1887

Abstract

The gene encodes a product necessary for the synthesis of the iron-chelating and transport molecule enterobactin (Ent); cells harbouring mutations fail to grow in iron-deficient environments. For unknown reasons, it has not been possible to identify the product. The nucleotide sequence of the region has now been determined. An open reading frame extending in the same direction as the adjacent gene and capable of encoding an approximately 24 kDa polypeptide was found; it contained a high percentage of rare codons and two possible translational start sites. Complementation data suggested that EntD proteins truncated at the carboxy terminus retain some activity. Two REP sequences were present upstream of and an IS sequence was observed downstream. RNA dot-blot hybridizations demonstrated that is transcribed from the strand predicted by the sequencing results. An recombinant plasmid was constructed and shown to express low amounts of a fusion protein of the anticipated size (approximately 125 kDa). The evidence suggests a number of possible explanations for difficulties in detecting the product. Sequence data indicate that if has its own promoter, it is weak; the REP sequences suggest that mRNA may be destabilized; and translation may be slow because of the frequency of rare codons and a possible unusual start codon (UUG). The data are also consistent with previous evidence that the product is unstable.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-135-11-3043
1989-11-01
2021-10-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/135/11/mic-135-11-3043.html?itemId=/content/journal/micro/10.1099/00221287-135-11-3043&mimeType=html&fmt=ahah

References

  1. Adler H. I., Fischer W. D., Cohen A., Hardigree A. A. 1967; Miniature Escherichia coli cells deficient in DNA. Proceedings of the National Academy of Sciences of the United States of America 57:321–326
    [Google Scholar]
  2. Arnow L. E. 1937; Colorimetric determinations of the components of 3,4-dihydroxyphenylalanine- tyrosine mixtures. Journal of Biological Chemistry 118:531–537
    [Google Scholar]
  3. Bagg A., Neilands J. B. 1987; Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiological Reviews 51:509–518
    [Google Scholar]
  4. Berg C. M., Shaw K. J., Vender H., Broucha-Mankiewicz M. 1979; Physiological characterization of polar Tn5-induced isoleucine-valine auxo- trophs in Escherichia coli K-12: evidence for an internal promoter in the ilvOGEDA operon. Genetics 93:309–310
    [Google Scholar]
  5. Biggen M. D., Gibson T. J., Hong G. F. 1983; Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proceedings of the National Academy of Sciences of the United States of America 80:3963–3965
    [Google Scholar]
  6. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513–1523
    [Google Scholar]
  7. De Bruijn F. J., Lupski J. R. 1984; The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids - a review. Gene 27:131–149
    [Google Scholar]
  8. Casadaban M. J., Chou J., Cohen S. N. 1980; In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. Journal of Bacteriology 143:971–980
    [Google Scholar]
  9. Chong P., Hui I., Loo T., Gillam S. 1985; Structural analysis of a new GC-specific insertion element IS186. FEBS Letters 192:47–52
    [Google Scholar]
  10. Coderre P. E., Earhart C. F. 1984; Characterization of a plasmid carrying the Escherichia coli K-12 entD, fepA, fes and entF genes. FEMS Microbiology Letters 25:111–116
    [Google Scholar]
  11. Cohen S. N., Chang A. C. Y., Hsu L. 1972; Nonchromosomal antibiotic resistance in bacteria:genetic transformation of Escherichia coli by R-factor DNA. Proceedings of the National Academy of Sciences of the United States of America 69:2110–2114
    [Google Scholar]
  12. Dale R. M. K., McClure B. A., Horchins J. P. 1985; A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the com mitochondrial 18s rDNA. Plasmid 13:31–40
    [Google Scholar]
  13. Dunn J. J., Studier F. W. 1983; Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. Journal of Molecular Biology 166:477–535
    [Google Scholar]
  14. Earhart C. F. 1987; Ferrienterobactin transport in Escherichia coli. In Iron Transport in Microbes, Plants, and Animals pp. 67–84 Winkelmann G., Vander Helm D., Neilands J. B. Edited by Weinheim: VCH Verlagsgesellschaft;
    [Google Scholar]
