Archives of Microbiology

, Volume 181, Issue 4, pp 287–293 | Cite as

Cloning, identification and expression of an entE homologue angE from Vibrio anguillarum serotype O1

  • Qin Liu
  • Yue Ma
  • Haizhen Wu
  • Mingfei Shao
  • Haifeng Liu
  • Yuanxing Zhang
Original Paper

Abstract

Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, is synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulence plasmid pJM1. angE, as one of the NRPS genes, is responsible for selecting and activating 2,3-dihydroxybenzoic acid (2,3-DHBA), an important precursor in anguibactin synthesis, into 2,3-DHBA-AMP by adenylylation in the presence of ATP. In this work, an entE homologue, angE, was identified on pEIB1 (a pJM1-like plasmid) from virulent V. anguillarum serotype O1 strain MVM425. A recombinant clone carrying the complete angE was able to complement an Escherichia coli entE mutant. The angE-encoded protein was overexpressed in E. coli and purified by a three-step procedure. Purified AngE was then used to establish an in vitro enzymatic reaction in which its enzymatic activity of 1-(5′-monophosphate adenyl) 2,3-dihydroxybenzoic acid ligase (2,3-DHBA-AMP ligase) was proved using HPLC to detect AMP formation in the reaction mixture. Moreover, evidence at the level of both transcription and translation confirmed that angE was actively expressed in vivo in V. anguillarum MVM425, and interestingly, unlike many other iron-uptake-system-related genes, its expression is not induced by a low iron concentration in the surrounding environment.

Keywords

Vibrio anguillarrum pJM1-like plasmid Iron uptake system angE 2,3-DHBA-AMP ligase Anguibactin biosynthesis Gene expression 

