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The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

  • Garam Choi
    Footnotes
    Affiliations
    National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, and Center for Food Safety and Toxicology, Seoul National University, Seoul 08826, South Korea
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  • Kyung Ku Jang
    Footnotes
    Affiliations
    National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, and Center for Food Safety and Toxicology, Seoul National University, Seoul 08826, South Korea
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  • Jong Gyu Lim
    Affiliations
    National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, and Center for Food Safety and Toxicology, Seoul National University, Seoul 08826, South Korea
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  • Zee-Won Lee
    Affiliations
    National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, and Center for Food Safety and Toxicology, Seoul National University, Seoul 08826, South Korea
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  • Hanhyeok Im
    Affiliations
    National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, and Center for Food Safety and Toxicology, Seoul National University, Seoul 08826, South Korea
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  • Sang Ho Choi
    Correspondence
    To whom correspondence should be addressed. Tel.: 82-2-880-4857; Fax: 82-2-873-5095
    Affiliations
    National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, and Center for Food Safety and Toxicology, Seoul National University, Seoul 08826, South Korea
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  • Author Footnotes
    1 Both authors contributed equally to this study.
    2 Present address: Dept. of Microbiology, New York University School of Medicine, New York, NY 10016.
Open AccessPublished:March 13, 2020DOI:https://doi.org/10.1074/jbc.RA120.012724
      For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter PvvhBA, which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of PvvhBA, whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the PvvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

      Introduction

      Upon entering the host, enteropathogenic bacteria inevitably encounter drastic environmental changes and cope with harsh conditions raised by the host immune defense system. The ability to recognize environmental changes and produce appropriate virulence factors is essential for the pathogens to survive and develop diseases in the host (
      • Green J.
      • Rolfe M.D.
      • Smith L.J.
      Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide.
      ,
      • Fang F.C.
      • Frawley E.R.
      • Tapscott T.
      • Vázquez-Torres A.
      Bacterial stress responses during host infection.
      ). Accordingly, pathogens have evolved sophisticated mechanisms to regulate the expression of specific virulence genes in response to various host-derived signals (
      • Green J.
      • Rolfe M.D.
      • Smith L.J.
      Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide.
      ,
      • Fang F.C.
      • Frawley E.R.
      • Tapscott T.
      • Vázquez-Torres A.
      Bacterial stress responses during host infection.
      ). In an effort to understand the virulence gene regulation, numerous transcriptional regulators have been characterized (
      • Matson J.S.
      • Withey J.H.
      • DiRita V.J.
      Regulatory networks controlling Vibrio cholerae virulence gene expression.
      ). Notably, a number of transcriptional regulators use an iron-sulfur (Fe-S) cluster as a cofactor to sense environmental signals. Because the Fe-S cluster is easily disrupted under host-like conditions such as oxidative and nitrosative stress and iron starvation, it allows pathogens to promptly recognize and adapt to the host environment (
      • Py B.
      • Moreau P.L.
      • Barras F.
      Fe-S clusters, fragile sentinels of the cell.
      ,
      • Miller H.K.
      • Auerbuch V.
      Bacterial iron-sulfur cluster sensors in mammalian pathogens.
      ).
      The [2Fe-2S] containing transcriptional regulator IscR functions as a sensor of the cellular Fe-S cluster status and regulates Fe-S cluster biogenesis (
      • Schwartz C.J.
      • Giel J.L.
      • Patschkowski T.
      • Luther C.
      • Ruzicka F.J.
      • Beinert H.
      • Kiley P.J.
      IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins.
      ,
      • Giel J.L.
      • Nesbit A.D.
      • Mettert E.L.
      • Fleischhacker A.S.
      • Wanta B.T.
      • Kiley P.J.
      Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli.
      ). When the cellular Fe-S cluster level is sufficient to occupy IscR, [2Fe-2S]-IscR (holo-IscR) represses the isc operon (iscRSUA-hscBA-fdx) encoding IscR along with the proteins required for Fe-S cluster biogenesis. The [2Fe-2S] occupancy of IscR decreases under oxidative stress and iron starvation, resulting in apo-IscR lacking the clusters that relieves repression of the isc operon (
      • Giel J.L.
      • Nesbit A.D.
      • Mettert E.L.
      • Fleischhacker A.S.
      • Wanta B.T.
      • Kiley P.J.
      Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli.
      ,
      • Zheng M.
      • Wang X.
      • Templeton L.J.
      • Smulski D.R.
      • LaRossa R.A.
      • Storz G.
      DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide.
      ,
      • Outten F.W.
      • Djaman O.
      • Storz G.
      A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli.
      ,
      • Imlay J.A.
      Iron-sulphur clusters and the problem with oxygen.
      ,
      • Yeo W.S.
      • Lee J.H.
      • Lee K.C.
      • Roe J.H.
      IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins.
      ). As a result, the cellular apo-IscR protein level increases, and Fe-S cluster biogenesis is promoted (
      • Schwartz C.J.
      • Giel J.L.
      • Patschkowski T.
      • Luther C.
      • Ruzicka F.J.
      • Beinert H.
      • Kiley P.J.
      IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins.
      ,
      • Giel J.L.
      • Nesbit A.D.
      • Mettert E.L.
      • Fleischhacker A.S.
      • Wanta B.T.
      • Kiley P.J.
      Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli.
      ). IscR binds to two distinct Type 1 and Type 2 DNA motifs, depending upon the [2Fe-2S] occupancy of the protein (
      • Nesbit A.D.
      • Giel J.L.
      • Rose J.C.
      • Kiley P.J.
      Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation.
      ,
      • Rajagopalan S.
      • Teter S.J.
      • Zwart P.H.
      • Brennan R.G.
      • Phillips K.J.
      • Kiley P.J.
      Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity.
      ). Although holo-IscR binds to both Type 1 and Type 2 DNA motifs, apo-IscR exclusively binds to the Type 2 DNA motif (
      • Giel J.L.
      • Nesbit A.D.
      • Mettert E.L.
      • Fleischhacker A.S.
      • Wanta B.T.
      • Kiley P.J.
      Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli.
      ,
      • Yeo W.S.
      • Lee J.H.
      • Lee K.C.
      • Roe J.H.
      IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins.
      ,
      • Nesbit A.D.
      • Giel J.L.
      • Rose J.C.
      • Kiley P.J.
      Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation.
      ,
      • Rajagopalan S.
      • Teter S.J.
      • Zwart P.H.
      • Brennan R.G.
      • Phillips K.J.
      • Kiley P.J.
      Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity.
      ,
      • Wu Y.
      • Outten F.W.
      IscR controls iron-dependent biofilm formation in Escherichia coli by regulating type I fimbria expression.
      ).
      The opportunistic human pathogen Vibrio vulnificus is a causative agent of food-borne diseases ranging from gastroenteritis to life-threatening septicemia (
      • Oliver J.D.
      The biology of Vibrio vulnificus.
      ,
      • Baker-Austin C.
      • Oliver J.D.
      Vibrio vulnificus: new insights into a deadly opportunistic pathogen.
      ). It has been reported that V. vulnificus exploits various transcriptional regulators such as the cAMP receptor protein (CRP)
      The abbreviations used are: CRP
      cAMP receptor protein
      H-NS
      histone-like nucleoid-structuring protein
      M9G
      M9 minimal medium supplemented with 0.4% (w/v) glucose
      qRT-PCR
      quantitative RT-PCR
      DMEM
      Dulbecco's modified Eagle's medium
      l-NMMA
      l-NG-monomethyl arginine citrate
      DEA NONOate
      diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate
      DP
      2,2′-dipyridyl
      EMSA
      electrophoretic mobility shift assay
      6-FAM
      6-carboxyfluorescein
      Fur
      ferric uptake regulator
      LBS
      Luria-Bertani medium supplemented with 2% (w/v) NaCl
      RPKM
      reads per kilobase of transcript per million mapped sequence reads.
      , NanR, SmcR, and HlyU for the well-coordinated expression of its virulence factors (
      • Choi H.K.
      • Park N.Y.
      • Kim D.I.
      • Chung H.J.
      • Ryu S.
      • Choi S.H.
      Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus.
      ,
      • Kim B.S.
      • Hwang J.
      • Kim M.H.
      • Choi S.H.
      Cooperative regulation of the Vibrio vulnificus nan gene cluster by NanR protein, cAMP receptor protein, and N-acetylmannosamine 6-phosphate.
      ,
      • Kim S.M.
      • Park J.H.
      • Lee H.S.
      • Kim W.B.
      • Ryu J.M.
      • Han H.J.
      • Choi S.H.
      LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment, and its expression is induced by host cells.
      ,
      • Lee Z.W.
      • Kim B.S.
      • Jang K.K.
      • Bang Y.J.
      • Kim S.
      • Ha N.C.
      • Jung Y.H.
      • Lee H.J.
      • Han H.J.
      • Kim J.S.
      • Kim J.
      • Sahu P.K.
      • Jeong L.S.
      • Kim M.H.
      • Choi S.H.
      Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species.
      ). CRP and NanR recognize depletion of specific nutrients (
      • Kim B.S.
      • Hwang J.
      • Kim M.H.
      • Choi S.H.
      Cooperative regulation of the Vibrio vulnificus nan gene cluster by NanR protein, cAMP receptor protein, and N-acetylmannosamine 6-phosphate.
      ,
      • Green J.
      • Stapleton M.R.
      • Smith L.J.
      • Artymiuk P.J.
      • Kahramanoglou C.
      • Hunt D.M.
      • Buxton R.S.
      Cyclic-AMP and bacterial cyclic-AMP receptor proteins revisited: adaptation for different ecological niches.
      ), whereas SmcR senses increased cell density in the host to regulate virulence genes (
      • Kim S.M.
      • Park J.H.
      • Lee H.S.
      • Kim W.B.
      • Ryu J.M.
      • Han H.J.
      • Choi S.H.
      LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment, and its expression is induced by host cells.
      ,
      • Kim B.S.
      • Jang S.Y.
      • Bang Y.J.
      • Hwang J.
      • Koo Y.
      • Jang K.K.
      • Lim D.
      • Kim M.H.
      • Choi S.H.
      QStatin, a selective inhibitor of quorum sensing in Vibrio species.
      ). In addition, IscR and HlyU are preferentially induced by host cells or in septicemic patients, respectively, and also involved in activation of virulence genes in V. vulnificus (
      • Lee Z.W.
      • Kim B.S.
      • Jang K.K.
      • Bang Y.J.
      • Kim S.
      • Ha N.C.
      • Jung Y.H.
      • Lee H.J.
      • Han H.J.
      • Kim J.S.
      • Kim J.
      • Sahu P.K.
      • Jeong L.S.
      • Kim M.H.
      • Choi S.H.
      Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species.
      ,
      • Kim Y.R.
      • Lee S.E.
      • Kim C.M.
      • Kim S.Y.
      • Shin E.K.
      • Shin D.H.
      • Chung S.S.
      • Choy H.E.
      • Progulske-Fox A.
      • Hillman J.D.
      • Handfield M.
      • Rhee J.H.
      Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients.
      ,
      • Lim J.G.
      • Choi S.H.
      IscR is a global regulator essential for pathogenesis of Vibrio vulnificus and induced by host cells.
      ). Particularly, IscR also regulates numerous virulence genes in other pathogens such as Pseudomonas aeruginosa, Yersinia pseudotuberculosis, and Erwinia chrysanthemi, highlighting the importance of IscR for bacterial pathogenesis (
      • Rincon-Enriquez G.
      • Crété P.
      • Barras F.
      • Py B.
      Biogenesis of Fe/S proteins and pathogenicity: IscR plays a key role in allowing Erwinia chrysanthemi to adapt to hostile conditions.
      ,
      • Kim S.H.
      • Lee B.Y.
      • Lau G.W.
      • Cho Y.H.
      IscR modulates catalase A (KatA) activity, peroxide resistance and full virulence of Pseudomonas aeruginosa PA14.
      ,
      • Miller H.K.
      • Kwuan L.
      • Schwiesow L.
      • Bernick D.L.
      • Mettert E.
      • Ramirez H.A.
      • Ragle J.M.
      • Chan P.P.
      • Kiley P.J.
      • Lowe T.M.
      • Auerbuch V.
      IscR is essential for Yersinia pseudotuberculosis type III secretion and virulence.
      ).
      Among the virulence factors of V. vulnificus, a cytolysin/hemolysin VvhA is an extracellular pore-forming toxin essential for its hemolytic activity (
      • Gray L.D.
      • Kreger A.S.
      Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus.
      ,
      • Jeong H.G.
      • Satchell K.J.
      Additive function of Vibrio vulnificus MARTXVv and VvhA cytolysins promotes rapid growth and epithelial tissue necrosis during intestinal infection.
      ). VvhA is a product of the vvhA gene, which is cotranscribed with vvhB encoding a chaperone-like protein required for the production of active VvhA (
      • Choi H.K.
      • Park N.Y.
      • Kim D.I.
      • Chung H.J.
      • Ryu S.
      • Choi S.H.
      Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus.
      ,
      • Senoh M.
      • Okita Y.
      • Shinoda S.
      • Miyoshi S.
      The crucial amino acid residue related to inactivation of Vibrio vulnificus hemolysin.
      ). In the present study, we discovered that the vvhBA expression is highly induced in V. vulnificus exposed to murine blood and murine macrophage RAW 264.7 cells. To elucidate the regulatory mechanisms by which V. vulnificus increases vvhBA expression in host-like conditions, the exact role of IscR in vvhBA regulation was investigated. The vvhBA transcript and VvhA protein levels were compared in the WT and the isogenic iscR-deletion mutant (ΔiscR) under nitrosative stress and iron starvation. In addition, the combined effects of IscR, HlyU, and the histone-like nucleoid-structuring protein (H-NS) on vvhBA regulation were analyzed at the molecular levels (
      • Dorman C.J.
      H-NS: a universal regulator for a dynamic genome.
      ,
      • Elgaml A.
      • Miyoshi S.
      Role of the histone-like nucleoid structuring protein (H-NS) in the regulation of virulence factor expression and stress response in Vibrio vulnificus.
      ). Consequently, this study demonstrated that IscR is a sensor of host-derived nitrosative stress and iron starvation, and activates vvhBA transcription along with HlyU by relieving H-NS repression, contributing to the precise regulation of VvhA production during infection, which is essential for fitness and pathogenesis of V. vulnificus in the host.