  15. Elish M. E., Pierce J. R., Earhart C. F. 1988; Biochemical analysis of spontaneous fepA mutants of Escherichia coli. Journal of General Microbiology 134:1355–1364
    [Google Scholar]
  16. Elkins M. E., Earhart C. F. 1988; An Escherichia coli enterobactin cluster gene with sequence homology to trpE and pabB. FEMS Microbiology Letters 56:35–40
    [Google Scholar]
  17. Fairbanks G., Steck T. L., Wallace D. F. H. 1971; Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617
    [Google Scholar]
  18. Fleming T. P., Nahlik M. S., Neilands J. B., McIntosh M. A. 1985; Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12. Gene 34:47–54
    [Google Scholar]
  19. Von Gabain A., Belasco J. G., Schottel J. L., Chang A. C. Y., Cohen S. N. 1983; Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proceedings of the National Academy of Sciences of the United States of America 80:653–657
    [Google Scholar]
  20. Gold L. 1988; Posttranscriptional regulatory mechanisms in Escherichia coli. Annual Review of Biochemistry 57:199–233
    [Google Scholar]
  21. Greenwood K. T., Luke R. K. J. 1976; Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12: four polypeptides involved in the conversion of 2,3-dihydroxy benzoate to enterochelin. Biochimica et biophysica acta 454:285–297
    [Google Scholar]
  22. Greenwood K. T., Luke R. K. J. 1980; Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12, Salmonella typhimurium and Klebsiella pneumoniae. Biochimica et biophysica acta 614:185–195
    [Google Scholar]
  23. Greenwood K. T., Luke R. K. J. 1981; Regulation of enterochelin synthetase in Escherichia coli K-12. Biochimica et biophysica acta 660:371–374
    [Google Scholar]
  24. Howard-Flanders P., Simson E., Theriot L. 1964; A locus that controls filament formation and sensitivity to radiation in Escherichia coli K-12. Genetics 49:237–246
    [Google Scholar]
  25. Konigsberg W., Godson G. N. 1983; Evidence for use of rare codons in dnaG gene and other regulatory genes of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 80:687–691
    [Google Scholar]
  26. Kothary R. K., Jones D., Candido E. P. M. 1985; IS186: an Escherichia coli insertion element isolated from a cDNA library. Journal of Bacteriology 164:957–959
    [Google Scholar]
  27. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  28. Lawlor K. M., Payne S. M. 1984; Aerobactin genes in Shigella spp. Journal of Bacteriology 160:266–272
    [Google Scholar]
  29. Liu J., Duncan K., Walsh C. T. 1989; Nucleotide sequence of a cluster of Escherichia coli enterobactin biosynthetic genes: identification of entA and purification of its product 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase. Journal of Bacteriology 171:791–798
    [Google Scholar]
  30. Lugtenberg B., Meijers J., Peters R., Van der Hoek P., Van Alphen L. 1975; Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K-12 into four bands. FEBS Letters 58:254–258
    [Google Scholar]
  31. Lundrigan M. D., Kadner R. J. 1986; Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli: homology among outer membrane receptors which interact with TonB. Journal of Biological Chemistry 261:10797–10801
    [Google Scholar]
  32. Mackie G. A. 1981; Nucleotide sequence of the gene for ribosomal protein S20 and its flanking regions. Journal of Biological Chemistry 256:8177–8182
    [Google Scholar]
  33. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  34. McDonnell M. W., Simon M. N., Studier F. W. 1977; Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. Journal of Molecular Biology 110:119–146
    [Google Scholar]
  35. McDougall S., Neilands J. B. 1984; Plasmid- and chromosome-coded aerobactin synthesis in enteric bacteria: insertion sequences flank operon in plasmid-mediated systems. Journal of Bacteriology 159:300–305
    [Google Scholar]
  36. Meagher R. B., Tait R. C., Betlach M., Boyer H. W. 1977; Protein expression in E. coli minicells by recombinant plasmids. Cell 10:521–536
    [Google Scholar]
  37. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. 1984; Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Research 12:7035–7056
    [Google Scholar]
  38. Miller J. H. 1972 Experiments in Molecular Genetics p. 352 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  39. Nahlik M. S., Brickman T. J., Ozenberger B. A., McIntosh M. A. 1989; Nucleotide sequence and transcriptional organization of the Escherichia colienterobactin biosynthesis cistrons entB and entA. Journal of Bacteriology 171:784–790
