Functional versatility of Zur in metal homeostasis, motility, biofilm formation, and stress resistance in Yersinia pseudotuberculosis

ABSTRACT Zur (zinc uptake regulator) is a significant member of the Fur (ferric uptake regulator) superfamily, which is widely distributed in bacteria. Zur plays crucial roles in zinc homeostasis and influences cell development and environmental adaptation in various species. Yersinia pseudotuberculosis is a Gram-negative enteric that pathogen usually serves as a model organism in pathogenicity studies. The regulatory effects of Zur on the zinc transporter ZnuABC and the protein secretion system T6SS have been documented in Y. pseudotuberculosis. In this study, a comparative transcriptomics analysis between a ∆zur mutant and the wild-type (WT) strain of Y. pseudotuberculosis was conducted using RNA-seq. This analysis revealed global regulation by Zur across multiple functional categories, including membrane transport, cell motility, and molecular and energy metabolism. Additionally, Zur mediates the homeostasis not only of zinc but also ferric and magnesium in vivo. There was a notable decrease in 35 flagellar biosynthesis and assembly-related genes, leading to reduced swimming motility in the ∆zur mutant strain. Furthermore, Zur upregulated multiple simple sugar and oligopeptide transport system genes by directly binding to their promoters. The absence of Zur inhibited biofilm formation as well as reduced resistance to chloramphenicol and acidic stress. This study illustrates the comprehensive regulatory functions of Zur, emphasizing its importance in stress resistance and pathogenicity in Y. pseudotuberculosis. IMPORTANCE Bacteria encounter diverse stresses in the environment and possess essential regulators to modulate the expression of genes in responding to the stresses for better fitness and survival. Zur (zinc uptake regulator) plays a vital role in zinc homeostasis. Studies of Zur from multiple species reviewed that it influences cell development, stress resistance, and virulence of bacteria. Y. pseudotuberculosis is an enteric pathogen that serves a model organism in the study of pathogenicity, virulence factors, and mechanism of environmental adaptation. In this study, transcriptomics analysis of Zur’s regulons was conducted in Y. pseudotuberculosis. The functions of Zur as a global regulator in metal homeostasis, motility, nutrient acquisition, glycan metabolism, and nucleotide metabolism, in turn, increasing the biofilm formation, stress resistance, and virulence were reviewed. The importance of Zur in environmental adaptation and pathogenicity of Y. pseudotuberculosis was emphasized.


Genome-wide analysis of the genes regulated by Zur
In a preceding study, Zur was identified to downregulate the zinc transporter ZnuABC and concurrently upregulate T6SS4 (30), suggesting its potential multifunctionality.To delineate the comprehensive regulatory map of Zur, RNAs from both WT and ∆zur mutant strains in exponential phase were extracted and subjected to RNA-seq analysis (SRA accession: PRJNA1019653).The acquired data were subsequently processed, and the differentially expressed genes (DEGs) between the ∆zur mutant and the WT were identified at a P-value of <0.05 with a fold change >1.0.A total of 156 DEGs were identified: 110 DEGs were downregulated, and 46 were upregulated, in the ∆zur mutant based on the RNA-seq results (Table S2).Volcano plots and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis results are illustrated in Fig. 1A and  B. The DEGs were primarily clustered into 14 distinct pathways as per the KEGG pathway analysis, with membrane transport and cell motility emerging as the two predominant pathways.
Within the 41 DEGs classified under the membrane transport pathway, znuA (ypk_2140), znuB (ypk_2142), and znuC (ypk_2141) were observed to be downregula ted by Zur with fold-changes of 3.67, 1.54, and 1.60, respectively.This finding aligns with previous studies (30).T6SS4 genes, as well as 15 genes associated with T6SS1-3 and Tat system, exhibited upregulation, albeit with less pronounced differences; the fold-changes were all <1.0 (Table S3).To validate this regulatory pattern, qRT-PCR was conducted (Fig. S1).The transcriptional levels of T6SS1-4 and Tat genes were upregulated by Zur, consistent with RNA-seq analysis.This suggests that Zur might activate the expression of T6SS1-3 and Tat system in a manner analogous to T6SS4.

