Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Feb;175(3):589–596. doi: 10.1128/jb.175.3.589-596.1993

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster.

A Thariath 1, D Socha 1, M A Valvano 1, T Viswanatha 1
PMCID: PMC196193  PMID: 8423134

Abstract

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
589

Images in this article

593Image
on p.593

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnold L. D., Viswanatha T. The use of bis(mercaptoacetato-S,O)hydroxoiron(III) complex for the determination of hydroxamates. J Biochem Biophys Methods. 1983 Dec;8(4):307–320. doi: 10.1016/0165-022x(83)90005-2. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carbonetti N. H., Williams P. H. A cluster of five genes specifying the aerobactin iron uptake system of plasmid ColV-K30. Infect Immun. 1984 Oct;46(1):7–12. doi: 10.1128/iai.46.1.7-12.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gibson F., Magrath D. I. The isolation and characterization of a hydroxamic acid (aerobactin) formed by Aerobacter aerogenes 62-I. Biochim Biophys Acta. 1969 Nov 18;192(2):175–184. doi: 10.1016/0304-4165(69)90353-5. [DOI] [PubMed] [Google Scholar]
  7. Goh C. J., Szczepan E. W., Menhart N., Viswanatha T. Studies on lysine:N6-hydroxylation by cell-free systems of Aerobacter aerogenes 62-1. Biochim Biophys Acta. 1989 Mar 24;990(3):240–245. doi: 10.1016/s0304-4165(89)80040-6. [DOI] [PubMed] [Google Scholar]
  8. Griffiths E. Iron and bacterial virulence--a brief overview. Biol Met. 1991;4(1):7–13. doi: 10.1007/BF01135551. [DOI] [PubMed] [Google Scholar]
  9. Gross R., Engelbrecht F., Braun V. Genetic and biochemical characterization of the aerobactin synthesis operon on pColV. Mol Gen Genet. 1984;196(1):74–80. doi: 10.1007/BF00334095. [DOI] [PubMed] [Google Scholar]
  10. Gross R., Engelbrecht F., Braun V. Identification of the genes and their polypeptide products responsible for aerobactin synthesis by pColV plasmids. Mol Gen Genet. 1985;201(2):204–212. doi: 10.1007/BF00425661. [DOI] [PubMed] [Google Scholar]
  11. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  12. Herrero M., de Lorenzo V., Neilands J. B. Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions. J Bacteriol. 1988 Jan;170(1):56–64. doi: 10.1128/jb.170.1.56-64.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Konopka K., Bindereif A., Neilands J. B. Aerobactin-mediated utilization of transferrin iron. Biochemistry. 1982 Dec 7;21(25):6503–6508. doi: 10.1021/bi00268a028. [DOI] [PubMed] [Google Scholar]
  14. Kuo D. J., Jordan F. Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate. Biochemistry. 1983 Aug 2;22(16):3735–3740. doi: 10.1021/bi00285a003. [DOI] [PubMed] [Google Scholar]
  15. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Marolda C. L., Welsh J., Dafoe L., Valvano M. A. Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187. J Bacteriol. 1990 Jul;172(7):3590–3599. doi: 10.1128/jb.172.7.3590-3599.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Murray G. J., Clark G. E., Parniak M. A., Viswanatha T. Effect of metabolites on epsilon-N-hydroxylysine formation in cell-free extracts of Aerobacter aerogenes 62-1. Can J Biochem. 1977 Jun;55(6):625–629. doi: 10.1139/o77-090. [DOI] [PubMed] [Google Scholar]
  19. Parniak M. A., Jackson G. E., Murray G. J., Viswanatha T. Studies on the formation of N6-hydroxylysine in cell-free extracts of Aerobacter aerogenes 62-1. Biochim Biophys Acta. 1979 Jul 11;569(1):99–108. doi: 10.1016/0005-2744(79)90085-8. [DOI] [PubMed] [Google Scholar]
  20. Plattner H. J., Pfefferle P., Romaguera A., Waschütza S., Diekmann H. Isolation and some properties of lysine N6-hydroxylase from Escherichia coli strain EN222. Biol Met. 1989;2(1):1–5. doi: 10.1007/BF01116193. [DOI] [PubMed] [Google Scholar]
  21. Read S. M., Northcote D. H. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal Biochem. 1981 Sep 1;116(1):53–64. doi: 10.1016/0003-2697(81)90321-3. [DOI] [PubMed] [Google Scholar]
  22. Reeves R. H., Roth J. R. A recessive UGA suppressor. J Mol Biol. 1971 Mar 28;56(3):523–533. doi: 10.1016/0022-2836(71)90399-8. [DOI] [PubMed] [Google Scholar]
  23. Szczepan E. W., Kaller D., Honek J. F., Viswanatha T. Direct evidence for the participation of pyruvate in N-hydroxylation of lysine. FEBS Lett. 1987 Jan 26;211(2):239–242. doi: 10.1016/0014-5793(87)81444-8. [DOI] [PubMed] [Google Scholar]
  24. Valvano M. A., Crosa J. H. Aerobactin iron transport genes commonly encoded by certain ColV plasmids occur in the chromosome of a human invasive strain of Escherichia coli K1. Infect Immun. 1984 Oct;46(1):159–167. doi: 10.1128/iai.46.1.159-167.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Valvano M. A., Silver R. P., Crosa J. H. Occurrence of chromosome- or plasmid-mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichia coli K1. Infect Immun. 1986 Apr;52(1):192–199. doi: 10.1128/iai.52.1.192-199.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Waters V. L., Crosa J. H. DNA environment of the aerobactin iron uptake system genes in prototypic ColV plasmids. J Bacteriol. 1986 Aug;167(2):647–654. doi: 10.1128/jb.167.2.647-654.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Williams P. H. Novel iron uptake system specified by ColV plasmids: an important component in the virulence of invasive strains of Escherichia coli. Infect Immun. 1979 Dec;26(3):925–932. doi: 10.1128/iai.26.3.925-932.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  29. de Lorenzo V., Bindereif A., Paw B. H., Neilands J. B. Aerobactin biosynthesis and transport genes of plasmid ColV-K30 in Escherichia coli K-12. J Bacteriol. 1986 Feb;165(2):570–578. doi: 10.1128/jb.165.2.570-578.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. de Lorenzo V., Neilands J. B. Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli. J Bacteriol. 1986 Jul;167(1):350–355. doi: 10.1128/jb.167.1.350-355.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. von Heijne G. Transcending the impenetrable: how proteins come to terms with membranes. Biochim Biophys Acta. 1988 Jun 9;947(2):307–333. doi: 10.1016/0304-4157(88)90013-5. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES