Abstract

   Xenorhabdus nematophila has excellent potential for application in both
   medicine and agriculture due to its various active secondary
   metabolites. The transcriptional regulator OmpR negatively regulates
   Xenocoumacin 1 (Xcn1), which has wide antimicrobial activity. Here, we
   expressed and purified OmpR and verified its binding activities to
   promoters via an electrophoretic mobility shift assay. RNA sequencing
   was used to analyze the relevance and difference of differentially
   expressed genes between X. nematophila and its mutant ΔompR. Compared
   with the WT, 1127 differentially expressed genes were found in ΔompR,
   while 4150 co-expressed genes were detected. RT-qPCR data validated the
   RNA-seq results with 20 randomly selected genes. OmpR positively
   regulates the process of porphyrin metabolism, quorum sensing, β-Lactam
   resistance and glyoxylate and dicarboxylate metabolism, while
   negatively regulating the phosphotransferase system, two-component
   system and bacterial chemotaxis. OmpR indirectly regulates the
   biosynthesis of Xcn1 by positively regulating the process of glyoxylate
   metabolism, which consumes energy and precursors, and negatively
   regulates biomacromolecules biosynthesis, which provides energy and
   precursors. Overall, this work revealed the indirect effects of OmpR on
   the biosynthesis of Xcn1, serving as a foundation for future research
   into the intricate regulatory network of X. nematophila.

   Keywords: Xenorhabdus nematophila, transcriptome, xenocoumacin 1, OmpR,
   EMSA

1. Introduction

   Xenorhabdus nematophila, a Gram-negative bacterium classified as
   Morganellaceae, forms a mutualistic symbiosis with the entomopathogenic
   nematode Steinernema [[34]1,[35]2]. Both the bacterium Xenorhabdus and
   the nematode Steinernema generate a variety of specialized metabolites
   during the life cycle of the mutualistic symbiosis that act
   synergistically to ensure their proliferation and reproduction
   [[36]3,[37]4]. Xenorhabdus has excellent potential for application in
   both medicine and agriculture. Particularly, compounds produced by
   Xenorhabdus have a broad spectrum of biological activity, inhibiting
   the growth of bacteria [[38]5], fungi [[39]6], oomycetes [[40]7],
   acarine [[41]8], nematode [[42]9], insects [[43]10] and protozoa
   [[44]11].

   Xenocoumacin 1 (Xcn1) is a significant antimicrobial compound produced
   by X. nematophila and has wide prospects for application in sustainable
   agricultural development. In the biosynthetic gene cluster of Xcn1,
   xcnA-L genes contribute to Xcn1 biosynthesis and xcnMN genes act on
   Xcn1 to transform its analogs [[45]4,[46]12]. Acetyl coenzyme A and 1,
   3-bisphosphoglycerate are important precursors of Xcn1 biosynthesis
   [[47]4]. Seven transcriptional regulatory factors regulate Xcn1
   biosynthesis: FliZ [[48]13], Hfq [[49]2] and Lrp [[50]3] regulate Xcn1
   positively, while CpxR [[51]14], LeuO [[52]3], LrhA [[53]15] and OmpR
   [[54]16] regulate it negatively.

