Skip to main content
SpringerLink
Log in

VuuB and IutB reduce ferric-vulnibactin in Vibrio vulnificus M2799

  • Published:
BioMetals Aims and scope Submit manuscript

Abstract

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Boukhalfa H, Crumbliss AL (2002) Chemical aspects of siderophore mediated iron transport. Biometals 15:325–339

    CAS  PubMed  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram qualities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    CAS  PubMed  Google Scholar 

  • Butterton JR, Calderwood SB (1994) Identification, cloning, and sequencing of a gene required for ferric vibriobactin utilization by Vibrio cholerae. J Bacteriol 176:5631–5638

    CAS  PubMed  PubMed Central  Google Scholar 

  • Byers B, Arceneaux J (1998) Microbial iron transport: iron acquisition by pathogenic microorganisms. Met Ions Biol Syst 35:37–66

    CAS  PubMed  Google Scholar 

  • Cox CD (1980) Iron reductases from Pseudomonas aeruginosa. J Bacteriol 141:199–204

    CAS  PubMed  PubMed Central  Google Scholar 

  • Datta S, Crosa JH (2012) Identification and characterization of a novel outer membrane protein receptor required for hemin utilization in Vibrio vulnificus. Biometals 25:275–283

    CAS  PubMed  Google Scholar 

  • Griffiths GL, Sigel SP, Payne SM, Neilands JB (1984) Vibriobactin, a siderophore from Vibrio cholerae. J Biol Chem 259:383–385

    CAS  PubMed  Google Scholar 

  • Hantke K (2001) Iron and metal regulation in bacteria. Curr Opin Microbiol 4:172–177

    CAS  PubMed  Google Scholar 

  • Kawano H, Miyamoto K, Negoro M et al (2017) IutB participates in the ferric-vulnibactin utilization system in Vibrio vulnificus M2799. Biometals 30:203–216

    CAS  PubMed  Google Scholar 

  • Kawano H, Miyamoto K, Sakaguchi I et al (2013) Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799. Microb Pathog 65:73–81

    CAS  PubMed  Google Scholar 

  • Kawano H, Miyamoto K, Yasunobe M et al (2014) The RND protein is involved in the vulnibactin export system in Vibrio vulnificus M2799. Microb Pathog 75:59–67

    CAS  PubMed  Google Scholar 

  • Khan A, Singh P, Srivastava A (2018) Synthesis, nature and utility of universal iron chelator—Siderophore: a review. Microbiol Res 212–213:103–111

    PubMed  Google Scholar 

  • Kim C-M, Park R-Y, Park J-H et al (2006) Vibrio vulnificus vulnibactin, but not metalloprotease VvpE, is essentially required for iron-uptake from human holotransferrin. Biol Pharm Bull 29:911–918

    CAS  PubMed  Google Scholar 

  • Kim YR, Lee SE, Kook H et al (2008) Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells. Cell Microbiol 10:848–862

    CAS  PubMed  Google Scholar 

  • Kothary MH, Kreger AS (1987) Purification and characterization of an elastolytic protease of Vibrio vulnificus. J Gen Microbiol 133:1783–1791

    CAS  PubMed  Google Scholar 

  • Kwak JS, Jeong H-G, Satchell KJF (2011) Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection. Proc Natl Acad Sci USA 108:1645–1650

    CAS  PubMed  PubMed Central  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  PubMed  Google Scholar 

  • Li K, Chen WH, Bruner SD (2015) Structure and mechanism of the siderophore-interacting protein from the fuscachelin gene cluster of Thermobifida fusca. Biochemistry 54:3989–4000

    CAS  PubMed  Google Scholar 

  • Litwin CM, Byrne BL (1998) Cloning and characterization of an outer membrane protein of Vibrio vulnificus required for heme utilization: regulation of expression and determination of the gene sequence. Infect Immun 66:3134–3141

    CAS  PubMed  PubMed Central  Google Scholar 

  • Litwin CM, Calderwood SB (1993) Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange. J Bacteriol 175:706–715

    CAS  PubMed  PubMed Central  Google Scholar 

  • Litwin CM, Rayback TW, Skinner J (1996) Role of catechol siderophore synthesis in Vibrio vulnificus virulence. Infect Immun 64:2834–2838

    CAS  PubMed  PubMed Central  Google Scholar 

  • Matzanke BF, Anemüller S, Schünemann V et al (2004) FhuF, part of a siderophore-reductase system. Biochemistry 43:1386–1392

    CAS  PubMed  Google Scholar 

  • McKie AT, Barrow D, Latunde-Dada GO et al (2001) An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 291:1755–1759

    CAS  PubMed  Google Scholar 

  • Miethke M, Hou J, Marahiel MA (2011) The siderophore-interacting protein YqjH acts as a ferric reductase in different iron assimilation pathways of Escherichia coli. Biochemistry 50:10951–10964

    CAS  PubMed  Google Scholar 

  • Miyamoto K, Kosakai K, Ikebayashi S et al (2009) Proteomic analysis of Vibrio vulnificus M2799 grown under iron-repleted and iron-depleted conditions. Microb Pathog 46:171–177

    CAS  PubMed  Google Scholar 

  • Miyoshi N, Shimizu C, Miyoshi S, Shinoda S (1987) Purification and characterization of Vibrio vulnificus protease. Microbiol Immunol 31:13–25

    CAS  PubMed  Google Scholar 

  • Oh MH, Lee SM, Lee DH, Choi SH (2009) Regulation of the Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor protein and evaluation of its role in virulence. Infect Immun 77:1208–1215

