A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli
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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.
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.
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.
KeywordsFerric uptake regulator Protein expression Bidirectional promoter Bacterial two-hybrid CAS assay
Bacterial adenylate cyclase two-hybrid
Chrome azurol S
Ferric Uptake Regulator
Glyceraldehyde 3-phosphate dehydrogenase
Multiple cloning site
Open reading frame
SDS-polyacrylamide gel electrophoresis
Bacterial iron acquisition is tightly controlled in order to ensure adequate iron uptake to support cellular survival and growth while preventing an over-accumulation of iron leading to oxidative damage . To promote iron homeostasis, most genes involved in iron uptake mechanisms are only abundantly expressed under conditions of low intracellular iron, and are typically repressed when the cell is replete with iron. In Escherichia coli, one of the major regulators of iron homeostasis is the protein Fur (Ferric Uptake Regulator), a homodimeric protein with 17 kDa subunits [2, 3, 4]. Given its central role in regulating iron homeostasis and oxidative stress, Fur, along with the small RNA RyhB, are known virulence factors in a number of pathogenic bacterial species that require iron from host organisms . Fur, in the iron-bound holo form, binds tightly to a recognition site known as the Fur box. Although there is variation in Fur box sequences, they all share identity with a 19-bp consensus sequence 5′-(GATAATGAT(A/T)ATCATTATC)-3′ [6, 7]. In addition to its classical role as a repressor, holo-Fur has been reported to activate a number of gene targets [8, 9]. A recent genome-wide study has also reported that Fur regulates 82 genes in E. coli, both by apo- and holo-Fur activation and holo-Fur repression . In the classical holo-Fur repression mechanism, iron-bound Fur binds to a Fur box sequence that overlaps with, or is proximal to, promoters of iron-responsive genes, thus preventing their transcription . When intracellular iron is depleted, Fe2+ is released from Fur, causing conformational changes in the protein resulting in dissociation from the Fur box . This de-repression results in the up-regulation of Fur-controlled genes. Numerous genes are controlled by holo-Fur, including those that encode: (i) proteins involved in siderophore-mediated iron uptake , (ii) small RNAs such as ryhB that regulate bacterial iron uptake , (iii) some TCA cycle enzymes , (iv) superoxide dismutase [14, 16], and (v) Fur itself .
Reagents, plasmids, software and primers
Plasmids used in this study
Production of pFCF1 and pFCF2
A 489-bp DNA fragment (gBlock1; Additional file 2: Figure S1) with flanking NcoI and EcoRI sites containing: (i) the E. coli fepB/entC bidirectional promoter region, (ii) the FLAG tag sequence, (iii) the TEV protease cleavage site sequence, and (iv) the multiple cloning site (MCS) from pUT18C (Euromedex) was synthesized as a gBlock® (Integrated DNA Technologies, San Diego, California). This fragment was digested with NcoI and EcoRI (NEB) and cloned into pACYC184 linearized with the same restriction enzymes. The resulting vector was named pFCF1. In order to create pFCF1-entA, pFCF1-entE, and pFCF1-T25, E. coli entA and entE ORFs were PCR-amplified from pCA24N-based constructs as reported previously . The B. pertussis T25 fragment was PCR-amplified from pKT25 (Euromedex). PCR products were subcloned into the KpnI and EcoRI sites of the pFCF1 MCS.
A 903-bp DNA fragment (gBlock2; Additional file 3: Figure S2) containing: (i) the HA tag sequence and (ii) the kanamycin resistance (KanR) gene from pKT25 (Euromedex) was synthesized as a gBlock® (Integrated DNA Technologies, San Diego California). The ends of this fragment contained ~40-nucleotide regions that overlapped with corresponding sequences upstream and downstream of NcoI and ScaI sites, respectively, in pFCF1. The synthesized fragment was inserted into pFCF1 digested with NcoI and ScaI using the Gibson Assembly Master Mix (New England Biolabs) according to manufacturer’s protocol. The resulting vector was named pFCF2. The E. coli entA ORF was subcloned between KpnI and EcoRI sites of digested pFCF2 to generate pFCF2-entA. pFCF1 and pFCF2 constructs were verified by DNA sequencing (McGill University and Génome Québec Innovation Centre).