      Results

      Expression of vvhBA is induced upon exposure to murine blood and macrophages

      In an effort to identify virulence genes significantly induced in V. vulnificus upon invasion of the host bloodstream, transcriptomes of the bacteria exposed to murine blood or M9 minimal medium supplemented with 0.4% (w/v) glucose (M9G; negative control) were analyzed by RNA-seq. V. vulnificus exposed to murine blood differentially expressed 942 genes compared with that exposed to M9G; 491 genes were up-regulated and 451 genes were down-regulated (Dataset S1). Among the genes encoding extracellular toxins, expression of the vvhBA operon was the most elevated upon exposure to murine blood (about 26.0-fold; Fig. S1A), which was confirmed by quantitative RT-PCR (qRT-PCR) (about 81.1-fold; Fig. 1A). This result suggested that vvhBA is preferentially expressed in response to host-derived signals which exist in the murine bloodstream.
      Figure thumbnail gr1
      Figure 1Expression of vvhBA upon exposure to murine blood and RAW 264.7 cells. A, the genes induced in V. vulnificus upon exposure to murine blood were identified by RNA-seq analysis. vvhBA was selected as the most highly induced extracellular toxin-encoding gene, and its induction was confirmed by qRT-PCR. Each column represents the vvhBA transcript level in V. vulnificus exposed to murine blood relative to M9G (negative control). Error bars represent the S.E. calculated using DeSeq2 for RNA-seq and the S.D. for qRT-PCR. B, V. vulnificus was exposed to DMEM (negative control) or RAW 264.7 cells in the presence or absence of l-NMMA. The vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript level in the cells exposed to DMEM without l-NMMA was set to 1. Error bars represent the S.D. *, p < 0.05; ***, p < 0.0005; ns, not significant.
      Because VvhA-dependent cytotoxicity of V. vulnificus toward murine peritoneal macrophages has been reported (
      • Lee S.E.
      • Ryu P.Y.
      • Kim S.Y.
      • Kim Y.R.
      • Koh J.T.
      • Kim O.J.
      • Chung S.S.
      • Choy H.E.
      • Rhee J.H.
      Production of Vibrio vulnificus hemolysin in vivo and its pathogenic significance.
      ), we questioned whether the expression of vvhBA increases upon exposure to murine macrophage RAW 264.7 cells. As shown in Fig. 1B, V. vulnificus that was exposed to nitric oxide (NO)-producing RAW 264.7 cells increased vvhBA transcription compared with that exposed to Dulbecco's modified Eagle's medium (DMEM; negative control). Strikingly, the extent of increase in the vvhBA transcript level upon exposure to RAW 264.7 cells diminished significantly by addition of the NO synthase inhibitor l-NG-monomethyl arginine citrate (l-NMMA) (Fig. 1B). This result indicated that NO is one of the murine macrophage-derived signals that V. vulnificus senses to induce vvhBA expression. Taken together, the combined results showed that vvhBA expression is induced under host-like conditions in response to certain host-derived signals.