   The production of diverse active secondary metabolites is attributed to
   the environment in which X. nematophila exists. Two-component systems,
   including CpxA/CpxR and EnvZ/OmpR, play a crucial role in responding to
   nematodes and the environment. The sensor histidine kinases CpxA and
   EnvZ sense changes in pH and osmolarity, and phosphorylate the
   regulators CpxR and OmpR to govern target genes [[55]14,[56]17,[57]18].
   OmpR, a vital transcriptional regulator that governs over a hundred
   genes, is responsible for controlling antibiotic production and the
   formation of flagella and exoenzymes in X. nematophila [[58]19,[59]20].
   OmpR binds to the promoter region to regulate target genes directly.
   There is a binding site of OmpR in the promoter region of the opnP gene
   in X. nematophila [[60]21]. The ability of phosphorylated OmpR to bind
   to promoters was significantly higher than that of unphosphorylated
   OmpR [[61]22]. Crosstalk between CpxA and OmpR or between EnvZ and
   CpxR, might happen when there is a lack of cognate signaling partners
   [[62]23]. Interestingly, OmpR and CpxR occasionally bind to the same
   promoter simultaneously [[63]24]. Although CpxR and OmpR jointly
   regulate some physiological processes, whether there is a direct
   regulation between these two regulators is still unknown. OmpR
   negatively governs the production of Xcn1 by negatively regulating
   xcnA-L and positively regulating xcnMN expression [[64]16]. However, it
   is unclear which regulatory factor directly regulates the biosynthesis
   of Xcn1 by binding to the promoter region of the xcnA gene.

   In this work, the transcriptional regulation factor OmpR was purified,
   and the DNA-binding ability in promoters was determined. Furthermore,
   ompR gene deletion mutant strains were constructed from X. nematophila
   YL001, and differentially expressed genes were analyzed via enrichment
   analysis. In addition, a model for regulating Xcn1 biosynthesis via
   OmpR was preliminarily established.

2. Materials and Methods

2.1. Strains and Their Growth Conditions

   Xenorhabdus nematophila YL001 was isolated from its symbiotic nematode,
   Steinernema sp. YL001, which was obtained from the soil of Yangling,
   China [[65]25,[66]26]. The morphological and molecular characteristics
   of X. nematophila YL001 have been identified [[67]26]. Detailed
   information on the strains and plasmids utilized in this study is
   displayed in [68]Table 1.

Table 1.

   Bacterial strains and plasmids used in this study.
   Strains Relevant Characteristics Source
   Xenorhabdus nematophila
   YL001 Wild type; Amp^r Laboratory stock
   ΔompR ompR of X. nematophila YL001 was replaced by Km^r This study
   Escherichia coli
   S17-1λpir Donor strain for conjugations AngYuBio
   DH5α Strain for the reproduction of recombinant pET28a AngYuBio
   BL21(DE3) Strain for protein expression AngYuBio
   Plasmids
   pDM4 Suicide vector; Cm^r, sacB Laboratory stock
   pDM4-ompR-Km^r Recombinant plasmid pDM4 for ΔompR construction This
   study
   pET28a Source of Km^r gene; Vector for protein expression Laboratory
   stock
   pET28a-OmpR Protein expression vector of OmpR-N[6His] This study
   [69]Open in a new tab

   Note: Amp^r, Ampicillin resistance; Km^r, Kanamycin resistance; Cm^r,
   Chloramphenicol resistance.

   X. nematophila and E. coli were grown in Luria–Bertani (LB) medium at
   28 °C and 37 °C, respectively. Agar and antibiotics were supplied to
   the LB medium if desired. Kanamycin is used to culture E. coli
   containing vectors for heterologous expression of OmpR. Chloramphenicol
   is used to screen for E. coli containing suicide vectors for ompR
   knockout. Sucrose and kanamycin were used to screen for ΔompR.

2.2. DNA Manipulation

   Genomic DNA and plasmid were extracted individually using the Rapid
   Bacterial Genomic DNA Isolation Kit (Sangon, Shanghai, China) and the
   HiPure Plasmid EF Mini Kit (Magen, Foshan, China) according to the
   manufacturer’s instruction manual. Polymerase chain reaction (PCR)
   amplification was conducted with the help of Hieff Canace^® Plus
   High-Fidelity DNA Polymerase (Yeasen, Shanghai, China) and primers
   listed in [70]Tables S1–S3. The DNA fragments produced by PCR were
   purified utilizing the SanPrep Column DNA Gel Extraction Kit (Sangon,
   China). Recombinant plasmids were structured by the Hieff Clone^®
   Universal One Step Cloning Kit (Yeasen, China). Primers for PCR
   amplification were designed by Primer Premier 5.0 software [[71]27].
   Recombinant plasmids and mutant strains were verified by DNA sequencing
   (AuGCT, Beijing, China).