Zur regulates the expression of genes involved in metal homeostasis
Regulators from the Fur superfamily predominantly modulate the expression of genes related to metal homeostasis by sensing metal ion concentration in vivo.Structural analyses have shown that Zur in S. Typhimurium can bind Zn 2+ or Co 2+ in site 2 and that Zur in Caulobacter crescentus can cross-talk with Fur (38,39).While Fur is known to regulate the uptake of Mn 2+ in addition to Fe 2+ in Y. pseudotuberculosis (36), it remains to be determined whether Zur can sense and regulate other metal ions in Y. pseudotubercu losis.Among the top-ranked DEGs from RNA-seq analysis, the putative Fe 3+ transporter genes fhuB (ypk_2719), fhuC (ypk_2718), and fhuD (ypk_2720) were identified as being upregulated in ∆zur mutant, with fold-changes of 5.51, 6.68, and 8.81, respectively (Table S2).To verify the impact of Zur on fhuC transcription, qRT-PCR was conducted (Fig. 2A).The transcriptional level of fhuC in the ∆zur mutant increased by over 20-fold compared to the WT and the complemented strain.EMSA assays demonstrated that Zur binds directly to the promoter region of fhuBCD (Fig. 2B).Chromogenic reaction assays were utilized to test the ion-binding activity of Zur.With Fur serving as a control, Zur exhibited binding activity for both Fe 2+ and Fe 3+ (Fig. 2C).This suggests that Zur might regulate iron transporter FhuBCD by sensing the iron concentration.Subsequent analyses of intracellular metal ion concentrations of Y. pseudotuberculosis strains revealed significant increases in of Zn 2+ and Mg 2+ concentrations in the ∆zur mutant compared to the WT and the complemented strain (Fig. 2D).This aligns with observed upregulation of zinc transporter ZnuABC and magnesium transporter MgtE (ypk_1341, downregulated by Zur with a fold-change of 0.61 as indicated in Table S3) in ∆zur mutant in the RNA-seq data.However, iron concentrations decreased significantly in the ∆zur mutant.Several iron transporters exist in Y. pseudotuberculosis, including FeoABC, SitABCD, and IucABCD.Further examination of the RNA-seq data revealed three transporter genes (ypk_2538, ypk _2751, and ypk _3892) that were downregulated by Zur in mutant strain with fold-changes > 0.5 (Table S3).This could account for the decreased ion concentrations in the mutant.The growth assays under metal-limited conditions (by adding EDTA) demonstrated enhanced growth of Y. pseudotuberculosis in the absence of Zur when metal ions were scarce in the environment (Fig. 2E).In summary, Zur is pivotal in metal homeostasis in Y. pseudotuberculosis.

Zur regulates genes involved in nutrient acquisition
Apart from the DEGs involved in metal ion homeostasis in the membrane transport group, several DEGs primarily encode for nutrient transporters, including D-xylose, ribose, erythritol, maltose, arginine, glutamate/aspartate, and oligopeptide transporters (Table S2).qRT-PCR and EMSA were conducted to verify the transcriptional regulation by Zur of ypk_0057 (coding for D-xylose transporter, XylF), ypk_1611 (coding for ribose transporter, RbsB), and ypk_2067 (coding for oligopeptide transporter, OppD) (Fig. 3A  and B).The data revealed that Zur directly upregulates the transcription of these genes by binding to their promoters.The modulation of these nutrient transporter genes suggests a potential role of Zur in nutrient acquisition in Y. pseudotuberculosis.

