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. 1986 Jan;165(1):181–192. doi: 10.1128/jb.165.1.181-192.1986

Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12.

J W Coulton, P Mason, D R Cameron, G Carmel, R Jean, H N Rode
PMCID: PMC214387  PMID: 3079747

Abstract

The fusion-generating phage lambda plac Mu1 was used to produce fusions of lacZ to fhuA, the gene encoding the ferrichrome-iron receptor (FhuA protein) in the outer membrane of Escherichia coli K-12. Fusions to the fhuA gene in a delta (lac) strain were selected by their resistance to bacteriophage phi 80 vir. Ten independent (fhuA'-'lacZ) fusions were all Lac+ and were resistant to the lethal agents which require the FhuA protein as receptor, i.e., phi 80 vir, T5, T1, UC-1, and colicin M; none could utilize ferrichrome as the sole iron source. Specialized transducing phages were obtained by illegitimate excision from the chromosome of each of the fusion-bearing strains, and EcoRI fragments which encoded the fusions were subcloned into the high-copy plasmid pMLB524. Physical mapping of the fusion-containing plasmids confirmed the presence of three restriction sites which were also located on the chromosomal DNA of sequences near the fhuA gene. The direction of transcription of the fhuA gene was deduced from the direction of transcription of the (fhuA'-'lacZ) gene fusion. Identification of the chimeric proteins was made by both radiolabeling cells and immunoprecipitating the LacZ-containing proteins with antibody to beta-galactosidase and by preparing whole cell extracts from Lac+ cells containing the cloned gene fusions. Two sizes of (FhuA'-'LacZ) proteins were detected, 121 kDa and 124 kDa. The DNA sequences at the unique fusion joints were determined. The sequence information allowed us to identify three distinct fusion joints which were grouped as follows, type I fusions, 5'-ACT GCT CAG CCA A-3'; type IIa fusions, 5'-GCG GTT GAA CCG A-3'; and type IIb fusions: 5'-ACC GCT GCA CCT G-3'. To orient these fhuA fusion joints, the complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA. A single open reading frame was found which translated into a 747-amino acid polypeptide. The signal sequence of 33 amino acids was followed by a mature protein with a molecular weight of 78,992. Alignment of the amino acid sequence of the FhuA protein with the amino acid sequences presented for two other tonB-dependent receptor proteins in the outer membrane of E. coli showed an area of local homology at the amino terminus of all three proteins.