      IscR positively regulates vvhBA transcription

      Our previous microarray analysis predicted that the expression of vvhBA is up-regulated by IscR (
      • Lim J.G.
      • Choi S.H.
      IscR is a global regulator essential for pathogenesis of Vibrio vulnificus and induced by host cells.
      ). Consistent with this, vvhBA transcript and VvhA protein levels were reduced in ΔiscR and restored by complementation (Fig. 2, A and B). This result confirmed that IscR activates vvhBA expression mostly at the transcription level. Because IscR exists in two forms, holo- and apo-IscR, whose regulatory characteristics are distinct from each other (
      • Nesbit A.D.
      • Giel J.L.
      • Rose J.C.
      • Kiley P.J.
      Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation.
      ), we investigated whether apo-IscR activates vvhBA transcription. For this purpose, vvhBA transcript and VvhA protein levels were determined in the iscR3CA mutant of which the iscR coding region on the chromosome was replaced with iscR3CA encoding apo-locked IscR (
      • Lim J.G.
      • Bang Y.J.
      • Choi S.H.
      Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation.
      ). Notably, vvhBA transcript and VvhA protein levels significantly increased in the iscR3CA mutant compared with the WT and ΔiscR (Fig. 2, C and D), demonstrating that apo-IscR is able to activate vvhBA transcription in vivo. Moreover, the IscR3CA protein level in the iscR3CA mutant was higher than the IscR protein level in the WT, possibly due to derepression of the isc operon (Fig. 2D) (
      • Schwartz C.J.
      • Giel J.L.
      • Patschkowski T.
      • Luther C.
      • Ruzicka F.J.
      • Beinert H.
      • Kiley P.J.
      IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins.
      ,
      • Giel J.L.
      • Nesbit A.D.
      • Mettert E.L.
      • Fleischhacker A.S.
      • Wanta B.T.
      • Kiley P.J.
      Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli.
      ). This result suggested that the elevated IscR3CA level contributes to the increased vvhBA expression, because both holo- and apo-IscR bind to the Type 2 DNA motif with similar affinities in Escherichia coli (
      • Nesbit A.D.
      • Giel J.L.
      • Rose J.C.
      • Kiley P.J.
      Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation.
      ).
      Figure thumbnail gr2
      Figure 2The effect of iscR mutation on vvhBA expression. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. **, p < 0.005; ***, p < 0.0005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR or IscR3CA and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT (pJH0311) and WT, a WT; ΔiscR (pJH0311) and ΔiscR, an iscR-deletion mutant; ΔiscR (pJH0311::iscR), an iscR-complemented strain with pKK1531; iscR3CA, a strain expressing apo-locked IscR.

      IscR activates vvhBA by sensing nitrosative stress and iron starvation

      Because the Fe-S cluster is sensitive to nitrosative stress (
      • Py B.
      • Moreau P.L.
      • Barras F.
      Fe-S clusters, fragile sentinels of the cell.
      ), we examined whether IscR mediates the induction of vvhBA expression in response to NO. The vvhBA transcript and VvhA protein levels were significantly elevated by an NO donor DEA NONOate, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, in the WT (Fig. 3, A and B), consistent with Fig. 1B. In contrast, vvhBA transcript and VvhA protein levels were not increased in response to DEA NONOate in ΔiscR (Fig. 3, A and B), suggesting that IscR responds to host-derived nitrosative stress and activates vvhBA transcription.
      Figure thumbnail gr3
      Figure 3The effect of nitrosative stress and iron starvation on vvhBA and IscR expression. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3 and then exposed to 25 μm DEA NONOate for 20 min (A and B) or 50 μm DP for 10 min (C and D). A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT unexposed to DEA NONOate (A) or DP (C) were set to 1. Error bars represent the S.D. *, p < 0.05; ***, p < 0.0005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT, a WT; ΔiscR, an iscR-deletion mutant.
      Meanwhile, our observation that many genes essential for V. vulnificus to survive under iron starvation were up-regulated in murine blood (Fig. S1B) led us to investigate the effect of iron starvation on vvhBA expression. As shown in Fig. 3, C and D, vvhBA transcript and VvhA protein levels significantly increased in the presence of an iron chelator, 2,2′-dipyridyl (DP) in the WT, which was not apparent in ΔiscR. This result revealed that IscR recognizes iron starvation as another environmental change in the host and induces vvhBA expression. Strikingly, the IscR level in the WT increased upon exposure to DEA NONOate and DP (Fig. 3, B and D), implying that induction of vvhBA expression is attributed to increased IscR levels. Combined with the previous observations (Fig. 2, C and D), we propose a model in which IscR senses nitrosative stress and iron starvation and shifts to the apo-form, which leads to release of repression of the isc operon, elevation of apo-IscR level, and subsequent activation of vvhBA transcription.

      IscR binds downstream of PvvhBA to activate vvhBA

      To examine whether IscR directly binds to the vvhBA promoter PvvhBA, electrophoretic mobility shift assays (EMSAs) were performed. Because IscR was purified and used under aerobic conditions, most purified IscR would be in the apo-form (
      • Yeo W.S.
      • Lee J.H.
      • Lee K.C.
      • Roe J.H.
      IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins.
      ,
      • Lim J.G.
      • Bang Y.J.
      • Choi S.H.
      Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation.
      ). Addition of IscR to the radiolabeled DNA probe encompassing the PvvhBA regulatory region (−353 to +148 from the transcription start site of vvhBA (
      • Choi H.K.
      • Park N.Y.
      • Kim D.I.
      • Chung H.J.
      • Ryu S.
      • Choi S.H.
      Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus.
      )) resulted in two retarded bands in a concentration-dependent manner (Fig. 4A). This result indicated that at least two binding sites of IscR with different DNA-binding affinities are present in the PvvhBA regulatory region. The same unlabeled DNA fragment competed for IscR binding in a dose-dependent manner (Fig. 4A), confirming the specific binding of IscR to the PvvhBA regulatory region.
      Figure thumbnail gr4
      Figure 4Specific binding of IscR to PvvhBA and sequences of the PvvhBA regulatory region. A, a 501-bp DNA of the PvvhBA regulatory region (5 nm) was radiolabeled and then incubated with increasing amounts of IscR as indicated. For competition analysis, various amounts of the unlabeled DNA fragment were used as a self-competitor and added to a reaction mixture containing 5 nm radiolabeled DNA and 30 nm IscR. B1, a DNA-IscR complex; F, free DNA. B, the same DNA of the PvvhBA regulatory region (32.3 nm) was labeled with 6-FAM, incubated with increasing amounts of IscR as indicated, and then digested with DNase I. The regions protected by IscR are indicated by black boxes (ISCRB1, ISCRB2, ISCRB3). Nucleotide numbers shown are relative to the transcription start site of vvhBA, which was determined previously (
      • Choi H.K.
      • Park N.Y.
      • Kim D.I.
      • Chung H.J.
      • Ryu S.
      • Choi S.H.
      Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus.
      ). C, sequence analysis of the PvvhBA regulatory region. The transcription start site of vvhBA and the putative translational initiation codon of VvhB are indicated by solid and dashed bent arrows, respectively. The putative −10 and −35 regions are underlined and the putative ribosome-binding site (AGGA) is boldface. The binding sequences of IscR are shown with the black boxes as described above. The binding sequences of HlyU (HLYUB; a white box) and H-NS (HNSB1, HNSB2, HNSB3, HNSB4, HNSB5, HNSB6; gray boxes) were determined later in this study (, C and D). The consensus sequences of the IscR-binding Type 2 DNA motif are indicated above the V. vulnificus DNA sequences. W, A or T; Y, C or T; R, A or G; x, any nucleotide.
      To determine the precise location of the IscR-binding sites in the PvvhBA regulatory region, DNase I protection assays were performed using the same DNA probe labeled with 6-carboxyfluorescein (6-FAM). IscR protected two regions extending from +48 to +75 (ISCRB2, centered at +61.5) and +89 to +118 (ISCRB3, centered at +103.5), respectively, from DNase I digestion (Fig. 4B). Increasing IscR levels revealed an additional protected region extending from −5 to +24 (ISCRB1, centered at +10) (Fig. 4B). The sequence of ISCRB1, ISCRB2, and ISCRB3 showed about 83, 93, and 93% identity, respectively, to the consensus IscR-binding sequence of Type 2 DNA motif, which both holo- and apo-IscR can bind (Fig. 4C) (
      • Nesbit A.D.
      • Giel J.L.
      • Rose J.C.
      • Kiley P.J.
      Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation.
      ), supporting apo-IscR-mediated vvhBA activation (Fig. 2, C and D). Combined with the EMSA data (Fig. 4A), this result indicated that IscR binds directly to ISCRB2 and ISCRB3 with similar binding affinities, but binds relatively weakly to ISCRB1. It is noteworthy that all three binding sites of IscR are located downstream of PvvhBA (Fig. 4C), which is unusual for a positive regulator.

