Abstract
The gene coding for ferric enterobactin binding protein from E. coli O157:H7 was amplifi ed. This gene was cloned and expressed as C-terminal His (6)-tagged protein. The SDS-PAGE analysis of the total protein revealed only two distinct bands, with molecular masses of 31kDa and 34kDa. The Ni-NTA chromatography purifi ed FepB and the osmotically shocked periplasmic fraction of IPTG induced cells showed only a single band of 31 kDa. Polyclonal mouse antibody was raised against the recombinant protein during 4 weeks after immunization. Western blot analysis of the recombinant FepB with mouse antiserum revealeda single band of 31 kDa. Identification and purification of FepB helped reveal its appropriate molecular mass. Polyclonal antibody raised against the recombinant protein reacted with bacterial FepB. The recombinant protein FepB could have a protective effect against E. coli O157:H7 and might be useful as an effective vaccine.
Keywords: FepB, E. coli, Periplasmic binding protein, Ferric enterobactin
Full Text
The Full Text of this article is available as a PDF (621.2 KB).
References
- 1.Fischbach M.A., Lin H., Liu D.R., Walsh C.T. How pathogenic bacteria evade mammalian sabotage in the battle for iron. Nat Chem Biol. 2006;2:132–138. doi: 10.1038/nchembio771. [DOI] [PubMed] [Google Scholar]
- 2.Bullen J.J., Rogers H.J., Spalding P.B., Ward C.G. Natural resistance, iron and infection: a challenge for clinical medicine. J Med Microbiol. 2006;55:251–258. doi: 10.1099/jmm.0.46386-0. [DOI] [PubMed] [Google Scholar]
- 3.Cartron M.L., Maddocks S., Gillingham P., Craven C.J., Andrews S.C. Feo-transport of ferrous iron into bacteria. Biometals. 2006;19:143–157. doi: 10.1007/s10534-006-0003-2. [DOI] [PubMed] [Google Scholar]
- 4.Wandersman C., Delepelaire P. Bacterial iron sources: from siderophores to hemophores. Ann Rev Microbiol. 2004;58:611–647. doi: 10.1146/annurev.micro.58.030603.123811. [DOI] [PubMed] [Google Scholar]
- 5.Skaar E.P., Humayun M., Bae T., DeBord K.L., Schneewind O. Iron-source preference of Staphylococcus aureus infections. Science. 2004;305:1626–1628. doi: 10.1126/science.1099930. [DOI] [PubMed] [Google Scholar]
- 6.Bleuel C., Grosse C., Taudte N., Scherer J., Wesenberg D., Krauss G.J., Nies D.H., Grass G. TolC is involved in enterobactin effl ux across the outer membrane of Escherichia coli. J Bacteriol. 2005;187:6701–6707. doi: 10.1128/JB.187.19.6701-6707.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Postle K. Active transport by customized beta-barrels. Nat Struct Biol. 1999;6:3–6. doi: 10.1038/4869. [DOI] [PubMed] [Google Scholar]
- 8.Shea C.M., McIntosh M.A. Nucleotide sequence and genetic organization of the ferric enterobactin transport system: homology to other periplasmic binding proteindependent systems in Escherichia coli. Mol Microbiol. 1991;5:1415–1428. doi: 10.1111/j.1365-2958.1991.tb00788.x. [DOI] [PubMed] [Google Scholar]
- 9.Koster W. Iron(III) hydroxamate transport across the cytoplasmic membrane of Escherichia coli. Biol Met. 1991;4:23–32. doi: 10.1007/BF01135553. [DOI] [PubMed] [Google Scholar]
- 10.Alexander D., Zuberer D. Use of chromweazurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil Soils. 1991;12:39–45. doi: 10.1007/BF00369386. [DOI] [Google Scholar]
- 11.Hook-Barnard I.G., Brickman T.J., McIntosh M.A. Identifi cation of an AU-rich translational enhancer within the Escherichia coli fepB leader RNA. J Bacteriol. 2007;189:4028–4037. doi: 10.1128/JB.01924-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Sprencel C., Cao Z., Qi Z., Scott D.C., Montague M.A., Ivanoff N., Xu J., Raymond K.M., Newton S.M., Klebba P.E. Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB. J Bacteriol. 2000;182:5359–5364. doi: 10.1128/JB.182.19.5359-5364.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Ferreiros C., Criado M.T., Gomez J.A. The neisserial 37 kDa ferric binding protein (FbpA) Comp Biochem Physiol B Biochem Mol Biol. 1999;123:1–7. doi: 10.1016/S0305-0491(99)00044-9. [DOI] [PubMed] [Google Scholar]
- 14.Akhtar M.S., Qaisar A., Irfanullah J., Iqbal J., Khan B., Jehangir M., Nadeem M.A., Khan M.A., Afzal M.S., Ul-Haq I., Imran M.B. Antimicrobial peptide 99mTc-Ubiquicidin 29-41 as human infection-imaging agent: Clinical trial. J Nucl Med. 2005;46:567–573. [PubMed] [Google Scholar]