Abstract Flagella are essential for biofilm formation, adhesion, virulence, and motility. In this study, the deletion of argR resulted in defects in flagellar synthesis and reduced motility, nevertheless, the underlying mechanism by which ArgR regulated bacterial motility remained unclear. ChIP-Seq and RNA-Seq analysis revealed that ArgR regulated the expression of flagellar genes, concluding two-component system flrBC and multitudinous flagellar structure genes. Specifically, ArgR bound to the ARG box in the flrBC promoter, positively regulating flrBC expression, which in turn promoted flagellar synthesis and enhanced motility. Additionally, in the absence of arginine, ArgR inhibited the expression of diguanylate cyclase, leading to reduced c-di-GMP levels, thereby alleviating its inhibitory effect on motility. Thus, ArgR coordinated two distinct pathways to regulate flagellar assembly and motility, ultimately affecting adhesion, virulence, and biofilm formation. In summary, this study elucidates the molecular mechanism by which ArgR regulates motility, highlighting its crucial role in bacterial virulence and offering new insights for the prevention and control of pathogenic bacteria. Subject terms: Pathogens, Transcriptional regulatory elements, Biofilms __________________________________________________________________ Combined RNA-seq and CHIP-seq analysis revealed the regulatory mechanism of ArgR in bacterial motility of Aeromonas veronii. Introduction Flagella are the primary organelles of bacterial motility and are essential for bacterial chemotaxis, stress response, and adhesion. The flagellum is composed of a basal body, rod, extracellular hook and filament^[36]1. Flagellar synthesis consumes substantial intracellular protein resources, necessitating the tight and precise regulation of its production. V. cholerae flagellar genes are expressed within a four-tiered transcriptional hierarchy^[37]2, with the primary regulatory factor flrA at the top level (Class I genes). FlrA activates the transcription of Class II genes, including the two-component system flrBC, the σ^28 factor, and several basal body-associated proteins. The flrBC activates the transcription of Class III genes, which include the remaining basal body components, the hook, and the P/L rings. The σ^28 controls the transcription of Class IV genes, which encode the remaining flagellar proteins, motility subunits, and certain chemotaxis proteins^[38]3. The designated genes flrBC and fleSR exhibit high homology and perform the same function in the flagellar synthesis network^[39]4. The flrA/flrBC regulatory pathway is essential for motility^[40]5–[41]7. Aeromonas veronii is a rod-shaped, Gram-negative bacterium with strong adaptability and high pathogenicity to humans, fish, and terrestrial mammals^[42]8–[43]10. A. veronii is ubiquitously found in aquatic environments, where its flagella plays a crucial role in motility, facilitating both dispersion and transmission. This characteristic presents a significant threat to aquaculture and public health in China. Therefore, investigating the motility and pathogenic mechanisms of A. veronii is essential for developing strategies to prevent and control its spread. The genome of A.veronii C4 includes a four-tiered flagellar synthesis regulatory network for flagellar synthesis, mediated by FlrA, indicating its potential role in motility and virulence. ArgR (Arginine repressor) regulates arginine metabolism by acting as an arginine synthesis repressor^[44]11 and also serves as a global transcription factor that controls T3SS activation^[45]12, acid resistance^[46]13, anaerobic adaptation^[47]14, biofilm formation^[48]15, and other physiological processes. Additionally, ArgR is found to be associated with bacterial motility, though the molecular mechanism remains unknown^[49]16. Bacterial motility is influenced by flagellar synthesis and chemotaxis^[50]17,[51]18. Flagella serve as the primary organelles responsible for bacterial motility, and defects in flagellar synthesis or inhibition of rotation can lead to a loss of bacterial movement^[52]19,[53]20. Even with intact flagellar structures, bacterial motility is still controlled by chemotaxis^[54]21,[55]22. In A.veronii C4, deletion of argR resulted in defective flagella and reduced motility. We speculate that the diminished motility observed in the ΔargR strain is likely due to the absence of flagella; however, the mechanism by which argR influences flagellar synthesis remains unknown. Cyclic diguanylate (c-di-GMP) is a universal second messenger in bacteria, influencing various physiological processes including motility, biofilm formation, cell division, signal transduction, and differentiation^[56]23–[57]25. The c-di-GMP exerts its regulatory effects through various mechanisms, including binding to transcription factors and influencing transcription initiation^[58]26. Recently, c-di-GMP has been reported to inhibit the transcription of flrBC by binding to FlrA, thereby affecting the transcription of genes related to the hook, P-L ring, basal body, and hook-associated proteins (HAPs)^[59]27. In Pseudomonas aeruginosa, c-di-GMP inhibits flagellar synthesis by regulating FleQ which is homologous to FlrA^[60]4. Additionally, c-di-GMP directly binds to the PliZ domain-containing protein YcgR to inhibit bacterial motility^[61]28. ClpXP protease degrades σ^S in a c-di-GMP-dependent manner, affecting the expression of flagellar genes^[62]29. In summary, c-di-GMP is closely related to flagellar synthesis and function, with high levels of c-di-GMP inhibiting bacterial flagellar synthesis. In A. veronii, the molecular mechanisms by which ArgR influenced bacterial motility and virulence were explored using ChIP-seq and RNA-seq analyses. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses of the ChIP-seq and RNA-seq data showed enrichment in the flagellar assembly pathway, indicating that ArgR affects the expression of flagellar genes through direct or indirect mechanisms. In the absence of arginine, ArgR was found to directly bind to the flrBC promoter, positively regulating its expression and contributing to flagellar synthesis. Additionally, ArgR modulated diguanylate cyclase, reducing intracellular c-di-GMP levels and mitigating its inhibitory effect on flagellar synthesis. Collectively, this ArgR-regulated pathway influenced bacterial virulence through biofilm formation, cytotoxicity, and adhesion. Results ChIP-Seq and RNA-Seq revealed that ArgR affects the expression of flagellar genes The surface motility of Aeromonas veronii was reduced following the deletion of argR (ΔargR), but was restored to WT levels upon complementation with argR (ΔargR/P-argR) (Fig. [63]1b), similar to the phenotype observed in Pseudomonas putida with argR deletion when cultured in an arginine-free medium^[64]16. This suggests that the impact of argR on motility may also exist in A. veronii. Transmission electron microscopy revealed that the deletion of argR resulted in shorter flagella with reduced functional activity. In the absence of arginine, complementation of argR in ΔargR strain restored flagella to the WT state (Fig. [65]1a), suggesting that argR influences bacterial motility by affecting flagella synthesis. To investigate whether and how ArgR affects the transcription of flagellar genes, RNA-seq and ChIP-seq analyses were performed. RNA-seq indicated 607 genes differentially expressed following the deletion of argR, with 318 genes upregulated and 289 genes downregulated (Fig. S[66]1). The most enriched pathway was flagellar assembly (Fig. S[67]2), with 23 flagellar genes showing changes in transcription levels (Table [68]S1). Additionally, gene ontology (GO) functional annotation analysis revealed enrichment in cell motility, involving 27 genes (Fig. [69]1c). Transcriptional analysis showed that flagellar genes were significantly downregulated in ΔargR strain (Fig. [70]1d). RT-qPCR (Real-time quantitative PCR) validation results were consistent with the transcriptome sequencing data (Fig. [71]1e), indicating that argR uniformly affected the expression of flagellar genes. Fig. 1. Influence of ArgR on flagellar gene expression and surface motility in A. veronii. [72]Fig. 1 [73]Open in a new tab a Transmission electron microscopy images showing the morphology of flagella in wild-type strain (WT), argR mutant strain (ΔargR) and argR complemented strain (ΔargR/p-argR). b Motility distribution and diameter measurement of different strains on 0.3% LB semi-solid plates, no exogenous L-arginine was added. Data are expressed as mean ± SD (n = 3). Cultures grown overnight were diluted to an OD[600] of 1, 2 μL was spotted onto the center of the plate, the assay was repeated three times, with spotting on three plates in each case, and one representative image is shown for each strain. c Gene ontology (GO) analysis of differentially expressed genes between WT and ΔargR. The number in each term represents the quantity of differentially expressed genes. d Volcano plot illustrating the differential expression of flagellar synthesis-related genes identified in RNA-seq. Red dots represent significantly different flagellar genes, while black indicates no difference. e RT-qPCR analysis comparing the expression levels of flagellar genes between WT and ΔargR. The strains were cultured in M9 medium with glucose as the carbon source for 18 hours, no exogenous L-arginine was added. The validated genes all show significant differences (P < 0.05), determined by an unpaired t-test. f Scatter plot of differentially expressed genes from the top 20 KEGG pathways identified in ChIP-seq analysis. g Venn diagram showing the overlap of flagellar synthesis-related genes identified by RNA-seq and ChIP-seq. h Distribution of altered flagellar genes in the flagellar cascade regulation in the ΔargR strain. The genes enclosed in red boxes are all significantly downregulated in the RNA-seq analysis. The genes in the light blue box are Class II flagellar regulatory genes, while the genes in the green box are Class III flagellar structure genes. The red boxes indicate genes that are downregulated in the ΔargR strain, while the gray boxes indicate genes with unchanged expression in the ΔargR strain. The red “ד denotes the deletion of argR. Data are based on RNA-seq analysis. ChIP-Seq analysis was applied to investigate the regulatory mechanism of ArgR on flagellar genes. The ChIP-Seq data showed that ArgR primarily bound to the promoter-TSS region, accounting for 90.72% of the binding sites (Fig. S[74]3). A total of 819 potential ArgR binding targets were identified through ChIP-seq. KEGG functional enrichment analysis indicated that ArgR predominantly affected pathways such as flagellar assembly, bacterial chemotaxis, amino acid metabolism, two-component systems, and carbon metabolism. Among these, the flagellar assembly pathway exhibited the highest enrichment factor (Fig. [75]1f), with 16 flagellar genes being significantly enriched (Table [76]S2), in agreement with the RNA-seq data. Both ChIP-Seq and RNA-Seq analyses revealed four common flagellar synthesis genes affected by ArgR (Fig. [77]1g). The flagellar synthesis-related genes influenced by ArgR included the class II flagellar regulatory genes such as flrBC and the class III flagellar structure genes encoding the rod, P/L ring, hook, and rotor (Fig. [78]1h). These results indicate that ArgR directly or indirectly regulates the expression of flagellar genes and impacts motility, with flrBC likely being a key target. ArgR positively regulates flrBC RNA-seq revealed numerous differentially expressed flagellar structural genes, primarily including the gene clusters flgABCDEFGHL and flgM (Fig. [79]1h). These genes were not detected in ChIP-seq, indicating that they were not directly regulated by ArgR. The major flagellar regulatory factor flrA remained unchanged (Fig. [80]1h). Gene clusters flgABCDEFGHL and flgM, which are Class III flagellar genes, are typically regulated by the adjacent flrBC two-component system^[81]7. In A. veronii, flrB and flrC were co-expressed, and no promoter element was predicted upstream of flrC. Primers were designed to amplify the flrB-flrC intergenic region using cDNA as a template (Fig. S[82]4), indicating that flrB and flrC were co-transcribed from the upstream promoter of flrB. To investigate whether ArgR specifically bonds to the flrBC