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. 1989 Feb;171(2):775–783. doi: 10.1128/jb.171.2.775-783.1989

Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes.

B A Ozenberger 1, T J Brickman 1, M A McIntosh 1
PMCID: PMC209664  PMID: 2536681

Abstract

Biochemical analysis of the enzymatic activity catalyzing the conversion of chorismate to isochorismate in the enterobactin biosynthetic pathway attributed the reaction to the isochorismate synthetase enzyme, designated EntC. However, the lack of mutations defining this activity has hampered the precise identification of the entC structural gene. In this study, we engineered a stable insertion mutation into the chromosomal region between the enterobactin genes fepB and entE. This mutation disrupted the structural gene for a previously identified 44-kilodalton protein and eliminated production of 2,3-dihydroxybenzoic acid, the catechol precursor of enterobactin. The complete nucleotide sequence of this gene was determined and compared with the sequences of other genes encoding chorismate-utilizing proteins. The similarities observed in these comparisons not only indicated that the locus is entC but also supported the premise that these enzymes constitute a family of related proteins sharing a common evolutionary origin. In addition, in this and the accompanying paper (M. S. Nahlik, T. J. Brickman, B. A. Ozenberger, and M. A. McIntosh, J. Bacteriol. 171:784-790, 1989), evidence is presented indicating that the entA product is potentially a secondary factor in the chorismate-to-isochorismate conversion and that the prototypic entC lesion (entC401) resides in the structural gene for the EntA protein. Finally, polarity effects from the insertion mutation in entC on downstream biosynthetic genes indicated that this locus is the promoter-proximal cistron in an ent operon comprising at least five genes. Appropriate regulatory signals upstream of entC suggest that this operon is regulated by iron through interaction with the Fur repressor protein.