      HlyU and H-NS regulate vvhBA by directly binding to the PvvhBA regulatory region

      To further understand how IscR activates vvhBA transcription despite binding downstream of PvvhBA, we investigated whether IscR interacts with other transcriptional regulator(s) in the PvvhBA regulatory region. Among the previously proposed transcriptional regulators of vvhBA, the exact regulatory mechanisms of HlyU and H-NS have not been elucidated yet (
      • Kim Y.R.
      • Lee S.E.
      • Kim C.M.
      • Kim S.Y.
      • Shin E.K.
      • Shin D.H.
      • Chung S.S.
      • Choy H.E.
      • Progulske-Fox A.
      • Hillman J.D.
      • Handfield M.
      • Rhee J.H.
      Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients.
      ,
      • Elgaml A.
      • Miyoshi S.
      Role of the histone-like nucleoid structuring protein (H-NS) in the regulation of virulence factor expression and stress response in Vibrio vulnificus.
      ). Hence, the role of these two transcriptional regulators in vvhBA regulation was further examined. The vvhBA transcript and VvhA protein levels were reduced in the hlyU-deletion mutant (ΔhlyU), but elevated in the hns-deletion mutant (Δhns) (Fig. 5, A and B). The varied levels of vvhBA transcript and VvhA protein in the deletion mutants were restored by complementation (Fig. 5, C and D). This result confirmed that HlyU is a positive regulator, whereas H-NS is a negative regulator of vvhBA transcription.
      Figure thumbnail gr5
      Figure 5IscR and HlyU activate, but H-NS represses the vvhBA transcription. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. *, p < 0.05 relative to the WT; **, p < 0.005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT (pJH0311), a WT; ΔiscR (pJH0311), an iscR-deletion mutant; ΔhlyU (pJH0311), a hlyU-deletion mutant; Δhns (pJH0311), an hns-deletion mutant; ΔiscR (pJH0311::iscR), an iscR-complemented strain with pKK1531; ΔhlyU (pJH0311::hlyU), a hlyU-complemented strain with pZW1510; Δhns (pJH0311::hns), an hns-complemented strain with pGR1713.
      Next, EMSAs revealed that each transcriptional regulator binds specifically to the PvvhBA regulatory region (Fig. 6, A and B). Based on this result, the precise binding sites of HlyU and H-NS in the PvvhBA regulatory region were determined by DNase I protection assays. HlyU clearly protected the region extending from −128 to −114 (HLYUB, centered at −121) from DNase I digestion (Fig. 6C). In addition, the regions protected by H-NS extended from −119 to −109 (HNSB1, centered at −114), −98 to −76 (HNSB2, centered at −87), +16 to +26 (HNSB3, centered at +21), +60 to +74 (HNSB4, centered at +67), +87 to +99 (HNSB5, centered at +93), and +105 to +129 (HNSB6, centered at +117) (Fig. 6D). Notably, the binding sites of both IscR and HlyU overlapped with those of H-NS, which were located throughout the PvvhBA regulatory region (Fig. 4C). In addition, IscR and HlyU bound to the PvvhBA regulatory region at 30 nm, whereas H-NS showed binding at a much higher concentration of 200 nm (Figs. 4A, and 6, A and B). The combined results suggested that IscR and HlyU compete with H-NS for binding to the PvvhBA regulatory region and exhibit higher DNA-binding affinities.
      Figure thumbnail gr6
      Figure 6Specific binding of HlyU and H-NS to PvvhBA. A and B, a 501-bp DNA of the PvvhBA regulatory region (5 nm) was radiolabeled and then incubated with increasing amounts of HlyU (A) or H-NS (B) as indicated. For competition analysis, various amounts of the unlabeled DNA fragment were used as a self-competitor and added to a reaction mixture containing 5 nm radiolabeled DNA and 30 nm HlyU (A) or 300 nm H-NS (B). B2, a DNA-HlyU complex; B3, a DNA-H-NS complex; F, free DNA. C and D, the same DNA of the PvvhBA regulatory region (32.3 nm) was labeled with 6-FAM, incubated with increasing amounts of HlyU (C) or H-NS (D) as indicated, and then digested with DNase I. The regions protected by HlyU and H-NS are indicated by a white box (HLYUB) and gray boxes (HNSB1, HNSB2, HNSB3, HNSB4, HNSB5, HNSB6), respectively. Nucleotide numbers shown are relative to the transcription start site of vvhBA.

      IscR and HlyU outcompete H-NS by simultaneously binding to the PvvhBA regulatory region

      To determine whether IscR and HlyU compete with H-NS for binding, EMSAs were performed using reaction mixtures containing a fixed concentration of H-NS with various amounts of IscR. As the concentration of IscR increased, the DNA–H-NS complex (B3) was completely replaced by DNA–IscR complexes (B1) (Fig. 7A). This result indicated that IscR outcompetes H-NS that binds to the PvvhBA regulatory region. Similarly, EMSAs with a fixed concentration of H-NS and increasing amounts of HlyU revealed that HlyU also relieves H-NS binding to the PvvhBA regulatory region (Fig. 7B). Then, the effect of IscR and HlyU on the binding of each other to the PvvhBA regulatory region was examined. When IscR and HlyU were incubated together in the reaction mixture, the DNA–IscR–HlyU complexes (B4), as well as DNA–IscR (B1) and DNA–HlyU complexes (B2), were observed (Fig. 7C). This result showed that IscR and HlyU can bind simultaneously to the PvvhBA regulatory region, and they do not compete for binding sites. Taken together, the combined results suggested that IscR and HlyU bind simultaneously with strong DNA-binding affinities to alleviate H-NS binding from the PvvhBA regulatory region. Because these three transcriptional regulators did not regulate the expression of one another (Fig. S2), we hypothesized that IscR, together with HlyU, relieves H-NS repression of vvhBA transcription by binding downstream of PvvhBA.
      Figure thumbnail gr7
      Figure 7Interactions between IscR, HlyU, and H-NS in binding to PvvhBA. A and B, a 501-bp DNA of the PvvhBA regulatory region (5 nm) was radiolabeled and then incubated with a mixture of 200 nm H-NS and increasing amounts of either IscR (A) or HlyU (B) as indicated. C, the same radiolabeled DNA (5 nm) was incubated with a mixture of 30 nm HlyU and increasing amounts of IscR as indicated. B1, a DNA-IscR complex; B2, a DNA-HlyU complex; B3, a DNA-H-NS complex; B4, a DNA-IscR-HlyU complex; F, free DNA.