promoter, EMSA (Electrophoretic Mobility Shift Assay) was performed. In vitro binding of ArgR to the labeled probe PflrBC formed a retardation band upon the addition of ArgR protein (Fig. [83]2a). MST (Microscale Thermophoresis) also confirmed the binding of ArgR to the flrBC promoter, with a dissociation constant (Kd) of 392 ± 21.25 nM (Fig. [84]2b). The bacterial one-hybrid assay further validated the binding of ArgR to the flrBC promoter (Fig. [85]2c). Both RNA-seq and RT-qPCR demonstrated that ArgR positively regulated flrBC in vivo (Fig. [86]1e). The fusion reporter vector pDH-PflrBC-eGFP, co-transformed with the ArgR expression vector pTRG-ArgR, showed a significant increase in fluorescence, indicating that ArgR also positively regulated flrBC in vitro (Fig. [87]2d). The flrBC promoter was predicted to contain a conserved ARG box (-TGMATWWWWATNSN-) (Fig. [88]2e), where point mutations disrupted binding with ArgR, as confirmed by MST and EMSA assays (Fig. [89]2e, f). Similarly, the corresponding ARG box was conserved in the flrBC promoter of Vibrio cholerae and the fleSR promoter of Pseudomonas aeruginosa (Fig. S[90]5). These results indicate that ArgR directly binds to PflrBC and exerts positive regulatory effects, suggesting that this regulation may be conserved among bacteria. Fig. 2. Direct regulation of flrBC by ArgR and its influence on motility. [91]Fig. 2 [92]Open in a new tab a EMSA demonstrating the interaction between ArgR and the flrBC promoter. Lane 1 represents the 106 bp FAM-labeled flrBC promoter probe (PflrBC), lanes 2 to 5 contain a fixed amount of PflrBC and varying amounts of ArgR. The uncut original gel image can be found in Fig. S[93]7a. b MST (MicroScale Thermophoresis) analysis verifying the interaction between ArgR and the flrBC promoter. The experiment was repeated three times, and the binding affinity (Kd) was calculated. c Bacterial one-hybrid assay validating the interaction between ArgR and the flrBC promoter. d Fluorescence detection from co-transformation of the ArgR expression vector with the PflrBC-eGFP fluorescent reporter vector, the strain was cultured in M9 medium with glucose as the carbon source, and samples were taken at different time points to measure fluorescence values. Data are presented as mean ± SD (n = 3). The symbols* indicate P < 0.05, ** indicates P < 0.01, determined by one unpaired t-test. e Analysis of the flrBC promoter. The flrBC promoter was predicted by BPROM, with the −10 and −35 regions underlined, the ArgR binding site (ARG box) marked in red, and the ARG conserved binding motif highlighted in yellow. The uncut original gel image can be found in Fig. S[94]7b. f, g MST and EMSA verifying the conserved binding site of ArgR on PflrBC. The cold probe refers to the flrBC promoter without FAM labeling. h Transmission electron microscopy images illustrating the morphology of bacterial flagella, the scale bar represents 1 μm. i Motility assay results showing the effects of flrBC. Motility distribution and diameter measurement of different strains on 0.3% semi-solid plates, no exogenous L-arginine was added, data are presented as mean ± SD (n = 3), ** indicates P < 0.01, determined by an unpaired t-test. To verify the function of flrBC in flagellar synthesis, a double knockout strain of flrBC was constructed and subjected to motility test. The results showed that knockout of flrBC led to flagellar defects (Fig. [95]2g) and reduced motility (Fig. [96]2h, i), indicating that flrBC influences motility by affecting flagellar synthesis. This observation is consistent with the phenotype observed in Vibrio cholerae flrBC knockout strains^[97]30. The ΔargR and ΔflrBC strains exhibited similar phenotypes, which, combined with the positive regulatory relationship between argR and flrBC, suggests that argR positively regulates flrBC to affect on the motility of A. veronii C4. ArgR negatively regulates intracellular c-di-GMP levels The second messenger c-di-GMP is essential for controlling bacterial behavior and physiology. C-di-GMP has been reported to affect bacterial motility. The c-di-GMP directly binds to FleQ, a homolog of the regulator of flagellar synthesis FlrA, thereby inhibiting flagellar synthesis^[98]31. Additionally, c-di-GMP reduces flagellar rotation speed, thereby decreasing bacterial motility^[99]32. ChIP-seq analysis showed that ArgR directly bound to the promoter regions of four diguanylate cyclases (Table [100]1), with the highest enrichment observed for A.veroniiC4GL003492 ([101]GL003492) and A.veroniiC4GL000671 ([102]GL000671) (Fig. [103]3b). Diguanylate cyclase contains typical GGDEF domain. Structure predictions for the four diguanylate cyclases revealed GGDEF domains. [104]GL003492 contains multiple transmembrane domains, potentially involved in sensing external environmental signals, while [105]GL000671 lacks transmembrane domains and contains two GGDEF domains, suggesting its physiological function may be related to intracellular processes (Fig. [106]3a). MST further confirmed the binding of ArgR to these promoters (Fig. [107]3c). RT-qPCR demonstrated that the expression of [108]GL000671 was upregulated following argR deletion, indicating that ArgR directly binds to the [109]GL000671 promoter to repress its expression (Fig. [110]3d). To verify whether ArgR affected intracellular c-di-GMP levels, the c-di-GMP bioreporter, plasmid pCdrA::gfp^C was introduced into WT and ΔargR strains for fluorescence measurement. A significant increase in fluorescence was observed in ΔargR strain (Fig. [111]3e). LC-MS analysis of intracellular c-di-GMP levels corroborated these findings (Figs. [112]3f, S[113]6). These results suggest that ArgR inhibits the expression of diguanylate cyclase, thereby reducing intracellular c-di-GMP levels. Table 1. Four diguanylate cyclases identified from ChIP-seq analysis Gene ID ChIP-seq fold enrichment Description Position A.veroniiC4GL003492 9 Diguanylate cyclase promoter-TSS A.veroniiC4GL000671 6.25 Diuanylate cyclase promoter-TSS A.veroniiC4GL000724 1.4 Diuanylate cyclase promoter-TSS A.veroniiC4GL003433 1.2 Diuanylate cyclase promoter-TSS [114]Open in a new tab Genes annotated as guanylate cyclase were identified from the ChIP-seq