2.3. Protein Expression and Purification

   Protein expression and purification were conducted based on published
   methods [[72]28,[73]29]. In detail, fragments containing homologous
   arms for recombinant vector construction were amplified from the
   chromosomal DNA of X. nematophila YL001. Primers for heterologous
   expression of regulators are shown in [74]Table S1. Fragments of
   OmpR-N[6His] were cloned into pET28a plasmids that were cleaved using
   BamHI (Takara, Osaka, Japan) and NdeI (Takara, Osaka, Japan), creating
   pET28a-OmpR. Recombinant vectors were transformed into E. coli DH5α for
   reproduction. After DNA sequencing, recombinant plasmids were
   transformed into E. coli BL21 (DE3). E. coli BL21 (DE3) was cultured at
   37 °C and 200 rpm until an optical density up to 1 at 600 nm. After
   isopropyl-beta-D-thiogalactopyranoside (IPTG) was supplemented with
   concentrations of 0.5 mM, cultures were shaken overnight at 16 °C and
   180 rpm.

   Cells were collected by centrifugation and resuspended in lysis buffer
   (50 mM NaH[2]PO[4], 300 mM NaCl, pH 8.0). Subsequently, lysozyme (1
   mg/mL final), AEBSF (2 mM final), bestatin (0.13 mM final) and
   leupeptin (10 μM final) were added to the cell suspension and incubated
   on ice for 30 min. Cells were lysed by passage through the Ultrasonic
   cell pulverizer (Scientz Biotechnology, Ningbo, China) six times, and
   the lysate was centrifuged for 30 min at 4 °C and 10,000 rpm to collect
   the supernatant. The supernatant was subjected to BeyoGold™ His-tag
   Purification Resin (Beyotime, Shanghai, China) for protein binding.
   Purified protein was obtained by washing and elution with imidazole
   solutions in gradient concentrations (10~100 mM imidazole, 50 mM
   NaH[2]PO[4], 300 mM NaCl, pH 8.0). Eluted fractions were sampled and
   analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
   (SDS-PAGE). Purified OmpR-N[6His] was represented by SDS-PAGE and
   captured by Xiaomi 11 LE. Purified protein was concentrated using the
   Microsep Advance Centrifugal Device (Pall Corporation, Port Washington,
   NY, USA) at 8000 rpm and 4 °C and stored at −20 °C.

2.4. Electrophoretic Mobility Shift Assay (EMSA)

   EMSAs were carried out according to the reported method
   [[75]30,[76]31]. In detail, DNA probes were amplified from the
   chromosomal DNA of X. nematophila YL001 by PCR using the primers listed
   in [77]Table S2. Concentrations of purified protein and DNA probe were
   determined by the K5800 ultramicro spectrophotometer (KAIAO, Beijing,
   China). DNA probes, purified protein and 5× reaction buffer (250 mM
   Tris, 250 mM KCl, 0.5 mM DTT, 50 mM MgCl[2], 25% glycerol, pH 8.0) were
   incubated for 30 min at room temperature. The reaction mixture was
   supplemented with 5× EMSA loading buffer (Beyotime, Shanghai, China) to
   stop the reaction and subsequently analyzed by 6% native acrylamide gel
   electrophoresis that was performed at 90 V in 0.5× TBE buffer (45 mM
   Tris-HCl, 45 mM boric acid, pH 8.0) at 4 °C. Gels were dyed with Ultra
   GelRed (Vazyme, Nanjing, China), and the results were captured by
   GenoSens transilluminator (Clinx, Shanghai, China).