Zur promotes motility by upregulating flagellar gene expression
Both Fur and Zur have been observed to influence motility by regulating flagellar synthesis genes in Escherichia coli and Pectobacterium odoriferum (40,41).In our study, the cell motility-associated DEGs showed the most significant group (35 DEGs) in the KEGG pathways regulated by Zur in Y. pseudotuberculosis.The arrangement of flagellar genes in operons is depicted in Fig. 4A, with a corresponding heat map analysis presented in Fig. 4B.Both qRT-PCR and EMSA verified that Zur regulates these operons by directly binding to their promoters (Fig. 4C and D).These DEGs encode nearly the entire flagellar apparatus, encompassing biosynthetic, component, assembly proteins, and regulators (FlgM, FliA, and FliZ).As depicted in Fig. S2, Zur may also indirectly influence the biosynthesis and assembly of flagellar system through these regulators.To assess the impact of Zur on motility, a swimming motility assay was performed using WT, ∆zur mutant and the complemented strains.The swimming motility of ∆zur mutant strain was notably reduced compared to the WT and the complemented strain (Fig. 4E and F).

Zur participates in molecular pathways and energy metabolism in Y. pseudo tuberculosis
In addition to its influence on membrane transport and cell motility, Zur also regu lates genes associated with the metabolism of sugar, lipid, amino acids, nucleotide, protein, and energy (Fig. 1B).qRT-PCR and EMSA were used to confirm these DEGs in various pathways (Fig. 5A and B).Zur was observed to positively regulate several enzymes, such as peptidoglycan biosynthesis-related D-alanine-D-alanine ligase Ddl (YPK_3516), purine-nucleoside phosphorylase DeoD (YPK_3624), and deoxyribose-phos phate aldolase DeoC (YPK_3627), by directly binding to their promoters.This under scores Zur's regulatory influence on these molecular metabolic pathways.Moreover, Zur negatively regulated the large subunit ribosomal protein bL31-B (YPK_3210) and bL36-B (YPK_3211), which are implicated in both ribosome assembly during translation and zinc homeostasis (42,43).The fact that Zur affects numerous metabolic pathways emphasizes its role as a significant global regulator in Y. pseudotuberculosis.

Zur promotes biofilm formation and stresses resistance
Given Zur's regulatory activities across diverse metabolic processes, its influence on biofilm formation and stress resistance in various bacteria was explored (4,44).To test Zur's effect on biofilm formation, the biofilm of the WT, ∆zur mutant, and complemented strains were stained using crystal violet after 48 h of culture in M9 medium or supple mented with an additional monosaccharide.They were then quantified spectrophoto metrically, as shown in Fig. 6A.The biofilm production in M9 medium was minimal, with no significant disparity between the WT and mutant strains.However, the introduction of glucose and ribose to the medium increased biofilm formation.Remarkably, the biofilm formation of the ∆zur mutant strain was markedly diminished compared to both the WT and the complemented strains, especially in ribose-supplemented medium.This could potentially be attributed to Zur's positive regulation of monosaccharide transport and metabolism.Stress survival assays, involving exposure to chloramphenicol and acidic conditions, were also conducted (Fig. 6B and C).The survival rate of the ∆zur mutant strain showed a significant decrease under these stressors, suggesting that Zur enhances antibiotics and acid resistance in Y. pseudotuberculosis.

