC values.This observation was further supported by the growth curves, which showed that group I strains had better growth compared to group II strains.Based on these results, pslb65 from group I and pslbtw14 from group II were selected as representative strains for subsequent experiments.
To further understand the role of putative copper-tolerance genes, 24 deletion mutants were constructed in pslb65 and pslbtw14.Our results showed that the deletion of certain genes (detailed below) greatly decreased copper tolerance.In contrast, the deletion of cusA (Aave_0038), zneB (Aave_0039), cusB (Aave_4663), and gntR (Aave_2798) genes in pslb65 and pslbtw14 did not impact their copper-tolerance levels.Overall, pslb65 exhibited greater tolerance to copper stress compared to pslbtw14, and the deletion mutants of the same gene in pslb65 generally showed lower copper sensitivity compared to pslbtw14.
The deletion of the copA (Aave_0034) copZ (Aave_0033) genes resulted in a substantial reduction in copper tolerance in both group I and II strains of A. citrulli.Deleting cueR (Aave_0032) or cueO (Aave_1810) genes in pslbtw14 had a minor impact on copper tolerance, while deleting these genes in pslb65 reduced copper resistance by more than half.Therefore, we conclude that copA (Aave_0034), copZ (Aave_0033), cueR (Aave_0032), and cueO (Aave_1810) genes are major contributors to copper tolerance in A. citrulli.In particular, the deletion of the copA (Aave_0034) gene in the two representative strains led to almost complete loss of copper tolerance in the mutant strains, highlighting its core role in copper tolerance of A. citrulli.This finding aligns with the role of CopA in E. coli, where it functions as a central component of copper homeostasis under aerobic and anaerobic conditions and is responsible for cytoplasmic copper homeostasis [13].Thus, we speculate that the development of copA inhibitors could offer improved control of BFB.
The cusC-like (Aave_0387), cusA-like (Aave_0388), and cusB-like (Aave_0389) genes had SNPs between group I strain pslb65 and group II strain pslbtw14.Deletion of these genes slightly decreased copper tolerance in strains pslb65 and pslbtw14.This suggests that these genes have a minor impact on copper tolerance of A. citrulli.
The tolC (Aave_1811) gene had 2 SNPs between group I strain pslb65 and group II strain pslbtw14, and their proteins have one amino acid difference.Interestingly, deleting tolC (Aave_1811) in pslbtw14 did not affect copper tolerance, whereas its deletion in pslb65 noticeably reduced copper tolerance from 6.4 mM to 5.6 mM.This suggests that this gene may only be active in pslb65, and that group I strain pslb65 possesses more coppertolerance mechanisms than group II strain pslbtw14.In E. coli, the TolC protein is also involved in other efflux systems such as EmrAB-TolC or MacAB-TolC of the major facilitator superfamily (MFS) and ATP-binding cassette (ABC) superfamilies, respectively [31,32].Therefore, it is possible that genes related to TolC efflux pumps may play a role in copper tolerance in A. citrulli.After treatment with 4.0 mM copper, the expression of tolC gene in pslb65 was significantly upregulated, suggesting a crucial role in group I strain pslb65.This effect was more pronounced compared to cusC-like, cusA-like, and cusB-like genes.This indicates that pslb65 may have a more efficient mechanism for exporting periplasmic toxic ions to the extracellular system, potentially involving other efflux pumps that work in conjunction with TolC to form efflux pumps.
The qRT-PCR results showed that at a copper concentration of 2.0 mM, the expression levels of copper-tolerance-related genes in pslbtw14 were significantly upregulated, while the expression levels of cusC-like, cusA-like, cusB-like, and tolC in pslb65 were significantly downregulated.The results indicated distinct differences in the expression of copperrelated genes in pslb65 and pslbtw14 under copper stress.In addition, the expression levels of copper-tolerance-related genes in pslbtw14 treated with different copper concentrations indicated that at low and high copper concentrations, pslbtw14 seemed to rely on CueO to reduce the copper toxicity.However, the expression levels of copper-tolerance-related genes in pslb65 under different copper concentrations indicated that, at low copper concentrations, pslb65 relied on CueO to reduce the toxicity of copper, while at high copper concentrations, it mainly depended on the export of copper ions for detoxification.Overall, the varied expression of copper-tolerance-related genes in pslb65 and pslbtw14 suggests different strategies for coping with copper stress, potentially contributing to their differing copper sensitivities.
