Investigation of the Role of a Zinc Uptake Regulator (Zur) in the Virulence of Pectobacterium odoriferum

Pectobacterium spp. infect many horticultural crops worldwide and lead to serious crop losses. Zinc-uptake-regulator (Zur) proteins are present widely in prokaryotes and play an important role in pathogenicity. To uncover the role of Zur in P. odoriferum, we constructed mutant (ΔZur) and overexpression [Po (Zur)] strains of a Zur, and a virulence assay showed that the Po (Zur) was of significantly lower virulence, while the ΔZur displayed significantly increased virulence on Chinese cabbage compared to their respective control strains, wild-type P. odoriferum (Po WT) and P. odoriferum harboring an empty vector (Po (EV)) (p < 0.05). The growth curves of the ΔZur and Po (Zur) showed no obvious differences from those of the control strains. Comparative transcriptome analysis showed that Zur overexpression in P. odoriferum induced differentially expressed genes (DEGs) related to flagellum and cell motility, while mutating Zur resulted in DEGs mainly corresponding to divalent-metal-ion transport and membrane transport. Phenotypic experiments on the Po (Zur) showed that flagellum numbers and cell motility were reduced in comparison with the control, while those of the ΔZur did not change. Collectively, these results show that the Zur negatively regulates the virulence of P. odoriferum and might function via a dual mechanism dependent on dose.


Introduction
Zinc (Zn) is the second most abundant transition-metal element among all the essential trace elements in living organisms after iron [1]. It is a cofactor of many enzymes and helps to maintain the structural stability of even more proteins, including transcription factors. Zinc has both structural and functional roles in prokaryotes by binding to 5-6% of proteins [2]. Deficient zinc levels prevent normal cellular function, but excess intracellular zinc can lead to toxicity [3]. This is mainly because it binds to metalloproteins normally associated with other metals from the Irving-Williams series and impairs their function. This phenomenon is called mismetallation [4]. Therefore, intracellular-zinc concentrations must always be carefully regulated through zinc-homeostasis systems to control zinc uptake and release and maintain adequate cellular-zinc concentrations [5].
The zinc-uptake-regulator (Zur) protein belongs to the ferric-uptake-regulator (Fur) family of metalloregulatory proteins. The Zur is present widely in prokaryotes, and it plays an important role in regulating zinc homeostasis [6]. Two main zinc transporters for zinc uptake exist in most gram-negative bacteria: the znuABC, a high-affinity zinc-transport system and the low-affinity ZIP family Zn transporter ZupT [7]. znuABC is composed of the soluble periplasmic binding protein ZnuA, the high-permeability transmembrane protein ZnuB, and the ATPase ZnuC, and it is regulated by the Zur [8]. Corresponding to the critical role of the Zur in regulating zinc homeostasis, since the first report of the Zur in Escherichia coli [9], members of the Zur family have been studied in a wide range of bacteria,

Zur Plays A Negative Role in the Virulence of P. odoriferum BCS7
To identify the role of the Zur in the virulence of P. odoriferum BCS7, Zur-overexpression (Po (Zur)) and Zur-knockout (∆Zur) strains were constructed and used to inoculate Chinese cabbage petioles. The lesion area was measured at 24 h post-inoculation for each strain (including the control strains). Plants inoculated with Po (Zur) displayed a smaller lesion area compared to those inoculated with the control strain Po (EV) (p < 0.05), whereas the Zur mutant strain (∆Zur) induced a larger lesion area than the wild-type strain Po BCS7 (Po WT) (p < 0.05) (Figure 1a,b). Zur expression in the Po (Zur) and ∆Zur were confirmed using qPCR (Figure 1c,d). These results suggest that the Zur in P. odoriferum BCS7 plays a negative role in virulence. confirmed using qPCR (Figure 1c,d). These results suggest that the Zur in P. odoriferum BCS7 plays a negative role in virulence. (a) Virulence of Zur-overexpressing strain Po (Zur) was measured by examining the average area of the disease spot on Chinese cabbage, with the strain Po (EV) harboring an empty vector as control (n = 7). (b) Virulence of the mutant strain of the Zur (ΔZur) was measured by examining the average area of the disease spot on Chinese cabbage, with the wild-type strain (Po WT) as control (n = 7). (c,d) These show the relative expression of the Zur in the Po (Zur) and ΔZur compared to their control strains Po (EV) and Po WT, respectively. Each column shows the mean and standard deviation (n = 3). Significant differences with p < 0.05 (t-test) were labelled with an asterisk. All experiments were repeated three times independently, and similar trends were observed. The results of one experiment are shown.