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Selected References

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

  1. Benson S. A., Bremer E., Silhavy T. J. Intragenic regions required for LamB export. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3830–3834. doi: 10.1073/pnas.81.12.3830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benson S. A., Hall M. N., Silhavy T. J. Genetic analysis of protein export in Escherichia coli K12. Annu Rev Biochem. 1985;54:101–134. doi: 10.1146/annurev.bi.54.070185.000533. [DOI] [PubMed] [Google Scholar]
  3. Braun V., Hancock R. E., Hantke K., Hartmann A. Functional organization of the outer membrane of escherichia coli: phage and colicin receptors as components of iron uptake systems. J Supramol Struct. 1976;5(1):37–58. doi: 10.1002/jss.400050105. [DOI] [PubMed] [Google Scholar]
  4. Bremer E., Silhavy T. J., Weisemann J. M., Weinstock G. M. Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step. J Bacteriol. 1984 Jun;158(3):1084–1093. doi: 10.1128/jb.158.3.1084-1093.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broome-Smith J. K., Edelman A., Yousif S., Spratt B. G. The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12. Eur J Biochem. 1985 Mar 1;147(2):437–446. doi: 10.1111/j.1432-1033.1985.tb08768.x. [DOI] [PubMed] [Google Scholar]
  6. Coulton J. W., Mason P., DuBow M. S. Molecular cloning of the ferrichrome-iron receptor of Escherichia coli K-12. J Bacteriol. 1983 Dec;156(3):1315–1321. doi: 10.1128/jb.156.3.1315-1321.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coulton J. W., Naegeli H. U., Braun V. Iron supply of Escherichia coli with polymer-bound ferricrocin. Eur J Biochem. 1979 Aug 15;99(1):39–47. doi: 10.1111/j.1432-1033.1979.tb13228.x. [DOI] [PubMed] [Google Scholar]
  8. Coulton J. W. The ferrichrome-iron receptor of Escherichia coli K-12. Antigenicity of the fhuA protein. Biochim Biophys Acta. 1982 Jul 16;717(1):154–162. doi: 10.1016/0304-4165(82)90393-2. [DOI] [PubMed] [Google Scholar]
  9. Fecker L., Braun V. Cloning and expression of the fhu genes involved in iron(III)-hydroxamate uptake by Escherichia coli. J Bacteriol. 1983 Dec;156(3):1301–1314. doi: 10.1128/jb.156.3.1301-1314.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hall M. N., Schwartz M., Silhavy T. J. Sequence information within the lamB genes in required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12. J Mol Biol. 1982 Mar 25;156(1):93–112. doi: 10.1016/0022-2836(82)90461-2. [DOI] [PubMed] [Google Scholar]
  11. Hancock R. W., Braun V. Nature of the energy requirement for the irreversible adsorption of bacteriophages T1 and phi80 to Escherichia coli. J Bacteriol. 1976 Feb;125(2):409–415. doi: 10.1128/jb.125.2.409-415.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hantke K., Braun V. Functional interaction of the tonA/tonB receptor system in Escherichia coli. J Bacteriol. 1978 Jul;135(1):190–197. doi: 10.1128/jb.135.1.190-197.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hantke K. Identification of an iron uptake system specific for coprogen and rhodotorulic acid in Escherichia coli K12. Mol Gen Genet. 1983;191(2):301–306. doi: 10.1007/BF00334830. [DOI] [PubMed] [Google Scholar]
  14. Hantke K. Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant. Mol Gen Genet. 1981;182(2):288–292. doi: 10.1007/BF00269672. [DOI] [PubMed] [Google Scholar]
  15. Heller K., Kadner R. J. Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli. J Bacteriol. 1985 Mar;161(3):904–908. doi: 10.1128/jb.161.3.904-908.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kadner R. J., Heller K., Coulton J. W., Braun V. Genetic control of hydroxamate-mediated iron uptake in Escherichia coli. J Bacteriol. 1980 Jul;143(1):256–264. doi: 10.1128/jb.143.1.256-264.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  19. Lugtenberg B., Van Alphen L. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim Biophys Acta. 1983 Mar 21;737(1):51–115. doi: 10.1016/0304-4157(83)90014-x. [DOI] [PubMed] [Google Scholar]
  20. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  21. Neilands J. B. Microbial envelope proteins related to iron. Annu Rev Microbiol. 1982;36:285–309. doi: 10.1146/annurev.mi.36.100182.001441. [DOI] [PubMed] [Google Scholar]
  22. Nikaido H., Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. doi: 10.1128/mr.49.1.1-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Plastow G. S., Pratt J. M., Holland I. B. The ferrichrome receptor protein (tonA) of Escherichia coli is synthesised as a precursor in vitro. FEBS Lett. 1981 Aug 31;131(2):262–264. doi: 10.1016/0014-5793(81)80380-8. [DOI] [PubMed] [Google Scholar]
  24. Postle K., Good R. F. DNA sequence of the Escherichia coli tonB gene. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5235–5239. doi: 10.1073/pnas.80.17.5235. [DOI] [PMC free article] [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. 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]
  27. Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
  28. Silhavy T. J., Beckwith J. Isolation and characterization of mutants of Escherichia coli K12 affected in protein localization. Methods Enzymol. 1983;97:11–40. doi: 10.1016/0076-6879(83)97115-x. [DOI] [PubMed] [Google Scholar]
  29. Tommassen J., Leunissen J., van Damme-Jongsten M., Overduin P. Failure of E. coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm. EMBO J. 1985 Apr;4(4):1041–1047. doi: 10.1002/j.1460-2075.1985.tb03736.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Witholt B., Boekhout M., Brock M., Kingma J., Heerikhuizen H. V., Leij L. D. An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal Biochem. 1976 Jul;74(1):160–170. doi: 10.1016/0003-2697(76)90320-1. [DOI] [PubMed] [Google Scholar]

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