      IscR and HlyU alleviate H-NS repression to activate vvhBA additively in vivo

      To ascertain whether IscR and HlyU activate vvhBA by relieving H-NS repression in vivo, vvhBA transcript and VvhA protein levels were compared in the WT and various deletion mutants. Interestingly, vvhBA transcript and VvhA protein levels were comparable in Δhns, the iscR hns double-deletion mutant (ΔiscRΔhns), and the h-ns-deleted iscR3CA mutant (iscR3CAΔhns) (Fig. 8, A and B). This observation that IscR did not affect vvhBA transcription in the absence of H-NS suggested that IscR merely relieves H-NS repression. Furthermore, this result showed that IscR does not act as a roadblock for RNA polymerases in vivo despite its unusual binding sites on the PvvhBA regulatory region. Similarly, vvhBA transcript and VvhA protein levels in the hlyU hns double-deletion mutant (ΔhlyUΔhns) were comparable with those in Δhns (Fig. S3), indicating that HlyU also does not positively affect vvhBA transcription in the absence of H-NS. These results supported our hypothesis that IscR and HlyU relieve repression of vvhBA transcription by outcompeting H-NS for binding to the PvvhBA regulatory region.
      Figure thumbnail gr8
      Figure 8IscR relieves H-NS repression of vvhBA in cooperation with HlyU in vivo. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. *, p < 0.05; **, p < 0.005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR or IscR3CA, H-NS, HlyU, and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT, a WT; Δhns, an hns-deletion mutant; ΔiscRΔhns, an iscR hns double-deletion mutant; iscR3CAΔhns, an hns-deletion mutant expressing apo-locked IscR; ΔiscR, an iscR-deletion mutant; ΔhlyU, a hlyU-deletion mutant; ΔiscRΔhlyU, an iscR hlyU double-deletion mutant; iscR3CAΔhlyU, a hlyU-deletion mutant expressing apo-locked IscR.
      The relationship between IscR and HlyU in vvhBA regulation was also investigated in vivo. The vvhBA transcript and VvhA protein levels in the iscR hlyU double-deletion mutant (ΔiscRΔhlyU) were significantly lower than those in either ΔiscR or ΔhlyU (Fig. 8, C and D). This result revealed that these two transcriptional regulators have an additive effect in activating vvhBA transcription in vivo. The elevated IscR3CA level did not increase vvhBA transcript and VvhA protein levels in the hlyU-deleted iscR3CA mutant (iscR3CAΔhlyU) compared with those in either ΔiscR or ΔhlyU (Fig. 8, C and D), indicating that IscR alone cannot induce vvhBA expression to the WT level in the absence of HlyU. Taken together, the combined results demonstrated that both IscR and HlyU are required for complete release of H-NS repression, leading to full activation of vvhBA transcription in vivo.