Zur does not Affect the Growth of P. odoriferum BCS7
To determine whether the effect of the Zur on the virulence of P. odoriferum BCS7 was due to its effects on the bacterial growth, we compared the growth curve of the overexpression and mutant strains, Po (Zur) and ΔZur, and their corresponding control strains, Po (EV) and Po WT, in standard Luria-Bertani (LB) growth medium. The growth curve was monitored for 144 h until the bacteria entered the stationary phase and the growth of the four strains showed no obvious differences ( Figure 2). This indicates that the Zur has no effect on the growth of P. odoriferum BCS7 and that its regulatory role in virulence is probably effected through other mechanisms. (a) Virulence of Zuroverexpressing strain Po (Zur) was measured by examining the average area of the disease spot on Chinese cabbage, with the strain Po (EV) harboring an empty vector as control (n = 7). (b) Virulence of the mutant strain of the Zur (∆Zur) was measured by examining the average area of the disease spot on Chinese cabbage, with the wild-type strain (Po WT) as control (n = 7). (c,d) These show the relative expression of the Zur in the Po (Zur) and ∆Zur compared to their control strains Po (EV) and Po WT, respectively. Each column shows the mean and standard deviation (n = 3). Significant differences with p < 0.05 (t-test) were labelled with an asterisk. All experiments were repeated three times independently, and similar trends were observed. The results of one experiment are shown.

Zur does not Affect the Growth of P. odoriferum BCS7
To determine whether the effect of the Zur on the virulence of P. odoriferum BCS7 was due to its effects on the bacterial growth, we compared the growth curve of the overexpression and mutant strains, Po (Zur) and ∆Zur, and their corresponding control strains, Po (EV) and Po WT, in standard Luria-Bertani (LB) growth medium. The growth curve was monitored for 144 h until the bacteria entered the stationary phase and the growth of the four strains showed no obvious differences ( Figure 2). This indicates that the Zur has no effect on the growth of P. odoriferum BCS7 and that its regulatory role in virulence is probably effected through other mechanisms.

Figure 2.
Growth curve over 6 days (144 h) of ΔZur and Po (Zur). The assay was repeated three times independently, and similar trends were found. The results of one experiment are shown. Values are the mean ± SD (n = 3). Po WT: wild-type strain of Po BCS7, which was used as a control for ΔZur; Po (EV): Po BCS7 strain harboring an empty vector, which was used as a control for Po (Zur).

Comparative Transcriptome Analysis of Po (Zur) vs. Po (EV) and ΔZur vs. Po WT.
To identify the effect of the Zur on P. odoriferum at the transcript level, the transcriptomes of the four strains [Po (Zur), Po (EV), ΔZur, and Po WT], which were cultured in standard LB growth medium, were sequenced and two comparisons were performed-Po (Zur) vs. Po (EV) and ΔZur vs. Po WT. Each strain included three biological replicates, and a total of 30.09 Gb raw data and 29.89 Gb clean data were produced. Each sample contained 2.21-2.85 Gb clean data, with a Q20 quality score of 97.67-98.39% and a Q30 of 93.22-94.83% (Table S1). By aligning clean data onto the reference genome of P. odoriferum BCS7 and conducting a comparative gene expression analysis, genes with an adjusted pvalue (padj) < 0.05 and fold change (FC) ≥ 2 were identified as differentially expressed genes (DEGs). A total of 79 DEGs (22 upregulated and 57 downregulated) were identified in the comparison between Po (Zur) and Po (EV), while for ΔZur vs. Po WT, there were 72 DEGs in total, including 56 upregulated and 16 downregulated ones (Figure 3a,b; Tables S2 and S3). The Venn diagrams of the DEGs of these two comparisons showed that they had only four shared DEGs, while most DEGs were distinct between the two groups ( Figure 3c). Taken together, these results suggest that both gain-of-function and loss-offunction mutations of the Zur modulated the transcriptome of P. odoriferum, while the effect may be different.