The deletion of copper efflux regulator cueR gene significantly reduced the expression levels of copA, cueO, and copZ genes in ∆65-0032 under copper stress, while the expression levels of copZ genes in ∆14-0032 remained relatively stable.The downregulation of copper-tolerance-related genes was more pronounced in ∆65-0032 compared to ∆14-0032, indicating a potential divergence in the role of the cueR gene between these two strains.Some complementary strains restored copper tolerance to the wildtype level, while others showed increased or decreased tolerance compared to the wildtype.Previous research on E. coli has shown that the copper chaperone protein CopZ and the copper efflux protein CopA function together to remove excess copper ions from the cytoplasm [33].In A. citrulli, we hypothesized that CopZ binds to copper ions and transfers them to CopA for export.In the complementary strain ∆65-0033p65-0033, overexpression of the copZ (Aave_0033) gene resulted in an accumulation of copper chaperone CopZ carrying copper ions.However, the removal rate of copper ions from the cytoplasm was limited by CopA, resulting in a slower elimination of copper ions.This may explain why this complementary strain only partially restored its copper-tolerance level.On the other hand, the coppertolerance level of the complementary strain ∆65-0033p65-0033 restored to the level of wildtype strain pslbtw14, suggesting that overexpression of the copZ gene can only enable the strain to achieve a maximum copper-tolerance level of 2.8 mM.
Increasing the copy number of the copA (Aave_0034) gene has been shown to enhance the copper tolerance of strains in Acidithiobacillus thiooxidans [34].In this study, the copper tolerance of ∆14-0034p65-0034 was approximately double that of pslbtw14, likely due to the overexpression of the copA gene carried by the complementary plasmid p65-0034.However, the copper-tolerance level of ∆65-0034p65-0034 was similar to that of pslb65, suggesting that overexpression of the copA gene can only increase copper tolerance up to a maximum level of 6.4 mM.
Previous studies have suggested that CueR can interact with P-type ATPase (CopA) and multicopper oxidase (CueO) in A. citrulli [35].The presence of a typical palindrome motif in the promoter region of copA, which binds to CueR, suggests that CueR may positively regulate copA, resulting in an increase in copA gene copies [35].This increase enhances the active pumping of intracellular copper ions and improves copper tolerance.The higher copper-tolerance level observed in the complementary strain ∆14-0032p65-0032 compared to pslbtw14 may be due to the overexpression of the cueR (Aave_0032) gene.The copper-tolerance level of ∆65-0032p65-0032 was consistent with that of pslb65, suggesting that overexpression of the cueR (Aave_0032) gene can only achieve a maximum copper-tolerance level of 6.4 mM.
In addition, the complementary strain ∆65-1810p65-1810 exhibited enhanced copper tolerance compared to pslb65, which can be attributed to the overexpression of the cueO (Aave_1810) gene.CueO oxidizes highly toxic Cu + ions in the cytoplasm to less toxic Cu 2+ ions, reducing overall copper toxicity.Additionally, CueO also oxidizes certain chelating peptides containing reduced metal ions in the periplasmic space, further reducing the rate of Cu 2+ reduction [13].Consequently, the copper-tolerance level of ∆65-1810p65-1810 surpassed that of the wildtype strain.However, the copper-tolerance level of ∆14-1810p65-1810 remained similar to pslbtw14, suggesting that the impact of cueO (Aave_1810) on pslb65 is greater than on pslbtw14.
Overall, our study provides valuable insights into the copper tolerance of different strains of A. citrulli and identifies key genes associated with copper tolerance.These findings suggest that the differential copper sensitivity between the two groups of A. citrulli strains may be influenced by several factors, including additional mechanisms for copper ion removal, variations in the expression levels of copper-tolerance-related genes, and the complex genomic basis for copper tolerance.Further studies are needed to fully understand the mechanisms underlying copper tolerance in A. citrulli and its implications for pathogenicity.

