Abstract
The genes for transport and synthesis of the phenolate siderophore enterobactin are present on the chromosomes of both Ent+ and Ent- clinical isolates of Shigella flexneri. To determine why Ent- S. flexneri isolates fail to express a functional enterobactin system, the structure and expression of enterobactin genes were examined. Several alterations may be responsible for the inability of S. flexneri to express enterobactin. (i) The mRNA levels produced from the entC and fepB genes were not derepressed in low-iron media. (ii) DNA sequence analysis of the entC-fepB intergenic region revealed an 83-bp noncontiguous deletion in the putative fepB leader sequence. The deleted sequences are in a region which would be capable of forming extensive stem-and-loop structures. (iii) An amber codon in the 5' portion of the entC gene was also detected. (iv) An IS1 element, previously mapped to the Ent- S. flexneri enterobactin gene cluster, was found to lie within a potential transcriptional termination sequence in the entF-fepE intergenic region. (v) A mutation responsible for the inactivation of the entF gene was mapped to the entF coding region by using entF hybrid gene fusions. (vi) A comparison of outer membrane profiles from an E. coli strain harboring the cloned fepA gene from either an Ent+ or Ent- Shigella isolate revealed that the Ent- FepA protein is present in the outer membrane but at greatly reduced levels than that of the Ent+ FepA protein. This observation, along with additional studies, suggests that the Ent- FepA may be defective in translation and/or translocation.
Full text
PDF









Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ames G. F. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem. 1974 Jan 25;249(2):634–644. [PubMed] [Google Scholar]
- Bassford P. J., Jr, Silhavy T. J., Beckwith J. R. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979 Jul;139(1):19–31. doi: 10.1128/jb.139.1.19-31.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Betlach M. C., Boyer H. W. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. doi: 10.1016/0378-1119(77)90074-9. [DOI] [PubMed] [Google Scholar]
- Brawerman G. Determinants of messenger RNA stability. Cell. 1987 Jan 16;48(1):5–6. doi: 10.1016/0092-8674(87)90346-1. [DOI] [PubMed] [Google Scholar]
- Brickman T. J., Ozenberger B. A., McIntosh M. A. Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli. J Mol Biol. 1990 Apr 20;212(4):669–682. doi: 10.1016/0022-2836(90)90229-F. [DOI] [PubMed] [Google Scholar]
- Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
- Bryce G. F., Brot N. Studies on the enzymatic synthesis of the cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine in Escherichia coli. Biochemistry. 1972 Apr 25;11(9):1708–1715. doi: 10.1021/bi00759a028. [DOI] [PubMed] [Google Scholar]
- Calderwood S. B., Mekalanos J. J. Confirmation of the Fur operator site by insertion of a synthetic oligonucleotide into an operon fusion plasmid. J Bacteriol. 1988 Feb;170(2):1015–1017. doi: 10.1128/jb.170.2.1015-1017.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Elkins M. F., Earhart C. F. Nucleotide sequence and regulation of the Escherichia coli gene for ferrienterobactin transport protein FepB. J Bacteriol. 1989 Oct;171(10):5443–5451. doi: 10.1128/jb.171.10.5443-5451.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Griffiths E., Stevenson P., Hale T. L., Formal S. B. Synthesis of aerobactin and a 76,000-dalton iron-regulated outer membrane protein by Escherichia coli K-12-Shigella flexneri hybrids and by enteroinvasive strains of Escherichia coli. Infect Immun. 1985 Jul;49(1):67–71. doi: 10.1128/iai.49.1.67-71.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