Comparative Transcriptome Analysis of Po (Zur) vs. Po (EV) and ∆Zur vs. Po WT
To identify the effect of the Zur on P. odoriferum at the transcript level, the transcriptomes of the four strains [Po (Zur), Po (EV), ∆Zur, and Po WT], which were cultured in standard LB growth medium, were sequenced and two comparisons were performed-Po (Zur) vs. Po (EV) and ∆Zur vs. Po WT. Each strain included three biological replicates, and a total of 30.09 Gb raw data and 29.89 Gb clean data were produced. Each sample contained 2.21-2.85 Gb clean data, with a Q20 quality score of 97.67-98.39% and a Q30 of 93.22-94.83% (Table S1). By aligning clean data onto the reference genome of P. odoriferum BCS7 and conducting a comparative gene expression analysis, genes with an adjusted p-value (padj) < 0.05 and fold change (FC) ≥ 2 were identified as differentially expressed genes (DEGs). A total of 79 DEGs (22 upregulated and 57 downregulated) were identified in the comparison between Po (Zur) and Po (EV), while for ∆Zur vs. Po WT, there were 72 DEGs in total, including 56 upregulated and 16 downregulated ones (Figure 3a,b; Tables S2 and S3). The Venn diagrams of the DEGs of these two comparisons showed that they had only four shared DEGs, while most DEGs were distinct between the two groups ( Figure 3c). Taken together, these results suggest that both gain-of-function and loss-of-function mutations of the Zur modulated the transcriptome of P. odoriferum, while the effect may be different.

Zur Overexpression Negatively Affects the Flagellum and Cell Motility in P. odoriferum
To understand which functions of P. odoriferum were affected by Zur overexpression, gene-ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the obtained DEGs identified in Po (Zur) vs. Po (EV). In the GO enrichment analysis, DEGs in Po (Zur) vs. Po (EV) were associated with 15 functional groups belonging to two main categories: cellular components (CC) and biological processes (BP). In the CC category, DEGs were enriched in GO terms such as bacterial-type flagellum, cell projection, and bacterial-type flagellum part, and in the BP category, DEGs were enriched in eight functional groups, all of which were related to motility functions, including bacterial-type flagellum-dependent swarming motility, locomotion, and ciliumor flagellum-dependent cell motility (Figure 4a). In our KEGG pathway annotation results, the pathway term (level 2) of cell motility had the largest number of DEGs (20), followed by signal transduction (11) and membrane transport (10) (Figure 4b). A KEGG enrichment analysis showed that DEGs were largely enriched in the pathways of flagellar assembly, the two-component system, and bacterial chemotaxis ( Figure 4c). These analyses showed that Zur overexpression regulated the flagellum and cell motility in P. odoriferum.