Figure 1 .
Figure 1.Colony growth of Acidovorax citrulli strains pslb65, pslb9, pslbtw14, pslbtw38, pslbtw32, and Aac5 on KMB solid medium with varying copper concentrations after 6 days of incubation.Strains pslb65 and pslb9 belong to group I, while pslbtw14, pslbtw38, pslbtw32, and Aac5 belong to group II.The strains were cultured in KMB liquid medium at 28 °C and 220 r/min for 12 h.The bacterial suspensions were diluted to 3 × 10 4 CFU/mL and 10 µL of the bacterial suspension was spotted on KMB solid medium containing varying concentrations of CuSO4•5H2O.The picture shows the colony growth after 6 days of incubation.

Figure 2 .
Figure 2. Growth comparisons of A. citrulli wildtype strains under different Cu 2+ concentrations.The growth curves of the tested strains were measured at 4 mM Cu 2+ (a) and 6.4 mM Cu 2+ (b).Strains pslb65 and pslb9 belong to group I, while pslbtw14, pslbtw38, pslbtw32, and Aac5 belong to group II.Bacterial suspensions with an OD600 of 0.3 (equivalent to 3 × 10 8 CFU/mL) were mixed with KMB liquid medium containing copper in a ratio of 1:100.The mixture was incubated at 28 °C with shaking in a Bioscreen C. The OD600 values were measured every 2 h.

Figure 1 .
Figure 1.Colony growth of Acidovorax citrulli strains pslb65, pslb9, pslbtw14, pslbtw38, pslbtw32, and Aac5 on KMB solid medium with varying copper concentrations after 6 days of incubation.Strains pslb65 and pslb9 belong to group I, while pslbtw14, pslbtw38, pslbtw32, and Aac5 belong to group II.The strains were cultured in KMB liquid medium at 28 • C and 220 r/min for 12 h.The bacterial suspensions were diluted to 3 × 10 4 CFU/mL and 10 µL of the bacterial suspension was spotted on KMB solid medium containing varying concentrations of CuSO 4 •5H 2 O.The picture shows the colony growth after 6 days of incubation.

Figure 1 .
Figure 1.Colony growth of Acidovorax citrulli strains pslb65, pslb9, pslbtw14, pslbtw38, pslbtw32, and Aac5 on KMB solid medium with varying copper concentrations after 6 days of incubation.Strains pslb65 and pslb9 belong to group I, while pslbtw14, pslbtw38, pslbtw32, and Aac5 belong to group II.The strains were cultured in KMB liquid medium at 28 °C and 220 r/min for 12 h.The bacterial suspensions were diluted to 3 × 10 4 CFU/mL and 10 µL of the bacterial suspension was spotted on KMB solid medium containing varying concentrations of CuSO4•5H2O.The picture shows the colony growth after 6 days of incubation.

Figure 2 .
Figure 2. Growth comparisons of A. citrulli wildtype strains under different Cu 2+ concentrations.The growth curves of the tested strains were measured at 4 mM Cu 2+ (a) and 6.4 mM Cu 2+ (b).Strains pslb65 and pslb9 belong to group I, while pslbtw14, pslbtw38, pslbtw32, and Aac5 belong to group II.Bacterial suspensions with an OD600 of 0.3 (equivalent to 3 × 10 8 CFU/mL) were mixed with KMB liquid medium containing copper in a ratio of 1:100.The mixture was incubated at 28 °C with shaking in a Bioscreen C. The OD600 values were measured every 2 h.