data. The fold enrichment reflects the magnitude of ArgR binding, while the position indicates the specific ArgR binding site. Fig. 3. Regulation of diguanylate cyclase by ArgR and its effect on c-di-GMP levels. [115]Fig. 3 [116]Open in a new tab a Domains of diguanylate cyclase enriched from ChIP-seq were predicted by CD-search. The GGDEF domain indicates diguanylate cyclase activity. b ChIP-seq confirms the binding of ArgR to the promoter of the diguanylate cyclase gene. c MST confirm the binding of ArgR to the promoter of the diguanylate cyclase gene. d RT-qPCR analysis demonstrating the regulation of diguanylate cyclase expression by ArgR. Bacteria were inoculated at an OD of 0.02 and cultured overnight in M9 medium with the glucose as a carbon source, no exogenous L-arginine was added. e Measurement of intracellular c-di-GMP levels using a reporter plasmid. In the absence of arginine, strains (WT and ΔargR) carrying the c-di-GMP bioreporter plasmid (pCdrA::gfp^C) were cultured in M9 medium for 18 h, and then 1 OD of the culture was taken to measure fluorescence intensity (P < 0.05 by Student’s t-test). f, g Quantification of intracellular c-di-GMP levels by LC-MS. In the absence of arginine, experimental strains cultured in M9 for 20 hours, 50 mL of the culture was measured for OD and used for product extraction. Data are presented as mean ± SD (n = 3). The symbol *** indicates P < 0.001, determined by an unpaired t-test. h, i Motility and biofilm formation assays evaluating the effects of ArgR regulation. The experimental strains were cultured in M9, and 2 μL of the culture was spotted onto 0.3% LB semi-solid plates, no exogenous L-arginine was added, photographed after 6 h, with the experiment repeated three times. Biofilm formation was assessed using a 96-well plate after the strains were cultured in M9 medium with glucose as the carbon source for 24 hours, no exogenous L-arginine was added. Data are presented as mean ± SD (n = 3). The symbols * indicate P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, determined by an unpaired t-test. The diguanylate cyclase [117]GL000671 was directly regulated by ArgR, but it was unclear whether the expression of [118]GL000671 affected c-di-GMP levels. Overexpression of [119]GL000671 in WT strain resulted in a significant increase in c-di-GMP levels, indicating [120]GL000671 promotes c-di-GMP synthesis (Fig. [121]3f). To assess the effects of c-di-GMP on motility, it was found that overexpression of [122]GL000671 significantly reduced motility. Biofilm formation assays revealed increased biofilm formation following overexpression of [123]GL000671(Fig. [124]3h, i). These findings suggest that elevated c-di-GMP levels inhibit bacterial motility and promote biofilm formation. In summary, ArgR directly represses the expression of [125]GL000671, thereby lowering intracellular c-di-GMP levels and reducing its inhibitory effect on motility. ArgR did not alter the increase in c-di-GMP levels in response to exogenous L-arginine In the absence of exogenous arginine, ArgR coordinates the expression of flagellar and diguanylate cyclase genes, affecting motility. Related studies also indicate that ArgR retains functional activity when exogenous arginine is not added^[126]13,[127]14,[128]33. The addition of 10 mM L-arginine significantly increased the expression level of argR, although the change was modest (Fig. [129]4a). Following the addition of L-arginine, the expression level of [130]GL003492, [131]GL003433 and [132]GL000724 remained unchanged, while [133]GL000671 showed a downregulation (Fig. [134]4b). Furthermore, the introduction of 10 mM L-arginine significantly enhanced the levels of c-di-GMP in both the wild-type and argR knockout strains, indicating that arginine stimulates the synthesis and accumulation of c-di-GMP. In the absence of arginine, the c-di-GMP levels in the ΔargR strain were significantly higher than those in the wild-type (WT), indicating that ArgR negatively regulates the synthesis of c-di-GMP. After the addition of 10 mM L-arginine, the c-di-GMP levels in the ΔargR strain equal to those in the WT, suggesting that the changes in c-di-GMP content in the presence of exogenous arginine may not be solely dependent on ArgR (Fig. [135]4c). The addition of arginine did not alter the promoting effect of ArgR on motility (Fig. [136]4d), however, the addition of arginine appears to reduce the motility of the wild-type strain, possibly due to the increase in c-di-GMP levels. In Burkholderia cenocepacia, the addition of arginine also results in reduced motility^[137]34. Biofilm formation assays showed a similar trend, with the addition of 10 mM L-arginine significantly enhancing biofilm formation across all strains, particularly in the ΔargR and WT+pBBR-[138]GL000671 strains, which exhibited significantly higher biofilm levels than the wild-type (Fig. [139]4e). Taken together, these findings suggest that the arginine-mediated enhancement of c-di-GMP synthesis does not strictly rely on ArgR. Fig. 4. The addition of arginine increases the c-di-GMP levels and enhances biofilm formation. [140]Fig. 4 [141]Open in a new tab a RT-qPCR was used to measure the expression levels of argR. Bacteria were cultured in M9 medium for 18 h, both with and without the addition of 10 mM L-arginine (10 mM Arg), before detection. Data are presented as mean ± SD (n = 3). The symbol * indicates P < 0.05, determined by an unpaired t-test. b RT-qPCR was used to verify the effect of L-arginine addition on the expression of diguanylate cyclases. Data are presented as mean ± SD (n = 3). The symbol * indicates P < 0.05, determined by an unpaired t-test. c Measurement of intracellular c-di-GMP levels using a reporter plasmid. Strains (WT and ΔargR) carrying the c-di-GMP bioreporter plasmid (pCdrA::gfp^C) were cultured in M9 medium (with and without the addition of 10 mM L-arginine) for 18 h, and then 1 OD of the culture was taken to measure fluorescence intensity (P < 0.05 by two-way ANOVA). d The motility of the strains was assessed in LB semi-solid medium, both with and without the addition of 10 mM L-arginine. e The biofilm formation ability of each strain was assessed in M9 medium with glucose