Zur Overexpression Negatively Affects the Flagellum and Cell Motility in P. odoriferum
To understand which functions of P. odoriferum were affected by Zur overexpression, gene-ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the obtained DEGs identified in Po (Zur) vs. Po (EV). In the GO enrichment analysis, DEGs in Po (Zur) vs. Po (EV) were associated with 15 functional groups belonging to two main categories: cellular components (CC) and biological processes (BP). In the CC category, DEGs were enriched in GO terms such as bacterialtype flagellum, cell projection, and bacterial-type flagellum part, and in the BP category, DEGs were enriched in eight functional groups, all of which were related to motility functions, including bacterial-type flagellum-dependent swarming motility, locomotion, and cilium-or flagellum-dependent cell motility (Figure 4a). In our KEGG pathway annotation results, the pathway term (level 2) of cell motility had the largest number of DEGs (20), followed by signal transduction (11) and membrane transport (10) (Figure 4b). A KEGG enrichment analysis showed that DEGs were largely enriched in the pathways of flagellar assembly, the two-component system, and bacterial chemotaxis (Figure 4c). These analyses showed that Zur overexpression regulated the flagellum and cell motility in P. odoriferum. To confirm the DEGs identified in our comparative transcriptome analysis, 20 DEGs related to cell motility were selected, and their expression changes were validated using qPCR. The expression patterns of these 20 DEGs were consistent with those of the RNA-Seq analysis ( Figure 5), and thus the transcriptome data were confirmed to be reliable. In light of the possible roles of the Zur in the flagellum and cell motility of P. odoriferum, the exact effects were further determined using flagellum staining and a cell motility assay.
Flagellum staining and quantification of the four strains, Po (Zur), Po (EV), ∆Zur, and Po WT, showed that the Po (Zur) exhibited significantly less flagellum (none) in comparison with the control strain Po (EV) (1-2 flagella per bacterium cell) (p < 0.05), while the ∆Zur had no obvious difference in the number of flagella in comparison with the control strain Po WT, both having 1-2 flagella per bacterium cell (p > 0.05) (Figure 6a,b). These results indicate that Zur overexpression negatively affects the development of flagella in P. odoriferum, while depletion of the Zur does not.
Cell motility assays showed that the Po (Zur) had a remarkably reduced halo compared with its control strain, the Po (EV), which nearly swam to the edge of the medium plate (Figure 6c). The strain of the ∆Zur and its control strain, the Po WT, both swam to the full region of the medium plate and showed no obvious difference in swimming motility. The results suggest that Zur overexpression dramatically reduces cell motility in P. odoriferum, whereas Zur knockout does not affect cell motility. To confirm the DEGs identified in our comparative transcriptome analysis, 20 DEGs related to cell motility were selected, and their expression changes were validated using qPCR. The expression patterns of these 20 DEGs were consistent with those of the RNA-Seq analysis (Figure 5), and thus the transcriptome data were confirmed to be reliable. In light of the possible roles of the Zur in the flagellum and cell motility of P. odoriferum, the exact effects were further determined using flagellum staining and a cell motility assay.   (Figure 6a,b). These results indicate that Zur overexpression negatively affects the development of flagella in P. odoriferum, while depletion of the Zur does not.
Cell motility assays showed that the Po (Zur) had a remarkably reduced halo compared with its control strain, the Po (EV), which nearly swam to the edge of the medium plate (Figure 6c). The strain of the ΔZur and its control strain, the Po WT, both swam to the full region of the medium plate and showed no obvious difference in swimming motility. The results suggest that Zur overexpression dramatically reduces cell motility in P. odoriferum, whereas Zur knockout does not affect cell motility.

Zur Depletion Affects Metal-Ion Transport Rather Than the Flagellum and Cell Motility in P. odoriferum
Zur depletion did not show an effect on the flagellum and cell motility in P. odoriferum as Zur overexpression did ( Figure 6). To determine the effect of Zur depletion in P. odoriferum at the transcriptional level, GO and KEGG analyses were also conducted using the