2.5. Construction of Mutant Strains

   For recombinant suicide vector construction, fragments that contained
   homologous arms upstream and downstream of ompR were amplified
   individually from the chromosomal DNA of X. nematophila YL001 by PCR
   using the primers in [78]Table S3. Kanamycin-resistant fragments
   containing homologous arms were separately amplified from pET28a.
   Primers for the construction and verification of mutant strains are
   shown in [79]Table S3. The upstream, downstream and kanamycin-resistant
   fragment were connected and cloned into pDM4 plasmids that were cleaved
   using restriction endonuclease SphI (Takara, Japan) and SacI (Takara,
   Japan). Recombinant plasmids were transformed into E. coli S17-1λpir
   and conjugally transferred into the X. nematophila YL001
   [[80]14,[81]16]. Successful recombinants were identified in the LB
   medium containing sucrose, ampicillin and kanamycin.

2.6. RNA Isolation and Library Preparation

   RNA was extracted with Trizol after the bacteria used for RNA
   extraction were grown in the LB medium to the logarithmic growth phase
   at 28 °C and 180 rpm. Genomic DNA was disintegrated with the help of
   DNase Ⅰ without RNase. The integrity and quality of RNA were evaluated
   utilizing the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system
   (Agilent Technologies, Santa Clara, CA, USA). The purification of mRNA
   from total RNA was performed by using probes to remove rRNA.
   Subsequently, random hexamer primers and M-MuLV Reverse Transcriptase
   were employed to synthesize the first strand cDNA, and subsequently,
   RNaseH was used to degrade the RNA. The second strand of cDNA was
   synthesized using dUTP to replace the dTTP of dNTP as the raw material
   in the DNA polymerase I system. Blunt ends were produced by
   exonuclease/polymerase activities from the remaining overhangs.
   Adaptors with hairpin loop structures were ligated to prepare for
   hybridization after they had been adenylated on the 3′ ends of DNA
   fragments. Then, USER Enzyme was used to degrade the second strand of
   cDNA containing U. Purification of the library fragments with the
   AMPure XP system (Beckman Coulter, Brea, CA, USA) was performed to
   select the preferred cDNA fragments, which ranged from 370 to 420 bp in
   length. Products were purified by the AMPure XP system after PCR
   performed with Phusion High-Fidelity DNA polymerase, Universal PCR
   primers and Index (X) primers. Library quality was assessed on the
   Agilent Bioanalyzer 2100 system. As per the manufacturer’s
   instructions, the index-coded samples were clustered on a cBot Cluster
   Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia,
   San Diego, CA, USA). After cluster generation, the purified cDNA
   library was sequenced using the Illumina Novaseq platform, and then 150
   bp paired-end reads were generated.

2.7. Bioinformatics Analysis

   First, in-house perl scripts were used to process raw data (raw reads)
   in the fastq format. Clean data (clean reads) were obtained by removing
   reads containing adapters, reads containing N bases and low-quality
   reads from raw data. At the same time, Q20, Q30 and GC content of the
   clean data were calculated. All the downstream analyses were based on
   clean data of high quality. Next, Bowtie 2 (2.2.3) was used to map the
   clean reads to the X. nematophila YL001 transcriptome [[82]32]. The
   number of reads mapped to each gene was counted by HTSeq (version
   0.6.1) [[83]33]. And then the FPKM (Fragments Per Kilobase of
   transcript per Million mapped reads) of each gene was calculated based
   on the length of the gene and read counts mapped to this gene.
   Differential expression analysis of groups was performed using the
   DESeq R package (1.18.0) [[84]34]. Gene Ontology (GO) enrichment
   analysis of DEGs was implemented by the GOseq R package, in which gene
   length bias was corrected [[85]35]. The statistical enrichment of genes
   that are differentially expressed in KEGG pathways was tested using
   KOBAS software (3.0) [[86]36,[87]37].