- Imamoto F., Yanofsky C. Transcription of the tryptophan operon in polarity mutants of Escherichia coli. II. Evidence for normal production of tryp-mRNA molecules and for premature termination of transcription. J Mol Biol. 1967 Aug 28;28(1):25–35. doi: 10.1016/s0022-2836(67)80074-3. [DOI] [PubMed] [Google Scholar]
- Johnsrud L. DNA sequence of the transposable element IS1. Mol Gen Genet. 1979 Jan 31;169(2):213–218. doi: 10.1007/BF00271673. [DOI] [PubMed] [Google Scholar]
- 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]
- Lawlor K. M., Daskaleros P. A., Robinson R. E., Payne S. M. Virulence of iron transport mutants of Shigella flexneri and utilization of host iron compounds. Infect Immun. 1987 Mar;55(3):594–599. doi: 10.1128/iai.55.3.594-599.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lawlor K. M., Payne S. M. Aerobactin genes in Shigella spp. J Bacteriol. 1984 Oct;160(1):266–272. doi: 10.1128/jb.160.1.266-272.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Luke R. K., Gibson F. Location of three genes concerned with the conversion of 2,3-dihydroxybenzoate into enterochelin in Escherichia coli K-12. J Bacteriol. 1971 Aug;107(2):557–562. doi: 10.1128/jb.107.2.557-562.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- O'Brien I. G., Cox G. B., Gibson F. Enterochelin hydrolysis and iron metabolism in Escherichia coli. Biochim Biophys Acta. 1971 Jun 22;237(3):537–549. doi: 10.1016/0304-4165(71)90274-1. [DOI] [PubMed] [Google Scholar]
- O'Brien I. G., Gibson F. The structure of enterochelin and related 2,3-dihydroxy-N-benzoylserine conjugates from Escherichia coli. Biochim Biophys Acta. 1970 Aug 14;215(2):393–402. doi: 10.1016/0304-4165(70)90038-3. [DOI] [PubMed] [Google Scholar]
- Ozenberger B. A., Brickman T. J., McIntosh M. A. Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes. J Bacteriol. 1989 Feb;171(2):775–783. doi: 10.1128/jb.171.2.775-783.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Payne S. M., Niesel D. W., Peixotto S. S., Lawlor K. M. Expression of hydroxamate and phenolate siderophores by Shigella flexneri. J Bacteriol. 1983 Sep;155(3):949–955. doi: 10.1128/jb.155.3.949-955.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Payne S. M. Synthesis and utilization of siderophores by Shigella flexneri. J Bacteriol. 1980 Sep;143(3):1420–1424. doi: 10.1128/jb.143.3.1420-1424.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Perry R. D., San Clemente C. L. Siderophore synthesis in Klebsiella pneumoniae and Shigella sonnei during iron deficiency. J Bacteriol. 1979 Dec;140(3):1129–1132. doi: 10.1128/jb.140.3.1129-1132.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Pierce J. R., Earhart C. F. Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake. J Bacteriol. 1986 Jun;166(3):930–936. doi: 10.1128/jb.166.3.930-936.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pierce J. R., Pickett C. L., Earhart C. F. Two fep genes are required for ferrienterochelin uptake in Escherichia coli K-12. J Bacteriol. 1983 Jul;155(1):330–336. doi: 10.1128/jb.155.1.330-336.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pollack J. R., Neilands J. B. Enterobactin, an iron transport compound from Salmonella typhimurium. Biochem Biophys Res Commun. 1970 Mar 12;38(5):989–992. doi: 10.1016/0006-291x(70)90819-3. [DOI] [PubMed] [Google Scholar]
- 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]
- Schmitt M. P., Payne S. M. Genetics and regulation of enterobactin genes in Shigella flexneri. J Bacteriol. 1988 Dec;170(12):5579–5587. doi: 10.1128/jb.170.12.5579-5587.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Starlinger P. IS elements and transposons. Plasmid. 1980 May;3(3):241–259. doi: 10.1016/0147-619x(80)90039-6. [DOI] [PubMed] [Google Scholar]
- Warner P. J., Williams P. H., Bindereif A., Neilands J. B. ColV plasmid-specific aerobactin synthesis by invasive strains of Escherichia coli. Infect Immun. 1981 Aug;33(2):540–545. doi: 10.1128/iai.33.2.540-545.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]