Zur Depletion Affects Metal-Ion Transport Rather Than the Flagellum and Cell Motility in P. odoriferum
Zur depletion did not show an effect on the flagellum and cell motility in P. odoriferum as Zur overexpression did ( Figure 6). To determine the effect of Zur depletion in P. odoriferum at the transcriptional level, GO and KEGG analyses were also conducted using the DEGs identified in ∆Zur vs. Po WT. The GO enrichment analysis showed that DEGs were enriched in nine functional terms of the BP category, with divalent-metal-ion transport, response to metal ions, and the fatty acid catabolic process as the major ones (Figure 7a). The KEGG pathway classification showed that, in this comparison, the DEGs were associated with 21 pathway terms (level 2), mainly including membrane transport (13 DEGs), global and overview maps (12), cellular community-prokaryotes (7), and energy metabolism (6) (Figure 7b). In terms of specific pathways, the DEGs were mainly enriched in the pathways of ABC transporters and quorum sensing (Figure 7c). Notably, the DEGs related to metal-ion transport included BCS7_RS09460 (zinc ABC transporter substrate-binding protein ZnuA), BCS7_RS09455 (zinc ABC transporter ATP-binding protein ZnuC) and BCS7_RS09450 (zinc ABC transporter permease subunit ZnuB), which constitute the znu-ABC zinc-uptake system in prokaryotes [5]. These results suggest that, unlike Zur overexpression, Zur depletion affects pathways such as ion transport rather than the flagellum and cell motility in P. odoriferum, suggesting that the Zur affects the virulence of P. odoriferum via a dual mechanism.

Discussion
Zur was found in the genome of P. odoriferum. Research on Zur has shown that mu-

Discussion
Zur was found in the genome of P. odoriferum. Research on Zur has shown that mutating this gene decreases the virulence of many pathogenic bacteria, including S. enterica, X. campestris, X. oryzae pv. oryzae, and L. monocytogenes [16,20,23,26], except for S. aureus, whose Zur mutant did not show significantly different virulence [15]. To determine the precise function of the Zur in the virulence of P. odoriferum, Zur mutant and overexpression strains were constructed. The virulence assay showed that Zur overexpression significantly reduced the virulence of P. odoriferum on Chinese cabbage, while the Zur mutant exhibited increased virulence. The regulatory role of the Zur on the virulence of P. odoriferum differs from the Zur in previous reports. This indicates that the Zur may contribute to virulence through multiple mechanisms.
In S. enterica, the LD 50 of the Zur mutant increased approximately tenfold when mice were challenged intraperitoneally. The underlying reason for this virulence decrease could be related to the lower transcriptional expression of fliAZ in this mutant than in the wildtype strain, as the product of flagellar gene fliZ is necessary for the virulence of Salmonella serovar Typhimurium cells [16,35]. For X. campestris [20] and X. oryzae pv. oryzae [23], their Zur mutants showed virulence deficiency on host plants, and this could be explained by the decreased production of EPS, which is an important virulence factor in many pathogens and whose production shortage has been related to loss of virulence [23,36]. In our study, Zur overexpression in P. odoriferum reduced the number of flagella and attenuated the cell motility, whereas mutating it did not. The bacterial flagellum is one of the most important organelles. Developed on the bacterial cell surface, it enables bacteria to swim from unfavorable to favorable environments [37,38] and this structure also participates in bacterial pathogen infection through their roles in host cell adhesion, cell invasion, and the secretion of non-flagellar bacterial proteins that are involved in the virulence process [39]. A likely scenario here is that a large amount of the Zur inhibits the virulence of P. odoriferum by impacting on flagellum production and cell motility, yet the precise mechanism requires further investigation.
In contrast, the loss-of-function mutation of the Zur in P. odoriferum did not cause changes in flagellum production and cell motility, and comparative transcriptome analysis showed that DEGs induced by mutating the Zur were not related to flagellum and cell motility. They were mainly involved in metal-ion transport and ABC transporters (Figure 7). Interestingly, DEGs related to metal-ion transport included BCS7_RS09460 (encoding zinc ABC transporter substrate-binding protein ZnuA), BCS7_RS09455 (encoding zinc ABC transporter ATP-binding protein ZnuC), and BCS7_RS09450 (encoding zinc ABC transporter permease subunit ZnuB). These three genes were all upregulated in the ∆Zur, suggesting a negative regulating role of the Zur on the znuABC system in P. odoriferum. This result is unsurprising as the Zur has been reported to regulate zinc homeostasis via the znu-ABC zinc-uptake system in bacteria [6]. Zinc is an essential trace element in most organisms, but an excess of it can be toxic. Consequently, to maintain appropriate zinc levels in cells, bacteria have evolved both uptake and export systems that function in concert. Growing evidence has shown that the efficient uptake of divalent metals, including zinc, is critical to successful bacterial infection and virulence [40,41]. Accordingly, the znuABC zinc-uptake system, which belongs to the ABC transporter family, is found to be critical for bacterial pathogen virulence, such as full Salmonella virulence, in different animal models [42]. As the function of this system is negatively regulated by the Zur protein [9,20,43], it could also be that the increased virulence observed in our Zur mutant is due to the upregulation of its zinc-uptake-related znuABC genes. This hypothesis requires further investigation.
Taken together, our results indicate that Zur might play a negative role in regulating the virulence of P. odoriferum. Excessive Zur attenuated virulence-related flagellum and cell motility, while getting rid of the Zur might promote the virulence-related znuABC zinc-uptake system. Thus, the underlying mechanisms of the role of the Zur in virulence might be a dose-dependent strategy: a high expression of the Zur attenuates the flagellum and cell motility to reduce virulence, while a low expression of the Zur upregulates the zinc-uptake system of znuABC to enhance virulence. As a transcription factor, the Zur regulates the expression of its regulon genes by sensing the intracellular Zn concentrations, but it is important to know what controls the expression of the Zur. Previous studies have provided evidence that the Zur autoregulates its expression in the majority of bacterial species [44,45]. For example, in some species such as Bacillus anthracis, the Zur autoregulates its expression, being a part of an operon with the znuB and znuC [6]. It will be of great future interest to perform further experiments to demonstrate the detailed mechanism of the Zur in P. odoriferum virulence.