2.8. Reverse Transcription Quantitative PCR (RT-qPCR)

   Total RNA was extracted using the FastPure Cell/Tissue Total RNA
   Isolation Kit (Vazyme, Nanjing, China). HiScript III RT SuperMix for
   qPCR (Vazyme, China) was used to produce cDNA, and the FastHS SYBR QPCR
   mixture (ALLMEEK, Beijing, China) was used to amplify the tested genes.
   RT-qPCR amplifications were performed using the CFX96 Touch gene
   amplification instrument (BIO-RAD, Hercules, CA, USA). The relative
   expression levels were calculated using the 2^−ΔΔCt method [[88]38] and
   the housekeeping gene recA [[89]14] as the internal control to
   normalize the transcript levels of the tested genes.

3. Results and Discussion

3.1. Validation of Binding Activity of OmpR in Promoters

   Recombinant vector pET28a-OmpR was constructed and verified to express
   OmpR ([90]Figure 1a,b). OmpR was purified by imidazole solution and
   displayed by polyacrylamide gel electrophoresis ([91]Figure 1c). The
   binding activity of active OmpR was determined by an EMSA. The promoter
   of opnP ([92]Figure 2a) was the positive control according to published
   data [[93]21]. A previous study suggested that OmpR negatively
   regulated the production of Xcn1 [[94]16].

Figure 1.

   [95]Figure 1
   [96]Open in a new tab

   Heterologous expression of OmpR of X. nematophila YL001. (a) Vector
   containing ompR for OmpR labeled with 6 histidine. (b) Verification of
   recombinant vectors for protein expression by PCR using primers
   28a-F/R. (c) Purified protein OmpR-N[6His] (29.52 kDa) pointed by
   purple arrows.

Figure 2.

   [97]Figure 2
   [98]Open in a new tab

   Verification of OmpR binding to promoter of various genes. LPAP,
   lithium potassium acetyl phosphate. The numbers in brackets represent
   the position of DNA in the genome of X. nematophila YL001. (a) OmpR
   bound to the promoter of opnP. (b) OmpR did not bind to the promoter of
   xcnA. (c) OmpR did not bind to the promoter of lrhA. (d) OmpR did not
   bind to the promoter of leuO. (e) OmpR did not bind to the promoter of
   cpxR. (f) OmpR did not bind to the promoter of ompR.

   In this work, we found that OmpR was indirectly regulated because it
   could not bind to the promoter region of xcnA ([99]Figure 2b). It
   indicated that OmpR could not directly regulate the expression of the
   xcnA gene. OmpR did not bind the promoter of lrhA in this study
   ([100]Figure 2c) but bound to the promoter in Pantoea alhagi [[101]39],
   which indicates the different regulatory mechanisms of OmpR between X.
   nematophila and P. alhagi. LeuO, CpxR and OmpR were negative regulatory
   factors for Xcn1 production [[102]3,[103]14,[104]16]. Regretfully, OmpR
   did not directly regulate leuO ([105]Figure 2d) and cpxR ([106]Figure
   2e) because no binding to the promoter region probe was detected, which
   demonstrated that OmpR cannot directly regulate two regulatory factors,
   LeuO and CpxR. Moreover, OmpR did not directly regulate its gene
   expression, as no obstruction was detected ([107]Figure 2f).

3.2. Construction and Verification of Strains ΔompR

   To investigate the regulation mechanism of OmpR in X. nematophila on
   biosynthesis of Xcn1, ΔompR was constructed by replacing the ompR gene
   with the kanR box separately ([108]Figure 3a). Thus, 1038 bp fragments
   upstream of ompR, 941 bp kanamycin-resistant cassette fragments and 990
   bp fragments downstream of ompR were multiplied for the construction of
   the gene knockout vector ([109]Figure 3b). Internal and external
   primers were used to verify ΔompR ([110]Figure 3c). In ΔompR, no
   products were generated by primers ompR-in-F/R, and a longer fragment
   was generated by primers ompR-out-F/R compared to the wild type (wt).

Figure 3.