DISCUSSION
Zur is a pivotal regulator with an influence that extends beyond merely maintaining zinc homeostasis.The global regulatory activities of Zur across over 20 species have been analyzed through RNA-seq, microarray and ChiP analyses (40,41,(44)(45)(46).The suppres sion of zinc transporters, such as ZnuABC and ZinT, and the modulation of ribosomal proteins appear to be conserved across various bacteria (45).However, the exact roles of Zur can differ among species.Notably, within the Yersinia genus, the regulatory functions of Zur in Y. pestis differ from those in Y. pseudotuberculosis.Microarray expression studies identified 154 Zur-dependent genes of Y. pestis, comprising 90 upregulated genes and 64 downregulated genes.These genes are primarily involved in transporting, membrane protein synthesis, regulation, biosynthesis, and metabolism (47).Similar to its role in Y. pseudotuberculosis, Zur prominently regulates membrane transport proteins in Y. pestis, including those responsible for the uptake of zinc, iron, nickel, phosphate, amino acids, oligopeptide, and sugars.Furthermore, homologs of ribosomal proteins L31 and L36, as well as flagellar proteins, are subject to Zur's regulatory effects in both species.However, Zur has a more pronounced effect on taurine, urease, and iron uptake proteins in Y. pestis.In contrast, genes linked to sugar transport and motility are subject to greater regulation by Zur in Y. pseudotuberculosis than in Y. pestis.Additionally, a higher number of DEGs associated with molecular and energy metabolisms are observed in Y. pseudotuberculosis.Collectively, these findings suggest that while Zur operates as a global regulator, its specific roles can vary, making its function in Y. pseudotuberculosis distinct from that in other bacteria.
Zur can regulate the expression of numerous divalent metal ion transporters and binding proteins, aiding in vivo homeostasis of cations such as zinc, iron, nickel, manganese, calcium, and cobalt (27,29,44,48).In a prior study, Zur was demonstrated to downregulate the expression of zinc transporter gene znuABC by direct binding to its promoter using EMSA and β-Galactosidase analyses (30).RNA-seq analysis also revealed the downregulation of znuABC by Zur, with a subsequent increase in the zinc concentra tion in the ∆zur mutant strain, affirming its pivotal role in zinc uptake regulation.Contrary to the stable magnesium and iron concentration in the ∆zur mutant of Cupriavidus metallidurans (49), there were notable changes in the cation concentrations in the ∆zur mutant of Y. pseudotuberculosis (Fig. 2D).Eventhough the transcriptional change of magnesium transporter MgtE was modest (fold change of 0.6126), the magnesium concentration in mutant strain increased noticeably.This could be attributed to an accumulation of magnesium via the slightly upregulated transporters.Iron transport systems, such as Feo, Fec, Fep, Fbp, Fut, Sit, and Fhu, as well as aerobactin-mediated system, are orchestrated by both Fur and Zur, either directly or indirectly (37,45,50).In our research, only the ferric transporter FhuBCD was significantly downregulated by Zur in Y. pseudotuberculosis (Table S2).Contrasting with the increase of FhuBCD, the iron concentration dipped in the ∆zur mutant strain and spiked in the complementary strain (Fig. 2).This mirrors the regulatory role of Zur in iron as observed in Xanthomonas oryzae pv.oryzae (29).This phenomenon might stem from the function of other iron transport systems and role of the cross-talk between Zur and Fur in maintaining metal homeosta sis.Notably, the absence of Zur in Caulobater crescentus increases the zinc concentration, leading to the suppression of Fur-regulated genes (39).Additionally, elevated zinc level in vivo also trigger ZitB expression, promoting iron export in Streptomyces coelicolor (24).Hence, Zur can mediate the regulation of iron-associated genes either directly or indirectly.However, it remains unclear whether Zur tailors these genes by sensing iron.Our findings reveal Zur's iron-binding capacity in vitro (Fig. 2C), likely in the form of heme.Prior reports suggest that heme competes with Zn 2+ for binding site 3 of Zur, altering its DNA binding activity in Anabaena sp.PCC7120 (51,52).In Salmonella Typhimurium, Zur can bind cobalt at elevated concentration (38).The exact mechanism and implications of Zur's potential iron or heme binding warrant detailed structural analysis.
