Pakarian, Paknoosh and Pawelek, Peter D. (2016) A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli. BMC Biotechnology, 16 (1). p. 68. ISSN 1472-6750
Preview |
Text (application/pdf)
2MBpawelek-bmc-2016.pdf - Published Version Available under License Spectrum Terms of Access. |
Official URL: http://dx.doi.org/10.1186/s12896-016-0298-1
Abstract
Background
In the presence of sufficient iron, the Escherichia coli protein Fur (Ferric Uptake Regulator) represses genes controlled by the Fur box, a consensus sequence near or within promoters of target genes. De-repression of Fur-controlled genes occurs upon iron deprivation. In the E. coli chromosome, there is a bidirectional intercistronic promoter region with two non-overlapping Fur boxes. This region controls Fur-regulated expression of entCEBAH in the clockwise direction and fepB in the anticlockwise direction.
Results
We cloned the E. coli bidirectional fepB/entC promoter region into low-copy-number plasmid backbones (pACYC184 and pBR322) along with downstream sequences encoding epitope tags and a multiple cloning site (MCS) compatible with the bacterial adenylate cyclase two-hybrid (BACTH) system. The vector pFCF1 allows for iron-controlled expression of FLAG-tagged proteins, whereas the pFBH1 vector allows for iron-controlled expression of HA-tagged proteins. We showed that E. coli knockout strains transformed with pFCF1-entA, pFCF1-entE and pFBH1-entB express corresponding proteins with appropriate epitope tags when grown under iron restriction. Furthermore, transformants exhibited positive chrome azurol S (CAS) assay signals under iron deprivation, indicating that the transformants were functional for siderophore biosynthesis. Western blotting and growth studies in rich and iron-depleted media demonstrated that protein expression from these plasmids was under iron control. Finally, we produced the vector pFCF2, a pFCF1 derivative in which a kanamycin resistance (KanR) gene was engineered in the direction opposite of the MCS. The entA ORF was then subcloned into the pFCF2 MCS. Bidirectional protein expression in an iron-deprived pFCF2-entA transformant was confirmed using antibiotic selection, CAS assays and growth studies.
Conclusions
The vectors pFCF1, pFCF2, and pFBH1 have been shown to use the fepB/entC promoter region to control bidirectional in trans expression of epitope-tagged proteins in iron-depleted transformants. In the presence of intracellular iron, protein expression from these constructs was abrogated due to Fur repression. The compatibility of the pFCF1 and pFBH1 backbones allows for iron-controlled expression of multiple epitope-tagged proteins from a single co-transformant.
| Divisions: | Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry |
|---|---|
| Item Type: | Article |
| Refereed: | Yes |
| Authors: | Pakarian, Paknoosh and Pawelek, Peter D. |
| Journal or Publication: | BMC Biotechnology |
| Date: | 2016 |
| Funders: |
|
| Digital Object Identifier (DOI): | 10.1186/s12896-016-0298-1 |
| Keywords: | Ferric uptake regulator, Protein expression, Bidirectional promoter, Bacterial two-hybrid, CAS assay |
| ID Code: | 982267 |
| Deposited By: | Danielle Dennie |
| Deposited On: | 21 Mar 2017 17:43 |
| Last Modified: | 18 Jan 2018 17:54 |
References:
Andrews SC, Robinson AK, Rodríguez-Quiñones F. Bacterial iron homeostasis. FEMS Microbiol Rev. 2003;27:215–37.Hantke K. Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12. Mol Gen Genet. 1984;197:337–41.
Pohl E, Haller JC, Mijovilovich A, Meyer-Klaucke W, Garman E, Vasil ML. Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator. Mol Microbiol. 2003;47:903–15.
Fillat MF. The FUR, (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators. Arch Biochem Biophys. 2014;546:41–52.
Porcheron G, Dozois CM. Interplay between iron homeostasis and virulence: Fur and RyhB as major regulators of bacterial pathogenicity. Vet Microbiol. 2015;179:2–14.
de Lorenzo V, Wee S, Herrero M, Neilands JB. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol. 1987;169:2624–30.
Stojiljkovic I, Bäumler AJ, Hantke K. Fur regulon in Gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a fur titration assay. J Mol Biol. 1994;236:531–45.
Delany I, Rappuoli R, Scarlato V. Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis. Mol Microbiol. 2004;52:1081–90.
Nandal A, Huggins CC, Woodhall MR, McHugh J, Rodríguez-Quiñones F, Quail MA, Guest JR, Andrews SC. Induction of the ferritin gene (ftnA) of Escherichia coli by Fe(2+)-Fur is mediated by reversal of H-NS silencing and is RyhB independent. Mol Microbiol. 2010;75:637–57.
Seo SW, Kim D, Latif H, O’Brien EJ, Szubin R, Palsson BO. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli. Nat Commun. 2014;5:4910.
Baichoo N, Helmann JD. Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence. J Bacteriol. 2002;184:5826–32.
Pecqueur L, D’Autréaux B, Dupuy J, Nicolet Y, Jacquamet L, Brutscher B, Michaud-Soret I, Bersch B. Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and X-ray crystallography. J Biol Chem. 2006;281:21286–95.
Bagg A, Neilands JB. Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiol Rev. 1987;51:509–18.
Massé E, Gottesman S. A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A. 2002;99:4620–5.
Hantke K. Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism. Mol Gen Genet. 1987;210:135–9.
Troxell B, Fink RC, Porwollik S, McClelland M, Hassan HM. The Fur regulon in anaerobically grown Salmonella enterica sv. Typhimurium: identification of new Fur targets. BMC Microbiol. 2011;11:236.
de Lorenzo V, Herrero M, Giovannini F, Neilands JB. Fur (ferric uptake regulation) protein and CAP (catabolite-activator protein) modulate transcription of fur gene in Escherichia coli. Eur J Biochem. 1988;73:537–46.
Elkins MF, Earhart CF. Nucleotide sequence and regulation of the Escherichia coli gene for ferrienterobactin transport protein FepB. J Bacteriol. 1989;171:5443–51.
Brickman TJ, Ozenberger BA, McIntosh MA. Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli. J Mol Biol. 1990;212:669–82.
de Lorenzo V, et al. Mode of binding of the Fur protein to target DNA (Chapter 13). In: Iron transport in bacteria. Crosa JH, Mey AR, Payne SM, editors. Washington, D.C.: ASM Press; 2004.
Pakarian P, Pawelek PD. Subunit orientation in the Escherichia coli enterobactin biosynthetic EntA-EntE complex revealed by a two-hybrid approach. Biochimie. 2016;127:1–9.
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2:2006. 0008.
Payne SM. Detection, isolation, and characterization of siderophores. Methods Enzymol. 1994;235:329–44.
Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 1987;160:47–56.
Brzezinska M, Davies J. Two enzymes which phosphorylate neomycin and kanamycin in Escherichia coli strains carrying R factors. Antimicrob Agents Chemother. 1973;3:266–9.
Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A. 1998;95:5752–6.
Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978;134:1141–56.
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2:95–113.
Lin-Chao S, Chen WT, Wong TT. High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II. Mol Microbiol. 1992;6:3385–93.
Mead DA, Szczesna-Skorupa E, Kemper B. Single-stranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986;1:67–74.
Guan L, Liu Q, Li C, Zhang Y. Development of a Fur-dependent and tightly regulated expression system in Escherichia coli for toxic protein synthesis. BMC Biotechnol. 2013;13:25.
Repository Staff Only: item control page
Download Statistics
Download Statistics