Bacterial Strains, Plasmids, and Culture Conditions
All bacterial strains and plasmids used in this study are listed in Table S4. A rifampicinresistant strain, P. odoriferum BCS7 (and its derivatives), and Escherichia coli were grown on Luria-Bertani broth (LB) or LB agar (LA) plates. The growth temperatures for the Pectobacterium and E. coli strains were 28 • C and 37 • C, respectively. Antibiotics were used at the following concentrations when required: 100 µg/mL rifampin (Rif); 50 µg/mL kanamycin (Km); 50 µg/mL gentamicin (Gm); and 50 µg/mL streptomycin (Sm).

Construction of the P. odoriferum Mutant and the Zur-overexpressing Strain
The Zur loss-of-function mutant of P. odoriferum BCS7 (Po BCST) was constructed by using homologous recombination, as described previously [46], with minor modifications. Briefly, the upstream and downstream regions of the target gene Zur were amplified by using PCR with specifically designed primers (Table S5). A kanamycin cassette amplified from pET30a was ligated with these two fragments and cloned into pEX18Gm using an In-Fusion HD Cloning Kit (Takara, China) to yield a recombinant vector. This vector was transferred to Po BCS7 by means of conjugation using E. coli S17-1λpir. To select strains with gene deletion, transconjugants were plated on LA containing 10% sucrose, Rif, and Km, and the positive colonies were confirmed by means of PCR. To overexpress the Zur gene in the Po BCS7 strain, the open reading frame (ORF) of the Zur, together with its upstream promoter sequence (500 bp), were amplified using the genomic DNA of the Po BCS7 strain with the specific primers P-Zur-ORF-S1 and Zur-ORF-AS1, and the PCR products were ligated into the linearized plasmid of pBBRIMCS-5 using the In-Fusion HD Cloning Kit (Takara, China). The recombinant construct was transformed into E. coli DH5α and conjugated to Po BCS7 by triparental mating with E. coli S17-1λpir. The conjugants were selected on LA containing Rif and Gm and confirmed by means of PCR. The primers used for making the constructions are listed in Table S5.