Zur appears to facilitate the environmental adaptation and virulence of Y. pseudo tuberculosis by elevating the expression of proteins pivotal for nutrient acquisition, chemotaxis and motility, cell adhesion, and virulence factors (Table S2).Beyond the regulation of metal ion transport, various nutrient transport systems, including the xylose (YPK_0057-0059), ribose (YPK_1161 and YPK_1162), erythritol (YPK_1961-1963), maltose (YPK_0378 and YPK_0382), simple sugar (YPK_2408-2411), glutamate/aspar tate (YPK_3010), oligopeptide (YPK_2067 and YPK_2068), and nucleoside (YPK_1438 and YPK_3628) transport systems were upregulated by Zur.This suggests Zur's role in nutrient acquisition in Y. pseudotuberculosis.Although similar regulation of amino acid and oligopeptide transport systems was revealed, Zur did not upregulate any sugar transporter in Y. pestis (47).The quorum-sensing molecule AI-2 transport system (YPK_3651-3653) and the purine-binding chemotaxis protein Chew (YPK_1750) were upregulated by Zur, aiding environmental signal sensing and motility.Chemotaxis and flagellar motility are crucial for functions such as swimming, biofilm formation, and virulence (53).While various bacteria, including E. coli K-12, B. subtilis, C. metallidurans, P. odoriferum, and Y. pestis, have Zur-influenced chemotaxis and flagellar genes, the prominence of its role differs (16,27,41,47,49).For instance, only three genes (fliM, fliD, and flgD) involved in chemotaxis and motility were revealed to be notably regulated by Zur in Y. pestis (47).But 35 flagellar genes, excluding AI-2 transporter and Chew genes, were notably upregulated by Zur in Y. pseudotuberculosis, suggesting Zur's critical function in motility regulation.The swimming motility of the ∆zur mutant strain is markedly decreased in a swimming assay (Fig. 4E and F).Contrasting with P. odoriferum, where Zur reduces flagellar expression but does not alter motility in ∆zur (41), Zur in Y. pseudotuberculosis appears to have a wider regulatory impact.
Zur might influence acidic stress and antibiotics resistance through modulating membrane transporter, lipoprotein, cell wall plasticity, metabolism, and even biofilm formation (27,54,55).While biofilm formation is crucial for pathogen bacteria in terms of environmental adaptation and virulence, the mechanism of its formation and regulation remains enigmatic (56).Echoing findings in Bacillus anthracis and Anabaena sp.PCC7120, Zur in Y. pseudotuberculosis appears to promote biofilm formation (44,54).The enhanced sugar acquisition, glycan biosynthesis, and transmembrane transport mediated by Zur in Y. pseudotuberculosis likely support the production of extracellular polysaccharides, which are fundamental for biofilm structure.The upregulation of peptidoglycan biosynthetic pathway including YPK_3516, YPK_3517, YPK_3518, and YPK_3520 (Fig. 5; Table S2) could facilitate the integrity of cell wall.Thus, by regulating aspects like metal uptake, nutrient acquisition, biofilm formation, cell wall biosynthe sis, motility, virulence factors, filament, and T6SS (30), Zur could facilitate the stress resistance and pathogenicity of Y. pseudotuberculosis.
In conclusion, our study comprehensively analyzed the regulon of Zur in Y. pseudotu berculosis in this study.Beyond metal homeostasis, Zur is a global regulator influencing chemotaxis and motility, nutrient acquisition, glycan biosynthesis and metabolism, and nucleotide metabolism, in turn increasing stress resistance and virulence (Fig. 7).This research underscores Zur's overarching regulatory capacity and significance for stresses resistance and pathogenicity in Y. pseudotuberculosis.