References

  1. Actis LA, Fish W, Crosa JH, Kellerman K, Ellenberger SR, Jauser FM, Sanders LJ (1986) Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1). J Bacteriol 167:57–65PubMedGoogle Scholar
  2. Actis LA, Tolmasky ME, Farrell DH, Crosa JH (1988) Genetic and molecular characterization of essential components of the Vibrio anguillarum plasmid-mediated iron-transport system. J Biol Chem 263 (6):2853–2860PubMedGoogle Scholar
  3. Chen Q, Actis LA, Tolmasky ME, Crosa JH (1994) Chromosome-mediated 2,3-dihydroxybenzoic acid is a precursor in the biosynthesis of the plasmid mediated siderophore anguibactin in Vibrio anguillarum. J Bacteriol 176:4226–4234PubMedGoogle Scholar
  4. Chen Q, Wertheimer AM, Tolmasky ME, Crosa JH (1996) The AngR protein and the siderophore anguibactin positively regulate the expression of iron-transport genes in Vibrio anguillarum. Mol Microbiol 22:127–134PubMedGoogle Scholar
  5. Conchas RF, Lemos ML, Barja JL, Toranzo AE (1991) Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins. Appl Environ Microbiol 57:2956–2962Google Scholar
  6. Crosa J H (1980) A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron-sequestering system. Nature 284:566–568PubMedGoogle Scholar
  7. Crosa JH (1989) Genetics and molecular biology of siderophore mediated iron transport in bacteria. Microbiol Rev 53:517–530Google Scholar
  8. Crosa JH (1997) Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria. Microbiol Mol Biol Rev 67:319–336Google Scholar
  9. Crosa JH, Walsh CT (2002) Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66:223–249CrossRefPubMedGoogle Scholar
  10. Crosa JH, Hodges LL, Schiewe MH (1980) Curing of a plasmid is correlated with an attenuation of virulence in the marine fish pathogen Vibrio anguillarum. Infect Immun 27:897–902PubMedGoogle Scholar
  11. Farrell DH, Mikesell P, Actis LA, Crosa JH (1990) A regulatory gene, angR, of the iron uptake system of Vibrio anguillarum: similarity with phage P22 cro and regulation by iron. Gene 86:45–51PubMedGoogle Scholar
  12. Frederick M, Ausubel K, Brent R, Kingston R, Moore DD; Seidman JG, Smith JA, Struhl K (1995) Short protocols in molecular biology, 3rd edn. Wiley, New YorkGoogle Scholar
  13. Gehring AM, Bradley KA, Walsh CT (1997) Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate. Biochemistry 36:8495–8503CrossRefPubMedGoogle Scholar
  14. Holmstrøm1 K, Gram L (2003) Elucidation of the Vibrio anguillarum genetic response to the potential fish probiont Pseudomonas fluorescens AH2, using RNA-arbitrarily primed PCR. J Bacteriol 185:831–842CrossRefPubMedGoogle Scholar
  15. Koester WL, Actis LA, Waldbeser LS, Tolmasky ME, Crosa JH (1991) Molecular characterization of the iron transport system mediated by the pJM1 plasmid in Vibrio anguillarum 775. J Biol Chem 266:23829–23833PubMedGoogle Scholar
  16. Lorenzo MD, Stork M, Tolmasky ME, Actis LA, Farrell D, Welch TJ, Crosa LM, Wertheimer AM, Chen Q, Salinas P, Waldbeser L, Crosa JH (2003) Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775. J Bacteriol Oct: 5822–5830CrossRefGoogle Scholar
  17. Olsen JE, Larsen JL (1990) Restriction fragments length polymorphism of the Vibrio anguillarum virulence plasmid. Appl Environ Microbiol 56:3130–3132PubMedGoogle Scholar
  18. Pedersen K, Larsen JL (1995) Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes. Appl Environ Microbiol 61:2292-2296PubMedGoogle Scholar
  19. Pedersen K, Tiainen T, Larsen JL (1996) Plasmid profiles, restriction fragment length polymorphisms and O-serotypes among Vibrio anguillarum isolates. Epidemiol Infect 117:471–478PubMedGoogle Scholar
  20. Rusnak F, Faraci WS, Walsh C (1989) Subcloning, expression, and purification of the enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase: demonstration of enzyme-bound 2,3-dihydroxybenzoyl adenylate product. Biochemistry 28:6827–6833.PubMedGoogle Scholar
  21. Salinas PC, Crosa JH (1995) Regulation of angR, a gene with regulatory and biosynthetic functions in the pJM1 plasmid-mediated iron uptake system of Vibrio anguillarum. Gene 160:17–23CrossRefPubMedGoogle Scholar
  22. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56PubMedGoogle Scholar
  23. Staab JF, Elkins MF, Earhart CF (1989) Nucleotide sequence of the Escherichia coli entE gene. FEMS Microbiol Lett 59:15–20CrossRefGoogle Scholar
  24. Tolmasky ME, Crosa JH (1991) Regulation plasmid mediated iron transport and virulence in Vibrio anguillarum. Biol Methods 4:33–35Google Scholar
  25. Tolmasky ME, Actis LA, Toranzo A, Barja J, Crosa JH (1985) Plasmid mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain. J Gen Microbiol 131:1989–1997PubMedGoogle Scholar
  26. Tolmasky ME, Actis LA, Crosa JH (1988) Genetic analysis of the iron uptake region of the Vibrio anguillarum plasmid pJM1: molecular cloning of genetic determinants encoding a novel trans activator of siderophore biosynthesis. J Bacteriol 160:860–866Google Scholar
  27. Tolmasky ME, Actis LA, Crosa JH (1993) A single amino acid change in AngR, a protein encoded by pJM1-like virulence plasmids, results in hyper production of anguibactin. Infect Immun 61:3228–3233PubMedGoogle Scholar
  28. Tolmasky ME, Actis LA, Crosa JH (1994) A histidine decarboxylase gene encoded by the Vibrio anguillarum plasmid pJM1: histamine is a precursor in the biosynthesis of anguibactin. Mol Microbiol 15:87–95Google Scholar
  29. Welch TJ, Chai S, Crosa JH (2000) The overlapping angB and angG genes are encoded within the transacting factor region of the virulence plasmid in Vibrio anguillarum: essential role in siderophore biosynthesis. J Bacteriol 182:6762–6773CrossRefPubMedGoogle Scholar
  30. Wertheimer AM, Verweij W, Chen Q, Crosa LM, Nagasawa M, Tolmasky ME, Actis LA, Crosa JH (1999) Characterization of the angR gene of Vibrio anguillarum: essential role in virulence. Infect Immun 67:6496–6509PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Qin Liu
    • 1
  • Yue Ma
    • 1
  • Haizhen Wu
    • 1
  • Mingfei Shao
    • 1
  • Haifeng Liu
    • 1
  • Yuanxing Zhang
    • 1
  1. 1.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghai China

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