as the carbon source, both with and without the addition of 10 mM L-arginine. The bar chart displays the corresponding quantitative results. Data correspond to averages and standard deviations from three independent experiments with four technical replicates. The symbols * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 determined by two-way ANOVA. ArgR affects biofilm formation, cytotoxicity, and adhesion Bacterial flagella influence not only motility but also biofilm formation, adhesion, and virulence^[142]30,[143]35. The ΔflrA strain, which exhibited a complete loss of flagellar function, demonstrated reduced biofilm formation, cell adhesion capacity, and cytotoxicity, indicating the role of flagella in these processes (Fig. [144]5a–c). Although the ΔflrBC strain was partially deficient in flagella, its motility, biofilm formation, cell adhesion and cytotoxicity were also decreased (Fig. [145]5a–c). Similarly, the ΔargR strain showed reduced cell adhesion and cytotoxicity, comparable to the ΔflrA (Fig. [146]5a–c). The phenotypic similarity between ΔflrBC and ΔargR strains supports the role of ArgR in regulating flrBC. Additionally, biofilm formation was significantly increased in the ΔargR strain, potentially due to elevated intracellular c-di-GMP levels. In summary, flagella in A. veronii contribute to biofilm formation, cell adhesion, and virulence. ArgR coordinates the regulation of flagella expression and c-di-GMP synthesis, thereby modulating bacterial motility and affecting biofilm formation, cell adhesion, and virulence. Fig. 5. ArgR contributes to adhesion and virulence. [147]Fig. 5 [148]Open in a new tab a Biofilm formation assay of WT, ΔargR, ΔargR/P-argR, ΔflrBC and ΔflrA after 48 h of growth in M9 medium with glucose as the carbon source, no exogenous L-arginine was added. Data correspond to averages and standard deviations from three independent experiments with three technical replicates. b, c Adhesion and invasion assays of A. veronii to Caco-2 cells. The experimental strain was cultured in M9 medium with glucose as the carbon source for 24 h before infection. Data correspond to averages and standard deviations from three independent experiments with three technical replicates. The symbols * indicate P < 0.05, ** indicates P < 0.01, determined by an unpaired t-test. Discussion ArgR plays a fundamental role in bacterial motility and the structural components of motility, such as pili. In Pseudomonas aeruginosa, deletion of argR gene reduces surface motility^[149]16, although the underlying mechanism has not been fully elucidated. In Enterococcus faecalis strain OG1RF, ArgR displays a cascading regulatory role in pili synthesis by positively regulating ebpR^[150]36. In this study, deletion of argR in A. veronii C4 resulted in a reduced motility phenotype (Fig. [151]1b). Transmission electron microscopy revealed that the ΔargR strain exhibited motility defects and incomplete flagella compared to the WT strain (Fig. [152]1a), suggesting that ArgR affects the biosynthesis of polar flagella. Furthermore, we observed differences in morphology between the argR knockout strain and the wild-type. Vibrio cholerae regulates cell morphology via c-di-GMP to adapt to sessile and motile lifestyles^[153]37. So, we propose that the changes in c-di-GMP resulting from ArgR knockout may lead to alterations in bacterial morphology. The target genes involved in flagella synthesis regulated by ArgR were identified through a combination of ChIP-seq and RNA-seq analyses. On one hand, ArgR is closely associated with amino acid metabolism^[154]38. On the other hand, KEGG pathway enrichment analysis of ChIP-seq data indicated that ArgR regulates flagella assembly, involving 16 genes. KEGG pathway enrichment from RNA-seq data identified 23 flagella genes regulated by ArgR, all of which were downregulated in ΔargR strain. Additionally, GO enrichment analysis from RNA-seq data revealed changes in 27 genes associated with cell motility. Both ChIP-seq and RNA-seq results showed consistent changes in flagella genes, indicating a relationship between ArgR and flagella synthesis. Compared to the WT, the flagella synthesis-related genes with altered expression in ΔargR strain include the regulatory gene flrBC and the structural genes flgABCDEFGHIJKLM, fliA, fliEF, fliJK, and fliS. Notably, flgABCDEFGHIJKLM has been reported to be regulated by flrBC2, suggesting that ArgR directly regulates flrBC, which in turn regulates the flagella structural gene cluster. In Vibrio species, flagella synthesis is regulated by the four-tiered hierarchy^[155]5,[156]39. A. veronii C4, which exhibits a polar flagella phenotype, contains all the regulatory genes for flagella synthesis in its genome, suggesting a potentially similar regulatory network to that of Vibrio cholerae. RNA-seq analysis revealed that the expression level of the main regulator flrA and σ54 remained unchanged upon argR deletion, while flrBC expression was reduced 3–4 folds. Additionally, the downregulated flagella-related genes flgABCDEFGHIJKLM are class III genes regulated by flrC. Therefore, ArgR likely regulates downstream flagella expression by controlling flrBC. Bacterial one-hybrid assays, EMSA, and MST experiments demonstrated that ArgR directly interacts with the flrBC promoter, and RT-qPCR indicated that ArgR positively regulates flrBC (Fig. [157]2). The knockout of flrBC resulted in reduced bacterial motility and a flagella defect phenotype, confirming the role of flrBC in flagella synthesis, consistent with its regulatory role in Vibrio alginolyticus^[158]30. However, the deletion of flrBC in A. veronii did not completely eliminate the flagella, suggesting the presence of additional influencing factors. The second messenger c-di-GMP controls the switch between motile and sessile lifestyles in bacteria. In Salmonella, c-di-GMP interacts with the flagella-related regulatory protein YcgR, thereby affecting motility^[159]28. In Vibrio parahaemolyticus, c-di-GMP inhibits motility by regulating the expression of the laf gene cluster through the Scr regulatory system^[160]40. Elevated levels of c-di-GMP inhibit the expression of class III and class IV flagella genes^[161]41. The