Bacterial strains and growth conditions
The bacterial strains and plasmids utilized in this study can be found in Table S1.The growth conditions for the strains in this study were consistent with those employed in our previous research (30,57).Briefly, E. coli was grown in Luria-Bertani (LB) medium at 37°C, supplemented with the necessary antibiotics.Y. pseudotuberculosis YPIII strains were cultured either in Yersinia-Luria-Bertani (YLB) broth (composed of 1% tryptone, 0.5% yeast extract, and 0.5% NaCl) or M9 medium (comprising 6 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl, 1 mM MgSO 4 , 0.1 mM CaCl 2 , and 0.2% glucose) at 26°C, with appropriate antibiotics as needed.Specifically, nalidixic acid was added at a concentration of 15 µg/mL and kanamycin at 50 µg/mL.

Overexpression and purification of recombinant protein
The purification of Zur was carried out in accordance with the procedures established in our prior study (30).In brief, the BL21(DE3) bacteria with the pET28a-zur plasmid were grown at 37°C in LB medium until they reached an OD 600 of 0.5.Then, supplement with 0.5 mM IPTG induced at 24°C for 10 h.After harvesting, we disrupted the cells using sonication and purified the protein using His•Bind Ni-NTA resin (Novagen, Madison, WI).We confirmed the purified protein's high purity (>95%) using SDS-PAGE analysis and measured its concentration using the Bradford assay.

RNA-seq experiment
Y. pseudotuberculosis WT and ∆zur mutant strains were cultured in YLB medium to mid-logarithmic and normalized to OD 600 = 0.8.Bacterial cells were harvested by centrifugation (each group set ≥3 replicates).RNA extraction, library construction, and RNA sequencing were commissioned by BGI Genomics (Shenzhen, China).According to the relative level of expression between the two groups of samples, the differentially expressed genes can be divided into up-regulated genes and down-regulated genes.For the samples with biological replicates, DESeq2 was used to conduct differential expression analysis among sample groups, and the differential expression gene set between the two biological conditions was obtained: KEGG (Kyoto Encyclopedia of Genes and Genomes) functional enrichment was performed on the selected DEGs, and genes were classified according to their functions to achieve the purpose of gene annotation and classification.When P value < 0.05, significant functional enrichment was considered.

RNA extraction and quantitative real-time PCR
The Y. pseudotuberculosis strain was cultured in YLB medium in a shaker to OD 600 = 1.0, and the cells were harvested by centrifugation.Total RNA was isolated using the Steadypure Universal RNA Extraction Kit AG21017 (Accurate Biotech, Hunan, China) following the manufacturer's instructions.cDNA was synthesized by Evo M-MLV RT Reaction Mix Kit AG11728 (Accurate Biotech, Hunan, China).qRT-PCR was then per formed using the SYBR Green Pro Taq HS Premix (Accurate Biotech, Hunan, China) in the LightCycler 96 thermal cycler (Roche, USA).The 16S rRNA gene served as a housekeeping gene to normalize mRNA abundance.

Electrophoretic mobility shift assay
URD and DNA probes were amplified and purified on 1% agarose gel.URD was a 200 bp DNA amplified by qRT-PCR primers.Zur was incubated with 40 ng of non-specific URD and 40 ng of target promoter fragment in EMSA-binding buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 500 mM KCl, 2.5% glycerol) at room temperature for 20 min (58,59).The sample was then placed in a 6% native polyacrylamide gel in a 0.5 × Tris-borate-EDTA buffer at 100 V for 2 h at 4°C.The gel was stained with SYBR Safe DNA gel stain (Invitrogen, USA) and imaged with a fluorescent imaging system (Tanon 5200Multi, China) (60).

Determination of intracellular ion content
The intracellular ion content was assessed following established procedures as described in prior research (37,61).After reaching an optical density (OD 600 ) of 1.0 through cultivation, the bacteria were harvested.Add the lysis solution at a ratio of 5 mL/g to resuspend the bacterial precipitation, and then lysis at 4°C overnight.Centrifuge at 10,000 rpm for 30 min, add the supernatant in a 1:100 ratio to a 2% chromatographic grade nitric acid solution, after treatment for 12 h, centrifuge at 10,000 rpm for 30 min, and then extract the supernatant.Samples were measured by inductively coupled plasma mass spectrometry, and relative intracellular ion content was calculated using protein concentration.