      Discussion

      The V. vulnificus vvhBA operon encodes a well-studied extracellular pore-forming toxin VvhA, which exhibits powerful hemolytic and cytolytic activities (
      • Gray L.D.
      • Kreger A.S.
      Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus.
      ,
      • Lee S.E.
      • Ryu P.Y.
      • Kim S.Y.
      • Kim Y.R.
      • Koh J.T.
      • Kim O.J.
      • Chung S.S.
      • Choy H.E.
      • Rhee J.H.
      Production of Vibrio vulnificus hemolysin in vivo and its pathogenic significance.
      ). VvhA is actually expressed during infection and contributes to the severe intestinal tissue damage, subsequent invasion of V. vulnificus into the bloodstream, and dissemination of the pathogen to other organs in a murine infection model (
      • Jeong H.G.
      • Satchell K.J.
      Additive function of Vibrio vulnificus MARTXVv and VvhA cytolysins promotes rapid growth and epithelial tissue necrosis during intestinal infection.
      ,
      • Lee S.E.
      • Ryu P.Y.
      • Kim S.Y.
      • Kim Y.R.
      • Koh J.T.
      • Kim O.J.
      • Chung S.S.
      • Choy H.E.
      • Rhee J.H.
      Production of Vibrio vulnificus hemolysin in vivo and its pathogenic significance.
      ). In this study, RNA-seq analysis identified vvhBA as the most highly induced extracellular toxin-encoding gene in V. vulnificus upon exposure to murine blood (Fig. 1A and Fig. S1A). In addition, vvhBA expression increased upon exposure to NO-producing murine macrophage RAW 264.7 cells (Fig. 1B). These observations indicate that V. vulnificus recognizes certain host-derived signal(s) to induce vvhBA expression during infection.
      Our next concern was with the host-derived signal(s) that V. vulnificus senses to activate vvhBA. Interestingly, the Fe-S cluster containing transcriptional regulator IscR activated vvhBA transcription in response to nitrosative stress and iron starvation (Figure 2, Figure 3). This IscR-mediated activation of VvhA could be beneficial for V. vulnificus because the pathogen inevitably encounters host-derived nitrosative stress and iron starvation during infection (
      • Fang F.C.
      Antimicrobial reactive oxygen and nitrogen species: concepts and controversies.
      ,
      • Skaar E.P.
      The battle for iron between bacterial pathogens and their vertebrate hosts.
      ). In the host, NO is produced by immune cells, particularly by macrophages, as a primary antimicrobial agent (
      • Fang F.C.
      Antimicrobial reactive oxygen and nitrogen species: concepts and controversies.
      ). Simultaneously, free iron is depleted by the host iron-sequestering system to prevent outgrowth of invading pathogens (
      • Skaar E.P.
      The battle for iron between bacterial pathogens and their vertebrate hosts.
      ). Under these hostile conditions, the IscR-mediated induction of VvhA could disrupt the macrophages and components of the iron-sequestering system (
      • Lee S.E.
      • Ryu P.Y.
      • Kim S.Y.
      • Kim Y.R.
      • Koh J.T.
      • Kim O.J.
      • Chung S.S.
      • Choy H.E.
      • Rhee J.H.
      Production of Vibrio vulnificus hemolysin in vivo and its pathogenic significance.
      ,
      • Runyen-Janecky L.J.
      Role and regulation of heme iron acquisition in Gram-negative pathogens.
      ), thereby contributing to survival of V. vulnificus in the host.
      Then, we tried to figure out the molecular mechanism by which IscR activates vvhBA transcription. Intriguingly, IscR directly bound downstream of PvvhBA (Fig. 4), which is unusual for a positive regulator. In addition to IscR, HlyU and H-NS acted as positive and negative regulators for vvhBA transcription, respectively, by directly binding to the PvvhBA regulatory region (Figure 5, Figure 6). We verified that IscR and HlyU effectively relieve the H-NS binding with higher DNA-binding affinities (Fig. 7, A and B) and do not further affect vvhBA transcription in the absence of H-NS (Fig. 8, A and B, and Fig. S3). These results demonstrate that IscR and HlyU function as antirepressors against H-NS rather than direct activators for vvhBA transcription.
      H-NS binds to multiple AT-rich regions throughout the promoter, which possibly results in the formation of nucleoprotein filaments and subsequent RNA polymerase trapping (
      • Dorman C.J.
      H-NS: a universal regulator for a dynamic genome.
      ,
      • Liu M.
      • Naka H.
      • Crosa J.H.
      HlyU acts as an H-NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6.
      ). In a similar way, H-NS represses rtxA in V. vulnificus, but HlyU can disrupt the whole H-NS nucleoprotein complex by binding to a single site located far upstream of the rtxA promoter, resulting in antirepression of rtxA (
      • Liu M.
      • Naka H.
      • Crosa J.H.
      HlyU acts as an H-NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6.
      ). However, our study revealed that IscR and HlyU bind simultaneously to the PvvhBA regulatory region (Fig. 7C) and additively induce vvhBA transcription in vivo (Fig. 8, C and D). These results indicate that both antirepressors are required to bind upstream as well as downstream of PvvhBA for complete disruption of a possible H-NS nucleoprotein complex and antirepression of vvhBA. To our knowledge, this is the first report describing the additive mode of antirepression for gene activation.
      Because the Fe-S cluster is one of the most abundant enzymatic cofactors in fundamental cellular processes, the isc operon, iscRSUA-hscBA-fdx, is highly conserved in many bacteria (
      • Imlay J.A.
      Iron-sulphur clusters and the problem with oxygen.
      ). IscR binds downstream of the iscR promoter in V. vulnificus, and iscR, yadR, and yhgI promoters in E. coli to act as a negative regulator (
      • Giel J.L.
      • Rodionov D.
      • Liu M.
      • Blattner F.R.
      • Kiley P.J.
      IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli.
      ,
      • Lim J.G.
      • Park J.H.
      • Choi S.H.
      Low cell density regulator AphA upregulates the expression of Vibrio vulnificus iscR gene encoding the Fe-S cluster regulator IscR.
      ). In contrast, IscR binds upstream of the target promoters to positively regulate prx3 and gbpA in V. vulnificus, the suf operon and ydiU in E. coli, and the yscW-lcrF operon in Y. pseudotuberculosis (
      • Miller H.K.
      • Kwuan L.
      • Schwiesow L.
      • Bernick D.L.
      • Mettert E.
      • Ramirez H.A.
      • Ragle J.M.
      • Chan P.P.
      • Kiley P.J.
      • Lowe T.M.
      • Auerbuch V.
      IscR is essential for Yersinia pseudotuberculosis type III secretion and virulence.
      ,
      • Lim J.G.
      • Bang Y.J.
      • Choi S.H.
      Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation.
      ,
      • Giel J.L.
      • Rodionov D.
      • Liu M.
      • Blattner F.R.
      • Kiley P.J.
      IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli.
      ,
      • Jang K.K.
      • Gil S.Y.
      • Lim J.G.
      • Choi S.H.
      Regulatory characteristics of Vibrio vulnificus gbpA gene encoding a mucin-binding protein essential for pathogenesis.
      ). However, unlike these conserved and typical regulatory roles of IscR according to its binding sites, our results showed that V. vulnificus IscR atypically binds downstream of PvvhBA to activate vvhBA as an antirepressor.
      In general, transcriptional regulators that bind downstream of target promoters prevent the binding and progress of RNA polymerases as negative regulators (
      • Kim B.S.
      • Hwang J.
      • Kim M.H.
      • Choi S.H.
      Cooperative regulation of the Vibrio vulnificus nan gene cluster by NanR protein, cAMP receptor protein, and N-acetylmannosamine 6-phosphate.
      ). Nevertheless, a few positive regulators such as MetR, PhoP, DnaA, and Rns still bind downstream of their target promoters (
      • Cowan J.M.
      • Urbanowski M.L.
      • Talmi M.
      • Stauffer G.V.
      Regulation of the Salmonella typhimurium metF gene by the MetR protein.
      ,
      • Liu W.
      • Qi Y.
      • Hulett F.M.
      Sites internal to the coding regions of phoA pstS bind PhoP and are required for full promoter activity.
      ,
      • Qi Y.
      • Hulett F.M.
      PhoP-P and RNA polymerase σA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP-P activator sites within the coding region stimulate transcription in vitro.
      ,
      • Szalewska-Pa/lasz A.
      • Wegrzyn A.
      • B/laszczak A.
      • Taylor K.
      • Wegrzyn G.
      DnaA-stimulated transcriptional activation of oriλ: Escherichia coli RNA polymerase β subunit as a transcriptional activator contact site.
      ,
      • Munson G.P.
      • Scott J.R.
      Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator.
      ). Likewise, IscR binds downstream of PvvhBA and does not prevent vvhBA transcription even in the absence of H-NS (Fig. 8, A and B). One possible mechanism can be explained by the effect of other transcriptional regulators participating in vvhBA regulation. We previously reported that CRP activates vvhBA as a Class I activator (Fig. 9) (
      • Choi H.K.
      • Park N.Y.
      • Kim D.I.
      • Chung H.J.
      • Ryu S.
      • Choi S.H.
      Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus.
      ). Thus, CRP may strongly recruit RNA polymerases to PvvhBA, even though the transcription start site of vvhBA overlaps with ISCRB1 (Fig. 4C). Moreover, once RNA polymerases switch to elongation complexes, trailing elongation complexes could overcome an IscR-mediated roadblock by pushing the leading elongation complexes forward (
      • Epshtein V.
      • Toulmé F.
      • Rahmouni A.R.
      • Borukhov S.
      • Nudler E.
      Transcription through the roadblocks: the role of RNA polymerase cooperation.
      ). Taken together, the combined results led us to conclude that alleviation of H-NS binding from the PvvhBA regulatory region by IscR is much more effective for vvhBA transcription than the possible roadblock effect of IscR against the action of RNA polymerases.
      Figure thumbnail gr9
      Figure 9A proposed model for the regulation of vvhBA by multiple transcriptional regulators during host infection. Upon entering the host, V. vulnificus induces vvhBA expression in response to drastic environmental changes. IscR, along with HlyU, which is preferentially produced in the host (
      • Kim Y.R.
      • Lee S.E.
      • Kim C.M.
      • Kim S.Y.
      • Shin E.K.
      • Shin D.H.
      • Chung S.S.
      • Choy H.E.
      • Progulske-Fox A.
      • Hillman J.D.
      • Handfield M.
      • Rhee J.H.
      Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients.
      ), activates vvhBA by relieving H-NS repression by sensing nitrosative stress and iron starvation. Additionally, CRP activates vvhBA expression via Class I activation under certain nutrient-depleted conditions (
      • Choi H.K.
      • Park N.Y.
      • Kim D.I.
      • Chung H.J.
      • Ryu S.
      • Choi S.H.
      Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus.
      ,
      • Green J.
      • Stapleton M.R.
      • Smith L.J.
      • Artymiuk P.J.
      • Kahramanoglou C.
      • Hunt D.M.
      • Buxton R.S.
      Cyclic-AMP and bacterial cyclic-AMP receptor proteins revisited: adaptation for different ecological niches.
      ). Meanwhile, a repressive interaction of H-NS and Fur at PvvhBA would be relieved in response to the increase in temperature and iron starvation in the host, respectively (
      • Elgaml A.
      • Miyoshi S.
      Role of the histone-like nucleoid structuring protein (H-NS) in the regulation of virulence factor expression and stress response in Vibrio vulnificus.
      ,
      • Lee H.J.
      • Kim J.A.
      • Lee M.A.
      • Park S.J.
      • Lee K.H.
      Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases.
      ). Taken together, the transcriptional regulators integrate diverse host-derived signals to collaboratively regulate vvhBA transcription during infection. Solid lines indicate activation of vvhBA by positive regulators, whereas dashed lines show relieved repression of vvhBA by negative regulators in the host. The transcription start site of vvhBA and the putative translational initiation codon of VvhB are indicated by solid bent arrows. The putative −10 and −35 regions, and ribosome-binding site (RBS) are underlined. ISCRB, an IscR-binding site; HLYUB, a HlyU-binding site; HNSB, an H-NS-binding site; CRPB, a CRP-binding site; FURB, a Fur-binding site.
      Besides IscR, HlyU, and CRP, ferric uptake regulator (Fur) represses vvhBA transcription in the presence of iron by directly binding to PvvhBA (Fig. 9) (
      • Lee H.J.
      • Kim J.A.
      • Lee M.A.
      • Park S.J.
      • Lee K.H.
      Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases.
      ). Consistent with this, the vvhBA transcript level greatly increased in the fur-deletion mutant (Δfur) compared with that in the WT (Fig. S4). Although Fur might be the major regulator responsive to iron availability, the vvhBA transcript level in the iscR fur double-deletion mutant (ΔiscRΔfur) significantly decreased compared with that in Δfur (Fig. S4), demonstrating the distinct role of IscR in the elaborate regulation of vvhBA.
      Our current understanding of the transcriptional regulation of V. vulnificus vvhBA in the host environment is described in the legend to Fig. 9. IscR, together with HlyU, relieves H-NS repression of vvhBA by sensing nitrosative stress and iron starvation. Although the exact environmental signal affecting the regulatory activity of HlyU is still unknown, it is verified that HlyU is preferentially expressed in the host (
      • Kim Y.R.
      • Lee S.E.
      • Kim C.M.
      • Kim S.Y.
      • Shin E.K.
      • Shin D.H.
      • Chung S.S.
      • Choy H.E.
      • Progulske-Fox A.
      • Hillman J.D.
      • Handfield M.
      • Rhee J.H.
      Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients.
      ). CRP activates vvhBA possibly by sensing the depletion of specific nutrients (
      • Choi H.K.
      • Park N.Y.
      • Kim D.I.
      • Chung H.J.
      • Ryu S.
      • Choi S.H.
      Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus.
      ). At the same time, H-NS repression is weakened at elevated temperatures in the host (
      • Elgaml A.
      • Miyoshi S.
      Role of the histone-like nucleoid structuring protein (H-NS) in the regulation of virulence factor expression and stress response in Vibrio vulnificus.
      ), and the iron-Fur complex repression is also alleviated under iron starvation (
      • Lee H.J.
      • Kim J.A.
      • Lee M.A.
      • Park S.J.
      • Lee K.H.
      Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases.
      ), leading to increased vvhBA expression during infection. These transcriptional regulators take advantage of the multiple binding sites encompassing both downstream and upstream of PvvhBA for tight regulation of vvhBA in response to various host-derived signals.
      In summary, this study demonstrated that vvhBA, encoding a cytolysin/hemolysin of V. vulnificus, is preferentially expressed upon exposure to murine blood and macrophages. The positive regulator IscR activates vvhBA transcription by sensing host-derived nitrosative stress and iron starvation. IscR exerts its effect additively with another positive regulator HlyU by binding downstream of PvvhBA to relieve binding of the negative regulator H-NS from the PvvhBA regulatory region. The collaborative regulation by multiple global regulators allows more precise tuning of vvhBA expression through integrating various host-derived signals encountered during infection, and thereby enhances the overall success of V. vulnificus pathogenesis.