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Selected References

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  1. Bagg A., Neilands J. B. Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry. 1987 Aug 25;26(17):5471–5477. doi: 10.1021/bi00391a039. [DOI] [PubMed] [Google Scholar]
  2. Bagg A., Neilands J. B. Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiol Rev. 1987 Dec;51(4):509–518. doi: 10.1128/mr.51.4.509-518.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berg D. E., Schmandt M. A., Lowe J. B. Specificity of transposon Tn5 insertion. Genetics. 1983 Dec;105(4):813–828. doi: 10.1093/genetics/105.4.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  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. Fleming T. P., Nahlik M. S., McIntosh M. A. Regulation of enterobactin iron transport in Escherichia coli: characterization of ent::Mu d(Apr lac) operon fusions. J Bacteriol. 1983 Dec;156(3):1171–1177. doi: 10.1128/jb.156.3.1171-1177.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fleming T. P., Nahlik M. S., Neilands J. B., McIntosh M. A. Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12. Gene. 1985;34(1):47–54. doi: 10.1016/0378-1119(85)90293-8. [DOI] [PubMed] [Google Scholar]
  8. Goncharoff P., Nichols B. P. Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase. J Bacteriol. 1984 Jul;159(1):57–62. doi: 10.1128/jb.159.1.57-62.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grantham R., Gautier C., Gouy M., Jacobzone M., Mercier R. Codon catalog usage is a genome strategy modulated for gene expressivity. Nucleic Acids Res. 1981 Jan 10;9(1):r43–r74. doi: 10.1093/nar/9.1.213-b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Greenwood K. T., Luke R. J. Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12. Four polypeptides involved in the conversion of 2,3-dihydroxybenzoate to enterochelin. Biochim Biophys Acta. 1976 Dec 1;454(2):285–297. doi: 10.1016/0005-2787(76)90231-8. [DOI] [PubMed] [Google Scholar]
  11. Hudson G. S., Davidson B. E. Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12. J Mol Biol. 1984 Dec 25;180(4):1023–1051. doi: 10.1016/0022-2836(84)90269-9. [DOI] [PubMed] [Google Scholar]
  12. Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol. 1981 Sep 25;151(3):389–409. doi: 10.1016/0022-2836(81)90003-6. [DOI] [PubMed] [Google Scholar]
  13. Laird A. J., Young I. G. Tn5 mutagenesis of the enterochelin gene cluster of Escherichia coli. Gene. 1980 Nov;11(3-4):359–366. doi: 10.1016/0378-1119(80)90075-x. [DOI] [PubMed] [Google Scholar]
  14. McIntosh M. A., Earhart C. F. Coordinate regulation by iron of the synthesis of phenolate compounds and three outer membrane proteins in Escherichia coli. J Bacteriol. 1977 Jul;131(1):331–339. doi: 10.1128/jb.131.1.331-339.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Monk M., Kinross J. Conditional lethality of recA and recB derivatives of a strain of Escherichia coli K-12 with a temperature-sensitive deoxyribonucleic acid polymerase I. J Bacteriol. 1972 Mar;109(3):971–978. doi: 10.1128/jb.109.3.971-978.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nahlik M. S., Brickman T. J., Ozenberger B. A., McIntosh M. A. Nucleotide sequence and transcriptional organization of the Escherichia coli enterobactin biosynthesis cistrons entB and entA. J Bacteriol. 1989 Feb;171(2):784–790. doi: 10.1128/jb.171.2.784-790.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nahlik M. S., Fleming T. P., McIntosh M. A. Cluster of genes controlling synthesis and activation of 2,3-dihydroxybenzoic acid in production of enterobactin in Escherichia coli. J Bacteriol. 1987 Sep;169(9):4163–4170. doi: 10.1128/jb.169.9.4163-4170.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nichols B. P., van Cleemput M., Yanofsky C. Nucleotide sequence of Escherichia coli trpE. Anthranilate synthetase component I contains no tryptophan residues. J Mol Biol. 1981 Feb 15;146(1):45–54. doi: 10.1016/0022-2836(81)90365-x. [DOI] [PubMed] [Google Scholar]
  19. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  20. Ozenberger B. A., Nahlik M. S., McIntosh M. A. Genetic organization of multiple fep genes encoding ferric enterobactin transport functions in Escherichia coli. J Bacteriol. 1987 Aug;169(8):3638–3646. doi: 10.1128/jb.169.8.3638-3646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pettis G. S., Brickman T. J., McIntosh M. A. Transcriptional mapping and nucleotide sequence of the Escherichia coli fepA-fes enterobactin region. Identification of a unique iron-regulated bidirectional promoter. J Biol Chem. 1988 Dec 15;263(35):18857–18863. [PubMed] [Google Scholar]
  22. Pettis G. S., McIntosh M. A. Molecular characterization of the Escherichia coli enterobactin cistron entF and coupled expression of entF and the fes gene. J Bacteriol. 1987 Sep;169(9):4154–4162. doi: 10.1128/jb.169.9.4154-4162.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Queen C., Korn L. J. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):581–599. doi: 10.1093/nar/12.1part2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reznikoff W. S., Siegele D. A., Cowing D. W., Gross C. A. The regulation of transcription initiation in bacteria. Annu Rev Genet. 1985;19:355–387. doi: 10.1146/annurev.ge.19.120185.002035. [DOI] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schwyn B., Neilands J. B. Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 1987 Jan;160(1):47–56. doi: 10.1016/0003-2697(87)90612-9. [DOI] [PubMed] [Google Scholar]
  27. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  29. Young I. G., Langman L., Luke R. K., Gibson F. Biosynthesis of the iron-transport compound enterochelin: mutants of Escherichia coli unable to synthesize 2,3-dihydroxybenzoate. J Bacteriol. 1971 Apr;106(1):51–57. doi: 10.1128/jb.106.1.51-57.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. de Lorenzo V., Wee S., Herrero M., Neilands J. B. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol. 1987 Jun;169(6):2624–2630. doi: 10.1128/jb.169.6.2624-2630.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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