    CAS  PubMed  PubMed Central  Google Scholar 

  • Okujo N, Saito M, Yamamoto S et al (1994) Structure of vulnibactin, a new polyamine-containing siderophore from Vibrio vulnificus. Biometals 7:109–116

    CAS  PubMed  Google Scholar 

  • Oliver JD (2015) The Biology of Vibrio vulnificus. Microbiol Spectr 3: VE-0001–2014

  • Poch MT, Johnson W (1993) Ferric reductases of Legionella pneumophila. Biometals 6:107–114

    CAS  PubMed  Google Scholar 

  • Ratledge C, Dover LG (2000) Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54:881–941

    CAS  PubMed  Google Scholar 

  • Raymond KN, Dertz EA, Kim SS (2003) Enterobactin: an archetype for microbial iron transport. Proc Natl Acad Sci USA 100:3584–3588

    CAS  PubMed  PubMed Central  Google Scholar 

  • Saha M, Sarkar S, Sarkar B et al (2016) Microbial siderophores and their potential applications: a review. Environ Sci Pollut Res 23:3984–3999

    CAS  Google Scholar 

  • Schröder I, Johnson E, De Vries S (2003) Microbial ferric iron reductases. FEMS Microbiol Rev 27:427–447

    PubMed  Google Scholar 

  • Simpson LM, White VK, Zane SF, Oliver JD (1987) Correlation between virulence and colony morphology in Vibrio vulnificus. Infect Immun 55:269–272

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tacket CO, Brenner F, Blake PA (1984) Clinical features and an epidemiological study of Vibrio vulnificus infections. J Infect Dis 149:558–561

    CAS  PubMed  Google Scholar 

  • Tan W, Verma V, Jeong K et al (2014) Molecular characterization of vulnibactin biosynthesis in Vibrio vulnificus indicates the existence of an alternative siderophore. Front Microbiol 5:1–11

    PubMed  PubMed Central  Google Scholar 

  • Tanabe T, Naka A, Aso H et al (2005a) A novel aerobactin utilization cluster in Vibrio vulnificus with a gene involved in the transcription regulation of the iutA homologue. Microbiol Immunol 49:823–834

    CAS  PubMed  Google Scholar 

  • Tanabe T, Takata N, Naka A et al (2005b) Identification of an AraC-like regulator gene required for induction of the 78-kDa ferrioxamine B receptor in Vibrio vulnificus. FEMS Microbiol Lett 249:309–314

    CAS  PubMed  Google Scholar 

  • Timmerman MM, Woods JP (1999) Ferric reduction is a potential iron acquisition mechanism for Histoplasma capsulatum. Infect Immun 67:6403–6408

    CAS  PubMed  PubMed Central  Google Scholar 

  • Trindade IB, Silva JM, Fonseca BM et al (2019) Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatility. J Biol Chem 294:157–167

    CAS  PubMed  Google Scholar 

  • Tsuchiya T, Mitsuo E, Hayashi N et al (2007) Vibrio vulnificus damages macrophages during the early phase of infection. Infect Immun 75:4592–4596

    CAS  PubMed  PubMed Central  Google Scholar 

  • Vadas A, Monbouquette HG, Johnson E, Schröder I (1999) Identification and characterization of a novel ferric reductase from the hyperthermophilic Archaeon Archaeoglobus fulgidus. J Biol Chem 274:36715–36721

    CAS  PubMed  Google Scholar 

  • Wang S, Wu Y, Outten FW (2011) Fur and the novel regulator yqjI control transcription of the ferric reductase gene yqjH in Escherichia coli. J Bacteriol 193:563–574

    CAS  PubMed  Google Scholar 

  • Webster ACD, Litwin CM (2000) Cloning and characterization of vuuA, a gene encoding the Vibrio vulnificus ferric vulnibactin receptor. Infect Immun 68:526–534

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wyckoff EE, Allred BE, Raymond KN, Payne SM (2015) Catechol siderophore transport by Vibrio cholerae. J Bacteriol 197:2840–2849

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported in part by a Grant-in-Aid for the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, 2011–2015 (S1101031). We thank Prof. Shin-ichi Miyoshi for supplying the V. vulnificus clinical isolate strain M2799. We are grateful for helpful advice provided by Prof. Seiji Inoue, Prof. Rikiro Fukunaga, Dr Shinobu Fujii, Dr Takaji Sato, Dr Yasuko In, Dr Katsuhiko Minoura, and Dr Toshihiro Fujii at Osaka University of Pharmaceutical Sciences. We also thank Kate Fox, DPhil, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Author information

Authors and Affiliations

  1. Department of Microbiology and Infection Control, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka, 569-1094, Japan

    Naoko Okai, Katsushiro Miyamoto, Takahiro Tsuchiya, Jun Komano & Hiroshi Tsujibo

  2. Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka, 569-1094, Japan

    Koji Tomoo

  3. Laboratory of Hygienic Chemistry, College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime, 790-8578, Japan

    Tomotaka Tanabe & Tatsuya Funahashi

Authors
  1. Naoko Okai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katsushiro Miyamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Koji Tomoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takahiro Tsuchiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jun Komano
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tomotaka Tanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tatsuya Funahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hiroshi Tsujibo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Katsushiro Miyamoto.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Okai, N., Miyamoto, K., Tomoo, K. et al. VuuB and IutB reduce ferric-vulnibactin in Vibrio vulnificus M2799. Biometals 33, 187–200 (2020). https://doi.org/10.1007/s10534-020-00241-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10534-020-00241-5

Keywords

Search

Navigation