Ion-binding assay
Before conducting the experiment, two specific staining solutions were prepared: one is composed of a mixture of 0.75 mM Ferene S and 15 mM thioglycolic acid added to 2% (vol/vol) acetic acid, and the other is a solution without 15 mM mercaptoacetic acid.Mix Fe 2+ , Fe 3+ , Fur, Zur, and BSA with two staining solutions, dot blot on the nitrocellulose membrane, and observe the droplet color (62,63).In addition, the control group was mixed with dialysate and staining solution.

Biofilm formation assay
Biofilm formation was determined as described previously (60).In brief, bacterial cultures grown overnight were diluted 100-fold in 3 mL of fresh M9 medium (supplement with glucose or ribose), with the addition of appropriate antibiotics as needed.After 48 h of vertical incubation at 26°C with shaking at 150 rpm, the bacterial cultures were harvested following OD 600 measurement, and the test tubes were subsequently washed twice with fresh M9 medium.Cells adhering to the inner surfaces of the test tubes were stained with 0.1% crystal violet for 30 min and subsequently washed twice with M9.The dye bound to the cells was eluted using 4 mL of 95% ethanol, and the absorbance of the eluted solution was quantified at 590 nm using a microplate reader.

Stress survival assay
Take 1 mL of each bacterial solution (OD 600 = 1.0) and centrifuge at 4,000 rpm for 3 min to collect the bacteria.After rinsing with M9 medium three times, transfer to pH 4.0 and normal M9 medium in a 1:50 ratio (64).After being stressed at 26°C, 150 rpm for 1 h, apply to YLB plates containing nalidixic acid and kanamycin, and incubate at 30°C for 24 h (37,65).Chloramphenicol (5 µg/mL) stress was performed in the same manner as above.Count the colony-forming units (CFU) and calculate and analyze the survival rate.The survival rate is (CFU in the treatment group/CFU in the control group) ×100%.

FIG 1
FIG 1 RNA-seq analysis of the genes regulated by Zur.(A) Volcano plot illustrating gene expression analysis, with the x-axis representing the log2(∆zur/WT) fold change in gene expression and the y-axis showing the log10(Q-value) for statistical significance.Downregulated genes are represented on the left side in blue, while upregulated genes are on the right side in red.Each treatment includes three biological replicates.(B) KEGG pathway enrichment analysis of DEGs between the ∆zur mutant and the WT strain.

FIG 4
FIG 4 Zur promotes motility by upregulating the expression of flagellar genes.(A) Schematic illustration depicting the arrangement of flagellar genes within operons.(B) Heatmap of RNA-seq analysis.Heatmap was made by calculating log 2 (∆zur FPKM/WT FPKM).Different colors indicate different fold changes of gene expression.The darker the blue, the more significant the difference.FPKM, fragments per kilobase of transcript per Million mapped fragments.(C) qRT-PCR analysis of fliH (ypk_2393) mRNA levels.(D) EMSA was performed to analyze the interactions between His6-Zur and the promoters of flagellar-related genes (ypk_2391 p).Various amounts of Zur (0, 0.2, 0.4, and 0.8 µg), 50 ng DNA fragments and URD were used in each lane.(E and F) Zur regulates the swimming motility of Y. pseudotuberculosis.Overnight cultures of WT, mutant, and the complementary strains were diluted with fresh YLB medium to an OD 600 of 0.5 and spotted onto swimming plates (C), and the swimming halo diameters of each strain were measured (F).Data are shown as mean ± SEM (n ≥ 3).**P < 0.01; ***P < 0.001; ****P < 0.0001.

FIG 6
FIG 6 Zur promotes biofilm formation and stress resistance.(A) Effect of Zur on biofilm formation in M9 medium or supplemented with an additional monosaccharide.(B and C) Effect of Zur on stress resistance in Y. pseudotuberculosis.Survival rates of WT, ∆zur mutant, and the complemented strain ∆zur(zur) after challenging with 5 µg/mL chloramphenicol (B) or pH 4.0 (C) for 60 min were determined.Data are shown as mean ± SEM (n = 3).*P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.