Figure 2 .
Figure 2. Growth comparisons of A. citrulli wildtype strains under different Cu 2+ concentrations.The growth curves of the tested strains were measured at 4 mM Cu 2+ (a) and 6.4 mM Cu 2+ (b).Strains pslb65 and pslb9 belong to group I, while pslbtw14, pslbtw38, pslbtw32, and Aac5 belong to group II.Bacterial suspensions with an OD 600 of 0.3 (equivalent to 3 × 10 8 CFU/mL) were mixed with KMB liquid medium containing copper in a ratio of 1:100.The mixture was incubated at 28 • C with shaking in a Bioscreen C. The OD 600 values were measured every 2 h.

Figure 3 .
Figure 3. Colony growth of the tested strains on KMB solid medium with varying concentrations of copper.(a) Colony growth of representative Acidovorax citrulli group II wildtype and mutant strains.(b) Colony growth of representative A. citrulli group I wildtype and mutant strains.(c,d) Colony growth of mutant strains at lower copper concentrations.The strains were cultured in KMB liquid medium at 28 • C and 220 r/min for 12 h.The bacterial suspensions were diluted to 3 × 10 4 CFU/mL.Subsequently, 10 µL of the bacterial suspension was spotted on KMB solid medium amended with varying concentrations of CuSO 4 •5H 2 O.The picture shows the colony growth after 6 days of incubation.∆65-0032 represents the Aave_0032-gene-deletion mutant strain from pslb65.∆14-0032 represents the Aave_0032-gene-deletion mutant strain from pslbtw14.Similar notations apply to the other strains.

Figure 4 .
Figure 4.A schematic representation of the organization of copper-tolerance genes in the Acidovorax citrulli genome AAC00-1 (Genbank accession NC_008752.1).Genes with a major impact on copper tolerance are shown in blue; genes with a minor influence are shown in green.Genes that may not be directly associated with copper tolerance are shown in gray.The tolC (Aave_1811) gene shown in red exhibited a reduction in copper tolerance only when deleted in pslb65.The Cue system in E. coli is composed of CopA, CueO, and CueR, with copA and cueO being regulated by CueR.The Cus system is composed of CusA, CusB, CusC, and others.CopA primarily exports copper ions from the cytoplasm, while Cus system exports copper ions from the periplasm to maintain copper balance in the cell.CueO may oxidize toxic Cu + ions in the periplasm to the less toxic Cu 2+ ions.The mutant strain MIC results in A. citrulli suggest a similar copper-tolerance mechanism to that of E. coli.

Figure 4 .
Figure 4.A schematic representation of the organization of copper-tolerance genes in the Acidovorax citrulli genome AAC00-1 (Genbank accession NC_008752.1).Genes with a major impact on copper tolerance are shown in blue; genes with a minor influence are shown in green.Genes that may not be directly associated with copper tolerance are shown in gray.The tolC (Aave_1811) gene shown in red exhibited a reduction in copper tolerance only when deleted in pslb65.The Cue system in E. coli is composed of CopA, CueO, and CueR, with copA and cueO being regulated by CueR.The Cus system is composed of CusA, CusB, CusC, and others.CopA primarily exports copper ions from the cytoplasm, while Cus system exports copper ions from the periplasm to maintain copper balance in the cell.CueO may oxidize toxic Cu + ions in the periplasm to the less toxic Cu 2+ ions.The mutant strain MIC results in A. citrulli suggest a similar copper-tolerance mechanism to that of E. coli.

Figure 5 .
Figure 5. Colony growth of tested strains on KMB solid medium with varying concentrations of copper after 6 days of incubation.(a) Colony growth of representative Acidovorax citrulli group II strain pslbtw14 and its complementary strains.(b) Colony growth of representative A. citrulli group I strain pslb65 and its complementary strains.The notation p65-0032 represents pBBR1MCS-2carrying the Aave_0032 gene of pslb65, the complementary strain Δ14-0032p65-0032 represents the mutant strain Δ14-0032 carrying p65-0032, and the complementary strain Δ65-0032p65-0032 represents the mutant strain Δ65-0032 carrying p65-0032.Similar notations apply to the other strains.The strains were cultured in KMB liquid medium at 28 °C and 220 r/min for 12 h, and the bacterial suspensions were diluted to 3 × 10 4 CFU/mL.A 10 µL bacterial suspension was spotted on KMB solid medium mixed with varying concentrations of CuSO4•5H2O.The picture shows the colony growth after 6 days of culture.