principal flagella regulator FlrA and its homolog FleQ bind to c-di-GMP and inhibit transcriptional activity^[162]27,[163]31. In the absence of arginine, the deletion of argR resulted in significant downregulation of 23 flagella genes and reduced bacterial motility. Surprisingly, some diguanylate cyclases were identified in the ChIP-seq data, and the flrBC knockout strain did not completely lose motility, leading us to suspect that c-di-GMP also plays a role in ArgR-mediated motility. The elevated c-di-GMP levels observed in the ΔargR further confirmed our hypothesis. ArgR affects the intracellular c-di-GMP levels, but the precise molecular mechanism remains unclear^[164]14,[165]16. ChIP-seq data from A. veronii revealed that ArgR directly bound to the promoter regions of four diguanylate cyclase genes (Table [166]1). Nevertheless, RT-qPCR showed significant upregulation of only one diguanylate cyclase gene (Fig. [167]3d). Furthermore, LC-MS measurements showed a substantial increase in c-di-GMP levels in the ΔargR strain (Fig. [168]3f), suggesting that ArgR regulates diguanylate cyclase to modulate intracellular c-di-GMP levels. Our study confirmed that [169]GL000671 was likely the primary regulatory target of ArgR in A. veronii. Overexpression of [170]GL000671 elevated intracellular c-di-GMP levels (Fig. [171]3g), indicating that [172]GL000671 possesses diguanylate cyclase activity. Despite the increased intracellular c-di-GMP due to the exogenous supplement and overexpression of [173]GL000671, flagellar structure remained unaffected; however, motility was reduced. This suggests that c-di-GMP influences flagellar motor activity, although the specific targets regulated by c-di-GMP remain to be identified. This study demonstrates that in the absence of arginine, ArgR inhibits the expression of the diguanylate cyclase gene [174]GL000671, leading to a reduction in intracellular c-di-GMP levels. Similarly, in Pseudomonas aeruginosa, ArgR also suppresses the expression of the diguanylate cyclase gene cfcR, resulting in decreased intracellular c-di-GMP levels under arginine-free conditions^[175]16. In the context of arginine presence, there is limited research on the influence of ArgR on c-di-GMP levels. It has only been reported in Pseudomonas aeruginosa that ArgR is an essential factor for arginine-induced increases in c-di-GMP levels^[176]16. However, in this study, we found that the addition of arginine promotes the synthesis of c-di-GMP in both wild-type and ΔargR strains (Fig. [177]4c), indicating that in A. veronii, the c-di-GMP response to arginine is not solely dependent on ArgR. The biofilm phenotype further supports this conclusion (Fig. [178]4e). This appears to differ from previous reports, and we propose that other pathways may exist in A. veronii to respond to arginine and promote c-di-GMP synthesis. It has been reported that bacteria can enhance intracellular c-di-GMP levels through various mechanisms, such as the transmembrane protein RmcA, which senses exogenous arginine and activates diguanylate cyclase activity, leading to an increase in intracellular c-di-GMP^[179]42; arginine promotes the synthesis of putrescine, and the accumulation of putrescine can stimulate the production of c-di-GMP^[180]43; The L-arginine-binding protein ArtI senses arginine to activate the diguanylate cyclase STM1987, which promotes intracellular c-di-GMP levels. Additionally, c-di-GMP also affects the expression and activity of ArgR^[181]14,[182]16. Therefore, in A. veronii, ArgR may act as a balancing regulator of c-di-GMP in response to arginine and other stimuli, with varying intracellular c-di-GMP levels potentially prompting ArgR to perform distinct physiological functions. However, the precise molecular mechanisms require further investigation. In Helicobacter pylori, the attachment of flagella to a solid surface promotes the initiation of biofilm formation^[183]44. In A. veronii, deletion of flrA led to the loss of flagella and the reduction of biofilm formation ability (Fig. [184]5a). In contrast, the argR knockout strain, which exhibited flagellar defects, showed an increase biofilm formation (Fig. [185]5a), possibly due to the elevated intracellular c-di-GMP levels in ΔargR strain. Similarly, flagella contribute to bacterial virulence through enhanced adhesion and invasion^[186]45. The ΔargR strain exhibited the same virulence phenotype comparable to that of ΔflrA strain, suggesting that ArgR influences virulence by regulating flagella synthesis. Furthermore, ΔargR and ΔflrBC strains exhibited similar phenotypes and regulatory relationships, further underscoring the connection between ArgR and flagella. The mechanisms by which ArgR regulates bacterial motility and virulence are illustrated in Fig. [187]6. Specifically, ArgR directly activates the expression of Class II regulatory genes such as flrBC, which in turn promotes the expression of Class III flagellar structural genes. Additionally, ArgR interacts with diguanylate cyclase promoters to inhibit their expression, leading to reduced intracellular c-di-GMP levels and diminishing the inhibitory effect of c-di-GMP on motility. Thus, ArgR orchestrates the expression of flagellar genes and intracellular c-di-GMP levels, thereby influencing motility, biofilm formation, cell adhesion, and virulence in A. veronii. In conclusion, this study proposes a new mechanism through which ArgR regulates bacterial motility and virulence, providing a theoretical basis for the development of strategies to prevent and treat bacterial infections and for the identification of potential drug targets. Fig. 6. Schematic representation of ArgR-mediated regulation of physiological activities in A. veronii. [188]Fig. 6 [189]Open in a new tab ArgR regulates multiple physiological processes through two distinct pathways. First, ArgR directly interacts with the flrBC promoter to activate flagellar gene expression, thereby promoting motility. ArgR also inhibits the expression of guanylate cyclase, leading to reduced intracellular c-di-GMP levels and alleviating its inhibitory effects on motility. By coordinating these pathways, ArgR regulates flagellar assembly and motility, ultimately