      Experimental procedures

      Strains, plasmids, and culture conditions

      The strains and plasmids used in this study are listed under Table S1. Unless otherwise noted, the V. vulnificus strains were grown aerobically in Luria-Bertani (LB) medium supplemented with 2% (w/v) NaCl (LBS) at 30°C and their growth was monitored spectrophotometrically at 600 nm (A600). When required, 100 μg/ml of ampicillin was added to the medium. The murine macrophage RAW 264.7 cells were grown in DMEM containing 10% fetal bovine serum (VWR, Radnor, PA) and appropriate antibiotics (100 units/ml of penicillin G and 100 μg/ml of streptomycin (Gibco-BRL, Gaithersburg, MD)) in air supplemented with 5% CO2 at 37°C. To induce NO production, the RAW 264.7 cells were suspended in fresh DMEM containing 500 ng/ml of E. coli O111:B4 lipopolysaccharide (Sigma) and 1 mml-arginine (Sigma) (
      • Walker G.
      • Pfeilschifter J.
      • Kunz D.
      Mechanisms of suppression of inducible nitric-oxide synthase (iNOS) expression in interferon (IFN)-γ-stimulated RAW 264.7 cells by dexamethasone: evidence for glucocorticoid-induced degradation of iNOS protein by calpain as a key step in post-transcriptional regulation.
      ,
      • Kim D.
      • Na E.J.
      • Kim S.
      • Kim J.S.
      • Jung Y.H.
      • Cao J.
      • Han H.J.
      • Bang I.S.
      • Yoo J.W.
      • Ha N.C.
      • Choi S.H.
      Transcriptomic identification and biochemical characterization of HmpA, a nitric oxide dioxygenase, essential for pathogenesis of Vibrio vulnificus.
      ).

      RNA purification and RNA-seq analysis

      To analyze transcriptomes differentially expressed upon exposure to murine blood, about 8 × 107 CFU of V. vulnificus were inoculated to 800 μl of murine blood or M9G (negative control). For two biological replicates, the V. vulnificus cells and murine blood were prepared from two independent colonies or mice, respectively, on the same day. The mixture was incubated at 37°C with rolling for 1 h and then centrifuged at 250 × g for 3 min to harvest supernatant containing bacterial cells. Total RNA from the V. vulnificus cells was isolated and quantified using an RNeasy® Mini Kit (Qiagen, Valencia, CA) and a NanoDrop Onec Microvolume UV-visible Spectrophotometer (Thermo Scientific, Waltham, MA), respectively. Strand-specific cDNA libraries were constructed and sequenced using HiSeq 2500 (Illumina, San Diego, CA) by LabGenomics (Seongnam, Gyeonggi, South Korea) as described previously (
      • Lee Z.W.
      • Kim B.S.
      • Jang K.K.
      • Bang Y.J.
      • Kim S.
      • Ha N.C.
      • Jung Y.H.
      • Lee H.J.
      • Han H.J.
      • Kim J.S.
      • Kim J.
      • Sahu P.K.
      • Jeong L.S.
      • Kim M.H.
      • Choi S.H.
      Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species.
      ). The raw sequencing reads were mapped to the V. vulnificus MO6–24/O reference genome (GenBankTM accession numbers: CP002469 and CP002470), and the expression level of each gene was calculated as reads per kilobase of transcript per million mapped sequence reads (RPKM) value using EDGE-pro version 1.3.1 (Estimated Degree of Gene Expression in PROkaryots) (
      • Magoc T.
      • Wood D.
      • Salzberg S.L.
      EDGE-pro: estimated degree of gene expression in prokaryotic genomes.
      ). The RPKM values were normalized and analyzed statistically using DeSeq2 version 1.26.0 to identify differentially expressed genes (greater than 2-fold change with p < 0.05) when exposed to murine blood (
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ). All manipulations for murine blood sampling were performed following the National Institutes of Health Guidelines for Humane Treatment and approved by the Animal Care and Use Committee of Seoul National University (SNU-170116-1).

      qRT-PCR

      Relative vvhBA transcript levels in the total RNA isolated from V. vulnificus grown under various environmental conditions were determined by qRT-PCR. In detail, V. vulnificus was grown in LBS to an A600 of 0.5 and then exposed to RAW 264.7 cells at a multiplicity of infection of 10 or DMEM (negative control) for 1 h in the presence or absence of 500 μml-NMMA (Sigma), which is a known NO synthase inhibitor (
      • Kim D.
      • Na E.J.
      • Kim S.
      • Kim J.S.
      • Jung Y.H.
      • Cao J.
      • Han H.J.
      • Bang I.S.
      • Yoo J.W.
      • Ha N.C.
      • Choi S.H.
      Transcriptomic identification and biochemical characterization of HmpA, a nitric oxide dioxygenase, essential for pathogenesis of Vibrio vulnificus.
      ,
      • Nathan C.F.
      • Hibbs Jr., J.B.
      Role of nitric oxide synthesis in macrophage antimicrobial activity.
      ). Moreover, V. vulnificus was exposed to 25 μm DEA NONOate (Cayman Chemical, Ann Arbor, MI) for 20 min or 50 μm DP (Sigma) for 10 min when necessary. cDNA was synthesized from 1 μg of the total RNA by using the iScriptTM cDNA synthesis kit (Bio-Rad). Real-time PCR amplification of the cDNA was performed by using the Chromo 4 real-time PCR detection system (Bio-Rad) with pairs of specific primers (Table S2) as described previously (
      • Jang K.K.
      • Lee Z.W.
      • Kim B.
      • Jung Y.H.
      • Han H.J.
      • Kim M.H.
      • Kim B.S.
      • Choi S.H.
      Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.
      ). Relative expression levels of the vvhBA transcript were calculated by using the 16S rRNA expression level as an internal reference for normalization (
      • Jang K.K.
      • Lee Z.W.
      • Kim B.
      • Jung Y.H.
      • Han H.J.
      • Kim M.H.
      • Kim B.S.
      • Choi S.H.
      Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.
      ).

      Generation and complementation of ΔiscR, ΔhlyU, Δhns, Δfur, ΔiscRΔhlyU, ΔiscRΔhns, ΔiscRΔfur, ΔhlyUΔhns, iscR3CAΔhlyU, and iscR3CAΔhns