Pathogenicity Assessment of P. odoriferum with Zur Depletion or Overexpression
The pathogenicity of Pectobacterium strains with Zur knockout (∆Zur) or overexpression [Po (Zur)] compared to their respective control strains, Po WT and Po (EV), was determined in vitro by macerating the petioles of Chinese cabbage. For the inoculation, we largely followed the method described by Li et al. [47], with minor modifications. Briefly, 3 µL bacterial suspension (2 × 10 8 CFU/mL) per site for each strain was used to inoculate Chinese cabbage at 2.5 mm depth, and the rot area for each petiole was measured 24 h later. The virulence of each strain was examined with nine petioles in the pathogenicity assessment (three petioles per Petri dish), and the experiment was repeated three times independently, with similar results.

Transcriptome Sequencing and Analysis
For our transcriptome sequencing, three replicates were included for each strain. Cells of the ∆Zur and Po (Zur) and their respective control strains, i.e., wild-type strain (Po WT) and empty-vector introduced strain [Po (EV)], were cultured in LB with Rif from a 1% dilution of their seed cultures (OD 600 = 1.0) overnight (16 h). One milliliter of the cultures was centrifuge-pelleted at 8000 rpm for 2 min and washed once with RNA-free water before total RNA extraction using TRIzol. The obtained total RNA was used to prepare sequencing libraries using the NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina ® (NEB, USA) following the manufacturer's recommendations, and the sequencing was performed on an Illumina NovaSeq 6000 platform. The raw reads of each sample were filtered to produce clean data by means of removing reads containing adapters or poly-N and low-quality reads. We mapped the clean data to the P. odoriferum BCS7 reference genome (CP009678.1) using STAR software (2.7.10b) [48] and obtained the counts of reads that were uniquely mapped to each gene model. Gene expression levels were normalized as fragments per kilobase of transcripts per million mapped reads (FPKM). DEGs were identified using the DESeq2 package in R [49]. Only genes with an adjusted p-value (padj) < 0.05 and an absolute value of log 2 (fold change) ≥ 1 were considered as DEGs. The DEGs were annotated using the GO and KEGG databases. GO enrichment was performed using clusterProfiler software (v3.18.0) with a p-value threshold of 0.05 in Fisher's exact test [50]. The KEGG enrichment was conducted with a hypergeometric test to analyze the enriched KEGG pathway terms of the DEGs, with the whole genome as the background.

Validation of mRNA-Seq by Quantitative Real-time PCR (qPCR)
Twenty DEGs related to cell motility were selected for validation by means of qPCR from the transcriptome data of Po (Zur) vs. Po (EV). The cDNA was prepared from the remaining total RNA after transcriptome sequencing by EasyScript One-step gDNA removal and cDNA synthesis supermix (TransGen Biotech, Beijing, China) and subjected to qPCR using TB Green ® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, China) as previously described [51]. RecA was used as the internal reference gene. The fold change in the relative gene expression of targets in samples of Po (Zur) compared with Po (EV) was analyzed using the 2 −∆∆Ct method [52]. All qPCR primers are listed in Table S5. The experiment was repeated three times independently, with three technical replicates each time.

Phenotypic Assays
Bacterial growth curves: cells of Po (Zur), Po (EV), ∆Zur, and Po WT were cultured overnight in LB medium with Rif for 12-16 h, and the cultures were adjusted to OD 600 = 1.0 using the original medium. Subsequently, cell suspensions were diluted with 20 mL LB medium containing Rif at a ratio of 1:100 for growth at 200 rpm, and the OD 600 value was monitored after incubation for 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144 h with an Eppendorf BioPhotometer Plus (Eppendorf, Germany). Flagella staining and observation: bacterial staining was conducted using the silver staining method as previously described [53], and flagella were observed under an optical microscope. Swimming motility assay: a single colony of Po (Zur), Po (EV), ∆Zur, and Po WT was cultured in the LB medium with Rif for 12-16 h to OD 600 = 1.0. The cell motility of each strain was visualized based on swimming haloes on an LA plate containing 0.3% agar 16 h after 3 µL of the cell suspension was patched onto it and incubated at 28 • C. The experiment was repeated three times with at least three replicates.

Statistical Analysis
A statistical analysis of data differences was performed using an independent t-test or one-way ANOVA in IBM SPSS Statistics 20.