affecting bacterial adhesion, virulence, and biofilm formation. Materials and methods Bacterial strains and culture conditions The strains and plasmids are listed in Table [190]S3. Aeromonas veronii C4 and its derivatives were cultured at 30 °C in Lysogeny Broth (LB) and M9 minimal medium (with glucose as the carbon source, unless otherwise specified, no L-arginine is added) supplemented with 50 μg/mL ampicillin. Escherichia coli was cultured in LB broth at 37 °C, with 50 μg/mL kanamycin and 0.1 mM IPTG supplemented to induce protein expression. Construction of plasmids, strains, and mutants The deletion mutant strains and complementation strains were constructed using A. veronii C4 as the parental strain. The deletion mutant strains (ΔargR, ΔflrBC, ΔflrA) were constructed by allelic exchange^[191]46. In brief, upstream and downstream homologous arms were amplified from A. veronii C4 genomic DNA and ligated into the pRE112 vector to construct the knockout vector. This vector was then introduced into A. veronii C4 by conjugation and selection was performed on 8% sucrose plates. Complementation strains were obtained by first recombining the corresponding genes into the pBBR plasmid, which was subsequently introduced into the gene knockout strains. The primers are listed in Table [192]S4. Transmission electron microscopy (TEM) The strains were inoculated in M9 medium (with glucose as the carbon source, no L-arginine added) and cultured until the logarithmic phase. The amount of 1 mL bacterial culture was collected, centrifuged, washed once with PBS buffer, and resuspended. The bacterial suspension was placed onto a copper grid and allowed to adhere for 1 min, followed by a single wash with 10 μL of sterile water. Next, 10 μL of 0.1% phosphate buffer was added to stain for 1 min. Excess liquid was removed with filter paper, and the copper grid was allowed to air dry naturally. The morphology of bacterial flagella was observed using a transmission electron microscope (HITACHI, Japan) operating at 80 kV. Chromatin immunoprecipitation-sequencing (ChIP-seq) ChIP was performed as previously described in ref. ^[193]47. The WT strain carrying pBBR-argR-flag was cultured in M9 medium with glucose as the carbon source, no L-arginine added. Cells were collected and cross-linked with 1% formaldehyde, washed with PBS, and lysed with SDS. DNA was sheared into 200–1000 bp fragments by sonication. The supernatant was used as Input. Target protein-DNA complexes were purified by immunoprecipitation, and the protein-DNA complexes were eluted. Input and IP samples were incubated overnight with 5 M NaCl to reverse protein-DNA cross-linking. RNase and protease were added to remove proteins and RNA. ChIP DNA was purified using phenol-chloroform extraction. After DNA extraction, end repair was performed, and an A base along with sequencing adapters were added. Target fragments were then recovered using magnetic beads and PCR-amplified to generate a sequencing library, which was subsequently sequenced on an Illumina sequencer. Quality control of the raw sequencing data was monitored using Trimmomatic software, which removed adapters and low-quality reads. Clean data were aligned to the reference genome. Using Input as the background, Macs2 was employed for peak calling in IP samples, utilizing the narrow peak mode for both peak calling and annotation. Peaks were considered significant if input number ≥2 and p-value < 0.05. Gene Ontology (GO) enrichment and pathway functional enrichment analyses were performed for peak-associated genes. MEME analysis was used to identify the ArgR binding motif^[194]48. Transcriptome sequencing (RNA-seq) Fresh bacterial cultures were inoculated into M9 medium (with glucose as the carbon source and no L-arginine added) and incubated with shaking at 150 rpm at 30 °C for 20 h. Cells were collected, and total RNA was extracted using the phenol/chloroform method. DNase I and the RiboZero Magnetic Kit were used to remove DNA and rRNA from the total RNA. The obtained RNA was reverse-transcribed to cDNA, followed by end repair and adapter addition. The cDNA was enriched by PCR amplification and sequenced using the HiSeq-Xten (Illumina, San Diego, CA, USA). The resulting RNA-seq raw data were assembled and aligned to the reference genome (GCF_026016025.1)^[195]49. Bowtie 2 (v2.2.5) was used to analyze mRNA expression^[196]50. Gene expression levels were measured using reads per kilobase of transcript per million mapped reads (RPKM), and statistical significance was corrected using the Benjamini–Hochberg false discovery rate (FDR). Differentially expressed genes were identified with a false discovery rate (FDR) of less than 0.05 and a fold change greater than two. The DESeq2 package in R was used to estimate fold changes and perform further analyses^[197]51. ArgR purification and electrophoretic mobility shift assay (EMSA) The argR gene was cloned into the pET-28a vector, and the resulting constructs were transformed into E. coli BL21. Cells were induced with 0.1 mM IPTG, collected, lysed by sonication, and the supernatant was used for ArgR purification via nickel affinity chromatography. EMSA was performed as described previously^[198]52. Primers with a 5’ FAM label were employed to amplify labeled probes via PCR. The protein, probe, and binding buffer were incubated at 37 °C for 1 hour. The samples were then separated on a 4.5% native gel by electrophoresis, and probes were detected using a multifunctional biomolecular imager (Typhoon FLA 9500). Microscale thermophoresis (MST) The experiment was performed according to the modified procedures in ref. ^[199]53. Fusion proteins containing the enhanced green fluorescent protein (eGFP) and ArgR were expressed and purified to be used as fluorescent probes, while PCR amplification products were purified to serve as ligand molecules. The fluorescent probes were diluted to 10 nM using MST reaction buffer (2 mM KH[2]PO[4], 8 mM Na[2]HPO[4], 350 mM NaCl, 0.05% Tween 20, pH 7.4), and the ligand molecules were serially diluted. Equal volumes of the fluorescent molecules and ligands were mixed and incubated for 1 hour. The reaction mixture was loaded onto MONT.115 capillary tube plates (Nanotemper Technologies, Germany) and measured using 2% LED excitation and medium MST power. Binding affinity between the probes and ligands was analyzed using MO. Affinity Analysis software (Nanotemper Technologies, Germany). Bacterial one-hybrid assay The target DNA sequence and the coding sequence of the target protein were cloned into the pBXcmT and pTGR plasmids, respectively. The resulting constructs were transformed into E. coli XL 1-Blue MRF’ reporter strains^[200]54. Co-transformants containing pBT-LGF2 and pTRG-Gal11P served as positive controls, while the empty vectors pTRG and pBT were used as negative controls. The colonies were plated on selective media containing 3-amino-1, 2, 4-triazole (3-AT), and incubated at 37 °C for 1 day, followed by 12 h at 30 °C. Fluorescence detection experiment The promoter of the flrBC gene was fused with eGFP to construct the fluorescence reporter vector pDH-PflrB-eGFP, which was co-transformed with the ArgR expression vector pTRG-ArgR into BL21 (DE3). The experimental strains were inoculated into LB medium and incubated at 37 °C with shaking. Bacteria were collected, resuspended in 200 μL PBS, and fluorescence was detected using a multi-mode microplate reader (Biotek, USA) with an excitation wavelength of 485 nm and an emission wavelength of 515 nm. Differences between the experimental and control groups were analyzed for significance using a One-Way ANOVA test. Quantitative real-time PCR (RT-qPCR) A. veronii was cultured at 30 °C until it reached the stationary phase, after which the cells were collected by centrifugation. Total bacterial RNA was extracted using an RNA extraction kit (Shengong, China). The cDNA was synthesized by reverse transcription of 500 ng total RNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Japan). The qRT-PCR was performed using the Recho LightCycler 96 system (Roche, Switzerland), with GAPDH as the internal reference gene. The relative expression levels of individual genes were calculated using the 2^^−ΔΔCT method. Liquid chromatography-mass spectrometry (LC-MS) The measurement was modified based on previous methods in ref. ^[201]55. We purchased the c-di-GMP standard (SML1228) from Merck for liquid chromatography-mass spectrometry detection. The cell pellets were resuspended in pre-cooled extraction buffer (methanol: acetonitrile, 40%: 40%: 20%), frozen at −80 °C for 1 h, and then centrifuged at high speed to collect the supernatant. The supernatant was dried using a vacuum freeze dryer and then dissolved in sterile water to obtain the c-di-GMP extract. The protein concentration in the pellet was determined using the Bradford method for normalization. The extracted c-di-GMP was centrifuged at high speed for 10 min, and the supernatant was used for LC-MS analysis. Indirect quantification of c-di-GMP The sensor plasmid pCdrA::gfp^C characterizes intracellular c-di-GMP levels^[202]56. The sensor plasmid was constructed and introduced into both WT and ΔargR strains, which were cultured in M9 medium until reaching stationary phase. Cells were collected, washed with PBS, and resuspended. A 200 μL aliquot of the cell suspension was transferred to a black 96-well plate, and fluorescence intensity was measured at excitation/emission wavelengths of 485/515 nm using a multifunctional microplate reader. Optical density at 600 nm (OD[600]) was measured for normalization. The fluorescence intensity was divided by OD[600] to calculate relative fluorescence intensity. Biofilm and motility assays Each strain was cultured in m9 medium overnight and transferred to a 96-well plate at an OD[600] of 0.05. After incubation for 36 hours with no shaking at 30 °C. The culture was washed with PBS buffer, stained with 200 μL of 1% crystal violet dye at room temperature for 10 min, washed, dried at 55 °C, and treated with 200 μL of 33% glacial acetic acid. The mixture was incubated at 37 °C for 30 min, and absorbance was measured at 595 nm using a microplate reader to quantify biofilm content. A semi-solid medium (1 g tryptone, 0.5 g NaCl, 0.5 g agar powder, 0.3 g yeast extract, dissolved in 0.1 L) was prepared. One μL of culture was spotted onto the medium, which was then allowed to stand at room temperature for 20 min and incubated at 30 °C for 4 h. Swarming assay was observed and photographed, and the motility diameter was measured to evaluate motility. Caco-2 adhesion and invasion assay Each strain was cultured overnight, the bacterial cells were collected and resuspended to a concentration of 10^6 CFU/mL in Dulbecco’s Modified Eagle Medium (DMEM). Caco-2 cells were purchased from ATCC. Caco-2 cells were cultured in a 96-well plate until they were fully adhered. For the adhesion assay, the bacteria and Caco-2 cells were co-incubated at a multiplicity of infection (MOI) of 1:1 for 2 h. The cells were then washed three times with PBS to remove residual bacteria, and treated with 0.1% Triton X-100 for 20 min. The cell suspension was diluted 10-fold, plated onto LB agar containing ampicillin, and colonies were counted after 18 h of incubation. For the invasion assay, the bacteria and cells were co-incubated at an MOI of 1:1. Afterward, the cells were washed three times with PBS, treated with DMEM containing 100 μg/mL gentamicin, and incubated for 1 hour. Subsequently, 1/10 volume of CCK-8 solution was added, and the incubation continued for an additional 1 h. Absorbance was measured at OD[450] using a microplate reader. Statistical analysis Statistical analyses were performed using GraphPad Prism (version 6). Experiments were conducted in triplicate. Differences were analyzed using an unpaired two-tailed Student’s t-test. * p < 0.05 and ** p < 0.01 were considered statistically significant and highly significant, respectively. Reporting summary Further information on research design is available in the [203]Nature Portfolio Reporting Summary linked to this article. Supplementary information [204]Transparent Peer Review file^ (782.2KB, pdf) [205]Supplementary Material^ (1.9MB, docx) [206]42003_2024_7392_MOESM3_ESM.pdf^ (81.7KB, pdf) Description of additional supplementary data [207]Supplementary data^ (42.1MB, xlsx) [208]Reporting Summary^ (4.4MB, pdf) Acknowledgements