      ΔiscR JK093, the iscR3CA mutant JK128, and ΔhlyU ZW141 were previously constructed and used in this study (Table S1) (
      • Lim J.G.
      • Choi S.H.
      IscR is a global regulator essential for pathogenesis of Vibrio vulnificus and induced by host cells.
      ,
      • Lim J.G.
      • Bang Y.J.
      • Choi S.H.
      Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation.
      ,
      • Jang K.K.
      • Lee Z.W.
      • Kim B.
      • Jung Y.H.
      • Han H.J.
      • Kim M.H.
      • Kim B.S.
      • Choi S.H.
      Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.
      ). For construction of Δhns, the hns gene was inactivated in vitro by deletion of the ORF of hns (228 of 408 bp) using the PCR-mediated linker-scanning mutation method as described previously (
      • Jang K.K.
      • Gil S.Y.
      • Lim J.G.
      • Choi S.H.
      Regulatory characteristics of Vibrio vulnificus gbpA gene encoding a mucin-binding protein essential for pathogenesis.
      ). Briefly, the primer pairs HNS01-F and HNS01-R or HNS02-F and HNS02-R were used for amplification of the 5′- and 3′-amplicon, respectively (Table S2). The 228-bp deleted hns was amplified by PCR using a mixture of both amplicons as the template, and HNS01-F and HNS02-R as primers. The resulting product was ligated into SpeI-SphI–digested pDM4 to generate pEJ1502 (Table S1) (
      • Milton D.L.
      • O'Toole R.
      • Horstedt P.
      • Wolf-Watz H.
      Flagellin A is essential for the virulence of Vibrio anguillarum.
      ). Similarly, pJK1121 carrying the 292-bp deleted fur on pDM4 was constructed using the primers FUR01-F, FUR01-R, FUR02-F, and FUR02-R (Table S2). E. coli S17-1 λpir strain containing pEJ1502, pJK1121, or pZW1401 was used as a conjugal donor to V. vulnificus MO6–24/O, ΔiscR, ΔhlyU, the iscR3CA mutant to construct Δhns EJ151, Δfur JK114, ΔiscRΔhlyU KK1612, ΔiscRΔhns JK151, ΔiscRΔfur JK116, ΔhlyUΔhns JK152, iscR3CAΔhlyU KK1611, or iscR3CAΔhns KK1620, respectively (Table S1) (
      • Jang K.K.
      • Lee Z.W.
      • Kim B.
      • Jung Y.H.
      • Han H.J.
      • Kim M.H.
      • Kim B.S.
      • Choi S.H.
      Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.
      ,
      • Simon R.
      • Priefer U.
      • Puhler A.
      A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria.
      ). The conjugation and isolation of transconjugants were conducted using the method described previously (
      • Jang K.K.
      • Gil S.Y.
      • Lim J.G.
      • Choi S.H.
      Regulatory characteristics of Vibrio vulnificus gbpA gene encoding a mucin-binding protein essential for pathogenesis.
      ).
      To complement the mutations, pZW1510 carrying the hlyU gene on the broad host-range vector pJH0311 was used in this study (Table S1) (
      • Jang K.K.
      • Lee Z.W.
      • Kim B.
      • Jung Y.H.
      • Han H.J.
      • Kim M.H.
      • Kim B.S.
      • Choi S.H.
      Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.
      ,
      • Goo S.Y.
      • Lee H.J.
      • Kim W.H.
      • Han K.L.
      • Park D.K.
      • Lee H.J.
      • Kim S.M.
      • Kim K.S.
      • Lee K.H.
      • Park S.J.
      Identification of OmpU of Vibrio vulnificus as a fibronectin-binding protein and its role in bacterial pathogenesis.
      ). Similarly, the iscR and hns genes were amplified by PCR using pairs of specific primers listed in Table S2 and cloned into pJH0311 to create pKK1531 and pGR1713, respectively (Table S1). The plasmids were transferred into the appropriate mutants by conjugation as described above.

      Protein purification and Western blot analysis

      To overexpress IscR and HlyU, pJK0928 carrying the iscR gene on pET-22b(+) (Novagen, Madison, WI) and pYU1317 carrying the hlyU gene on pET-28a(+) (Novagen) were constructed previously and used in this study (Table S1) (
      • Lim J.G.
      • Choi S.H.
      IscR is a global regulator essential for pathogenesis of Vibrio vulnificus and induced by host cells.
      ,
      • Jang K.K.
      • Lee Z.W.
      • Kim B.
      • Jung Y.H.
      • Han H.J.
      • Kim M.H.
      • Kim B.S.
      • Choi S.H.
      Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.
      ). Similarly, the hns and ompU genes were subcloned into pET-28a(+) and pHIS-parallel1 using pairs of specific primers listed in Table S2 to create pKK1636 and pKK1615, respectively (Table S1) (
      • Sheffield P.
      • Garrard S.
      • Derewenda Z.
      Overcoming expression and purification problems of RhoGDI using a family of “parallel” expression vectors.
      ). Because injection of the VvhA protein is toxic to rabbit, the 5′-terminal region of vvhA (amino acids 1–261) was amplified by PCR using the primer pair VVHBA01-F and VVHBA01-R (Table S2), subcloned into pET-28a(+) to create pKK1643 (Table S1). The resulting His6-tagged IscR, HlyU, H-NS, OmpU, and truncated VvhA were expressed in E. coli BL21 (DE3) and purified by affinity chromatography according to the manufacturer's procedure (Qiagen).
      The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea). For Western blot analysis, V. vulnificus exposed to various environmental conditions were harvested and fractionated into cells and supernatants by centrifugation. The cells were lysed using B-PERTM Bacterial Protein Extraction Reagent with Enzymes (Thermo Fisher Scientific) and residual cell debris was removed by centrifugation to obtain clear cell lysates. The supernatants were filtered through a PuradiscTM 25-mm syringe filter (pore size 0.2 μm; GE Healthcare, Chicago, IL) and concentrated using Amicon Ultra-15 (cut-off 30 kDa; Millipore, Burlington, MA). The protein levels of IscR, HlyU, H-NS, and DnaK in the clear cell lysates or VvhA and OmpU in the supernatant concentrates were determined as described previously (
      • Lee Z.W.
      • Kim B.S.
      • Jang K.K.
      • Bang Y.J.
      • Kim S.
      • Ha N.C.
      • Jung Y.H.
      • Lee H.J.
      • Han H.J.
      • Kim J.S.
      • Kim J.
      • Sahu P.K.
      • Jeong L.S.
      • Kim M.H.
      • Choi S.H.
      Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species.
      ,
      • Jang K.K.
      • Gil S.Y.
      • Lim J.G.
      • Choi S.H.
      Regulatory characteristics of Vibrio vulnificus gbpA gene encoding a mucin-binding protein essential for pathogenesis.
      ).

      EMSA and DNase I protection assay

      For EMSAs, a 501-bp vvhBA promoter region (−353 to +148 from the transcription start site of vvhBA) was amplified by PCR using unlabeled VVHBA02-F and [γ-32P]ATP-labeled VVHBA02-R as primers (Table S2). The radiolabeled probe DNA was then incubated with purified IscR, HlyU, and H-NS for 30 min at 30°C in a 20-μl reaction mixture containing binding buffer (40 mm Tris-Cl (pH 7.9), 70 mm KCl, 1 mm DTT, and 100 μg of BSA) and 0.1 μg of poly(dI-dC) (Sigma) as a nonspecific competitor. For competition analysis, the same but unlabeled 501-bp DNA fragment was used as a self-competitor DNA. Electrophoretic analysis of the DNA-protein complexes was performed as described previously (
      • Lee Z.W.
      • Kim B.S.
      • Jang K.K.
      • Bang Y.J.
      • Kim S.
      • Ha N.C.
      • Jung Y.H.
      • Lee H.J.
      • Han H.J.
      • Kim J.S.
      • Kim J.
      • Sahu P.K.
      • Jeong L.S.
      • Kim M.H.
      • Choi S.H.
      Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species.
      ).
      Similarly, the same 501-bp vvhBA promoter region was amplified by PCR using unlabeled VVHBA02-F and 6-FAM-labeled VVHBA02-R as primers for DNase I protection assays (Table S2). The binding of IscR, HlyU, and H-NS to the labeled DNA was performed as described above, and DNase I digestion of DNA-protein complexes followed the procedures as described previously (
      • Jang K.K.
      • Lee Z.W.
      • Kim B.
      • Jung Y.H.
      • Han H.J.
      • Kim M.H.
      • Kim B.S.
      • Choi S.H.
      Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.
      ). The digested DNA products were precipitated with ethanol and eluted in sterilized H2O, and then analyzed using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA) with Peak ScannerTM Software version 1.0 (Applied Biosystems) (
      • Hwang S.H.
      • Park J.H.
      • Lee B.
      • Choi S.H.
      A regulatory network controls cabABC expression leading to biofilm and rugose colony development in Vibrio vulnificus.
      ).

      Data analysis

      Averages and S.D. were calculated from at least three independent experiments. Statistical analysis was performed by the Student's t-test using GraphPad Prism 7.0 (GraphPad Software).

      Data availability

      All data presented in this paper are contained within the manuscript and supporting information. The raw data of RNA-seq analysis were deposited in NCBI Sequence Read Archive (SRA) database under accession numbers PRJNA560127.

      Author contributions

      G. C., K. K. J., J. G. L., and S. H. C. conceptualization; G. C., K. K. J., J. G. L., and Z.-W. L. resources; G. C., K. K. J., J. G. L., Z.-W. L., and H. I. formal analysis; G. C., K. K. J., and J. G. L. validation; G. C., K. K. J., and J. G. L. investigation; G. C., K. K. J., and H. I. visualization; G. C. and K. K. J. writing-original draft; S. H. C. supervision; S. H. C. funding acquisition; S. H. C. writing-review and editing.

      Acknowledgments

      We thank Eun Jung Na and Bityeoul Kim for kindly providing Δhns EJ151 and the HlyU-expressing plasmid pYU1317, respectively. We are also grateful to all members of the Choi laboratory for valuable discussions and technical support.

      Supplementary Material

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