   [111]Figure 3
   [112]Open in a new tab

   Construction and verification of strains ΔompR. (a) Graphical overview
   of gene knockout, including primers (yellow arrow) and location of ompR
   (cyan box) and kanR (red box). (b) Fragments for construction of ΔompR.
   Up: upstream of ompR (1038 bp); KanR: kanamycin-resistant cassette (941
   bp); Down: downstream of ompR (990 bp). (c) Identification of the ΔompR
   with internal and external primers: 454 bp PCR products were generated
   by ompR-in-F/R with wild type (wt) as template, while no products were
   generated with ΔompR as template; 1118 bp PCR products were generated
   by primers ompR-out-F/R with wt as template; 1424 bp PCR products were
   generated with ΔompR as template.

3.3. Quantitative Analysis and Differential Expression Analysis of Genes

   The Pearson correlation R^2 between samples of strains is displayed,
   illustrating that replicates of samples within groups were reliable
   ([113]Figure 4a). The two-dimensional plot reveals that different
   individuals from the same group cluster together through principal
   component analysis (PCA), demonstrating that the sum of technical and
   biological variation is significantly smaller than the changes between
   ΔompR and WT ([114]Figure 4b). In this experiment, 4150 co-expressed
   genes were detected ([115]Figure 4c). Compared with the WT (|log2 Fold
   Change| > 0, p adj < 0.05), a total of 526 up-regulated genes and 601
   down-regulated genes were detected in ΔompR ([116]Figure 4d). Randomly,
   twenty significantly differentially expressed genes (DEGs) were
   selected to perform an RT-qPCR experiment for validating the data of
   RNA-Seq ([117]Figure 4e). The RT-qPCR results of the DEGs were in
   accordance with the results observed in the RNA-seq data.

Figure 4.

   [118]Figure 4
   [119]Open in a new tab

   Correlation and differentially expressed genes analysis based on
   RNA-Seq. (a) Correlation of gene expression levels between X.
   nematophila YL001 (WT) and ΔompR. (b) Principal component analysis
   (PCA) of gene expression levels between WT and ΔompR. (c) Venn diagram
   of co-expressed genes between WT and ΔompR. (d) Volcano map of
   differentially expressed genes between ΔompR and WT. (e) Differentially
   expressed genes of RNA-Seq in ΔompR were validated by RT-qPCR.

3.4. Enrichment Analysis of Differentially Expressed Genes in ΔompR

   From the GO enrichment analysis results, the top 30 enriched terms were
   selected to draw a bar chart for display. Among the decreased
   expression genes in ΔompR, nine terms of biological process, including
   metabolic and biosynthetic processes of cobalamin, vitamin and
   tetrapyrrole, and two terms of molecular function, including pyridoxal
   phosphate binding and vitamin B6 binding, were significantly (p < 0.05)
   enriched ([120]Figure 5a). Among the increased expression genes in
   ΔompR, 33 terms of biological process, including the regulation of
   biological process, chemotaxis, response to external stimulus and the
   regulation of macromolecule metabolic process, among others, and 2
   terms of molecular function, including metal ion binding, were
   significantly (p < 0.05) enriched ([121]Figure 5b).

Figure 5.

   [122]Figure 5
   [123]Open in a new tab

   Gene Ontology (GO) enrichment analysis of differentially expressed
   genes. Abscissa represents the GO term that was significantly enriched,
   and ordinate represents the significance level of the GO term enriched.
   A higher value of ordinate indicates that the term was enriched more
   significantly. Different colors represent the three subclasses:
   biological process (BP, red), cellular component (CC, green) and
   molecular function (MF, blue). (a) Enriched terms of decreased genes in
   the ΔompR. (b) Enriched terms of increased genes in the ΔompR.

   The top 20 enriched pathways were selected to draw a dot diagram to
   display the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   enrichment analysis of the DEGs. Decreased expression genes in ΔompR
   were enriched in four pathways: porphyrin metabolism, including 22
   genes; quorum sensing, including 22 genes; beta-Lactam resistance,
   including 11 genes; and glyoxylate and dicarboxylate metabolism,
   including 13 genes ([124]Figure 6a). Increased expression genes in
   ΔompR were enriched in three pathways: the phosphotransferase system,
   including 8 genes; the two-component system, including 24 genes; and
   bacterial chemotaxis, including 9 genes ([125]Figure 6b).

Figure 6.

   [126]Figure 6
   [127]Open in a new tab

   KEGG pathway enrichment analysis of differentially expressed genes. The
   abscissa is the ratio of the number of DEGs annotated to the KEGG
   pathway and the total number of DEGs, and the ordinate is the KEGG
   pathway. The number of DEGs was displayed by the size of the dots. The
   color of the dots gradually changes from purple to red, indicating that
   the pathway was enriched more significantly. (a) KEGG pathway
   enrichment analysis of down-regulated genes in ΔompR. (b) KEGG pathway
   enrichment analysis of up-regulated genes in ΔompR.

   The biosynthesis of Xcn1 required energy and precursor
   1,3-bisphosphoglycerate [[128]4]. OmpR negatively regulated Xcn1 in X.
   nematophila [[129]16]. GO enrichment found that the macromolecule
   biosynthetic process, including 41 genes, was negatively regulated by
   OmpR. It is presumed that OmpR regulates biosynthesis of Xcn1 by
   negatively regulating biomacromolecules biosynthesis ([130]Figure 5b).
   KEGG pathway enrichment analysis found that down-regulated genes were
   significantly enriched in the glyoxylate and dicarboxylate metabolism
   pathway in ΔompR. Glyoxylate and dicarboxylate metabolism pathway
   consumed the energy and Xcn1 biosynthesis precursors. Therefore, we
   supposed that OmpR regulates the biosynthesis of Xcn1 by positively
   regulating the process of glyoxylate metabolism ([131]Figure 7).

Figure 7.

   [132]Figure 7
   [133]Open in a new tab

   OmpR regulates biosynthesis of Xcn1 indirectly in X. nematophila. Green
   arrows represent positive regulation, and red T-shaped arrows represent
   negative regulation.

4. Conclusions

   OmpR positively regulates the process of porphyrin metabolism, quorum
   sensing, β-Lactam resistance and glyoxylate and dicarboxylate
   metabolism, while negatively regulating the phosphotransferase system,
   the two-component system and bacterial chemotaxis. OmpR indirectly
   regulates the biosynthesis of Xcn1 by positively regulating the process
   of glyoxylate metabolism, which consumes energy and precursors, and
   negatively regulates biomacromolecules biosynthesis, which provides
   energy and precursors ([134]Figure 7).

Supplementary Materials

   The following supporting information can be downloaded at
   [135]https://www.mdpi.com/article/10.3390/microorganisms13061360/s1,
   Table S1. Primers for protein expression; Table S2. Primers for
   electrophoretic mobility shift assay; Table S3. Primers for
   construction and verification of mutant strains. Supplementary Excels:
   Enrichment analysis results of differentially expressed genes in ΔompR.
   [136]microorganisms-13-01360-s001.zip^ (46.7KB, zip)

Author Contributions

   Data curation, Y.H. and X.Z.; Formal analysis, Y.H. and X.Z.; Funding
   acquisition, S.Z. and Y.W.; Investigation, X.Z. and M.H.; Methodology,
   Y.H., S.Z. and G.T.; Project administration, Y.W.; Software, G.T.;
   Supervision, Y.W.; Validation, X.Z. and M.H.; Writing—original draft,
   Y.H.; Writing—review & editing, Y.H., M.H. and S.Z. All authors have
   read and agreed to the published version of the manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The original contributions presented in this study are included in the
   article/[137]Supplementary Material. Further inquiries can be directed
   to the corresponding authors.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This work was funded by the National Natural Science Foundation of
   China (32072474) acquired by Yonghong Wang and funded by the
   Independent Innovation Fund of Agricultural Science and Technology of
   Ningxia Hui Autonomous Region (NGSB-2021-10-04) acquired by Shujing
   Zhang.

Footnotes

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References