    [Google Scholar]
  40. Newbury S. F., Smith N. H., Robinson E. C., Hiles I. D., Higgins C. F. 1987a; Stabilization of translationally active mRNA by prokaryotic REP sequences. Cell 48:297–310
    [Google Scholar]
  41. Newbury S. F., Smith N. H., Higgins C. F. 1987b; Differential mRNA stability controls relative gene expression within a polycistronic operon. Cell 511131–1143
    [Google Scholar]
  42. Perez-Casal J. F., Crosa J. H. 1984; Aerobactin iron uptake sequences in plasmid ColV-K30 are flanked by inverted IS1-like elements and replication regions. Journal of Bacteriology 160:256–265
    [Google Scholar]
  43. Pettis G. S., McIntosh M. A. 1987; Molecular characterization of the Escherichia coli enterobactin cistron entF and its coupled expression to the fesgene. Journal of Bacteriology 169:4154–4162
    [Google Scholar]
  44. Pettis G. S., Brickman T. J., McIntosh M. A. 1988; Transcriptional mapping and nucleotide sequence of the Escherichia coli fepA-fes enterobactin region. Journal of Biological Chemistry 263:18857–18863
    [Google Scholar]
  45. Poulis M. I., Shaw D. C., Campbell H. D., Young I. G. 1981; In vitro synthesis of the respiratory NADH dehydrogenase of Escherichia coli. Role of UUG as initiation codon. Biochemistry 20:4178–4185
    [Google Scholar]
  46. Reddy P., Peterkofsky A., McKenney K. 1985; Translational efficiency of the Escherichia coli adenylate cyclase gene: mutating the UUG initiation codon to GUG or AUG results in increased gene expression. Proceedings of the National Academy of Sciences of the United States of America 825656–5660
    [Google Scholar]
  47. Roy A., Haziza C., Danchin A. 1983; Regulation of adenylate cyclase synthesis in Escherichia coli: nucleotide sequence of the control region. EMBO Journal 2:791–797
    [Google Scholar]
  48. Rupprecht K. R., Gordon G., Lundrigan M. D., Gayda R. C., Markovitz A., Earhart C. F. 1984; ompT: Escherichia coli K-12 structural gene for protein a (3b). Journal of Bacteriology 153:1104–1106
    [Google Scholar]
  49. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74:5463–5468
    [Google Scholar]
  50. Schwyn B., Neilands J. B. 1987; Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry 160:47–56
    [Google Scholar]
  51. Sharp P. M., Li W. H. 1986; Codon usage in regulatory genes in Escherichia coli does not reflect selection for ‘rare’ codons. Nucleic Acids Research 14:7737–7749
    [Google Scholar]
  52. Shine J., Dalgarno L. 1974; The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proceedings of the National Academy of Sciences of the United States of America 71:1342–1346
    [Google Scholar]
  53. Studier F. W., Moffatt B. A. 1986; Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology 189:113–130
    [Google Scholar]
  54. Sugimura K., Higashi N. 1988; A novel outer-membrane-associated protease in Escherichia coli. Journal of Bacteriology 170:3650–3654
    [Google Scholar]
  55. Sugimura K., Nishihara N. 1988; Purification, characterization and primary structure of Escherichia coli protease VII with specificity for paired basic residues: identity of protease VII and OmpT. Journal of Bacteriology 170:5625–5632
    [Google Scholar]
  56. Tinoco I., Borer P. N., Dengler B., Levine M. D., Uhlenbeck O. C., Crothers D. M., Gralla J. 1973; Improved estimation of secondary structure in ribonucleic acids. Nature New Biology 246:40–41
    [Google Scholar]
  57. White B. A., Bancroft F. C. 1982; Cytoplasmic dot hybridization - simple analysis of relative mRNA levels in multiple small cell or tissue samples. Journal of Biological Chemistry 257:8569–8572
    [Google Scholar]
  58. Woodrow G. C., Young I. G., Gibson F. 1979; Biosynthesis of enterochelin in Escherichia coli K-12: separation of the polypeptides coded for by the entD, E, F and G genes. Biochimica et biophysica acta 582:145–153
    [Google Scholar]
  59. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp8 and pUC19 vectors. Gene 33:103–119
    [Google Scholar]
  60. Yin K. C., Blinkowa A., Walker J. R. 1986; Nucleotide sequence of the Escherichia coli gene dnaZX. Nucleic Acids Research 14:6541–6549
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-135-11-3043
Loading
/content/journal/micro/10.1099/00221287-135-11-3043
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error