Figure 5 .
Figure 5. Colony growth of tested strains on KMB solid medium with varying concentrations of copper after 6 days of incubation.(a) Colony growth of representative Acidovorax citrulli group II strain pslbtw14 and its complementary strains.(b) Colony growth of representative A. citrulli group I strain pslb65 and its complementary strains.The notation p65-0032 represents pBBR1MCS-2 carrying the Aave_0032 gene of pslb65, the complementary strain ∆14-0032p65-0032 represents the mutant strain ∆14-0032 carrying p65-0032, and the complementary strain ∆65-0032p65-0032 represents the mutant strain ∆65-0032 carrying p65-0032.Similar notations apply to the other strains.The strains were cultured in KMB liquid medium at 28 • C and 220 r/min for 12 h, and the bacterial suspensions were diluted to 3 × 10 4 CFU/mL.A 10 µL bacterial suspension was spotted on KMB solid medium mixed with varying concentrations of CuSO 4 •5H 2 O.The picture shows the colony growth after 6 days of culture.

Figure 6 .
Figure 6.The expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 when exposed to different copper concentrations.The control group (CK) received no copper treatment.The rpoB gene was used as an internal reference gene.Each treatment had three replicates, and the experiment was conducted three times.Error bars represent standard errors of the means, and asterisks denote significant differences (p < 0.05, two-way ANOVA test, Dunnett's multiple comparisons test) (a,b).

Figure 7 .
Figure 7.The expression levels of copper-tolerance-related genes of Δ14-0032 and Δ65-0032 following treatment with 1.6 mM copper.The rpoB gene was used as an internal reference gene.Each treatment had three replicates, and the experiment was conducted three times.Error bars represent standard errors of the means, and asterisks denote significant differences (p < 0.05, two-way ANOVA test, Dunnett's multiple comparisons test).(a) The expression levels of copper-tolerance-related genes in

Figure 6 .
Figure 6.The expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 when exposed to different copper concentrations.The control group (CK) received no copper treatment.The rpoB gene was used as an internal reference gene.Each treatment had three replicates, and the experiment was conducted three times.Error bars represent standard errors of the means, and asterisks denote significant differences (p < 0.05, two-way ANOVA test, Dunnett's multiple comparisons test) (a,b).

Figure 6 .
Figure 6.The expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 when exposed to different copper concentrations.The control group (CK) received no copper treatment.The rpoB gene was used as an internal reference gene.Each treatment had three replicates, and the experiment was conducted three times.Error bars represent standard errors of the means, and asterisks denote significant differences (p < 0.05, two-way ANOVA test, Dunnett's multiple comparisons test) (a,b).

Figure 7 . 7 .
Figure 7.The expression levels of copper-tolerance-related genes of Δ14-0032 and Δ65-0032 following treatment with 1.6 mM copper.The rpoB gene was used as an internal reference gene.Each treatment had three replicates, and the experiment was conducted three times.Error bars represent standard errors of the means, and asterisks denote significant differences (p < 0.05, two-way ANOVA test, Dunnett's multiple comparisons test).(a) The expression levels of copper-tolerance-related genes in 7. The expression levels of copper-tolerance-related genes of ∆14-0032 and ∆65-0032 following treatment with 1.6 mM copper.The rpoB gene was used as an internal reference gene.Each treatment had three replicates, and the experiment was conducted three times.Error bars represent standard errors of the means, and asterisks denote significant differences (p < 0.05, two-way ANOVA test, Dunnett's multiple comparisons test).(a) The expression levels of copper-tolerance-related genes in pslbtw14 and ∆14-0032 when exposed to 1.6 mM copper.(b) The expression levels of coppertolerance-related genes in pslb65 and ∆65-0032 when exposed to 1.6 mM copper.

Table 1 .
Primers used for quantitative real-time PCR.