Production of pFBH1
A 522-bp DNA fragment (gBlock3; Additional file 4: Figure S3) containing: (i) the E. coli fepB/entC bidirectional promoter region, (ii) the HA tag sequence, (iii) the TEV protease cleavage site sequence, and (iv) the multiple cloning site (MCS) from pUT18C (Euromedex) was synthesized as a gBlock® (Integrated DNA Technologies, San Diego California). The ends of this fragment contained 25-nucleotide regions that overlapped with corresponding sequences upstream and downstream of EcoRI and SalI sites, respectively, in pBR322. The fragment was inserted into pBR322 digested with EcoRI and SalI using the Gibson Assembly Master Mix (New England Biolabs) according to the manufacturer’s protocol. The resulting vector was named pFBH1. The E. coli entB ORF was subcloned into the KpnI and EcoRI sites of the pFBH1 MCS. The pFBH1 construct was verified by DNA sequencing (McGill University and Génome Québec Innovation Centre).
All plasmid constructs and empty vector controls were transformed into respective E. coli BW25113 (F−, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ − , rph-1, Δ(rhaD-rhaB)568, hsdR514) knockout strains  that have been modified to remove the kanamycin resistance gene as reported previously . Strains transformed with, pFCF1, pFCF1-entA, and pFCF1-entE were plated onto LB agar containing 12.5 μg/ml tetracycline. Strains transformed with pFCF2, pFCF2-entA were plated onto LB agar containing 12.5 μg/ml tetracycline and 50 μg/ml kanamycin. Strains transformed with pFBH1 or pFBH1-entB were plated onto LB agar containing 100 μg/ml ampicillin. All plates were incubated overnight at 37 °C. Overnight cultures (LB broth with appropriate antibiotic) from colony picks were diluted 1:1000 in 1× modified M9 medium  and 12.5 μg/ml tetracycline with or without 50 μg/ml kanamycin or 100 μg/ml ampicillin. Minimal medium cultures were grown at 37 °C overnight. CAS-agar plates supplemented with appropriate antibiotics were prepared according to Payne et al. . CAS plates were spotted with 1 μL overnight cultures and incubated at 37 °C for approximately 16 h. Presences of orange halos were indicative of enterobactin biosynthesis . Each CAS assay was performed in triplicate.
Single colony picks of transformants used for CAS assays were used to inoculate LB broth supplemented with appropriate antibiotics. Overnight cultures were diluted 1:100 in LB plus antibiotics and then grown at 30 °C until they reached an A600 of 1.00. Cultures were centrifuged for 1 min at 21,000 × g and cell pellets were resuspended in 1× modified M9 medium such that all cultures were diluted to an equivalent cell density (A600 = 1.00). Cultures for growth measurements were then prepared by 1:1000 dilution into 1× modified M9 medium plus 50 μM 2,2′-dipyridyl containing appropriate antibiotic. Diluted cultures were incubated at 30 °C for 16 h with agitation. Cell densities were measured as A600 values. Growth experiments were performed in triplicate.
Additional growth studies of pFCF2-entA transformants were performed to demonstrate that the kanamycin resistance gene in this construct was under Fur control. Colony picks from the E. coli BW25113 entA − strain transformed with pFCF2-entA were used to inoculate 3 ml of LB broth containing 12.5 μg/ml tetracycline and 50 μg/ml kanamycin. Overnight cultures incubated at 30 °C were diluted 1:100 in LB broth containing 12.5 μg/ml tetracycline and 50 μg/ml kanamycin and then grown at 30 °C until an A600 of 1.0 was reached. Cultures were centrifuged for 1 min at 21,000 × g and cell pellets were resuspended in 1× modified M9 medium such that all were diluted to an equivalent cell density (A600 = 1.00). Cultures for growth measurements were then prepared by 1:1000 dilution into one of the following: (i) 1× modified M9 medium plus 50 μM 2,2′-dipyridyl containing 12.5 μg/ml tetracycline, (ii) 1× modified M9 medium plus 50 μM 2,2′-dipyridyl containing 12.5 μg/ml tetracycline and 50 μg/ml kanamycin, (iii) LB broth containing 40 μM FeSO4, 12.5 μg/ml tetracycline, 0.2 % glucose, and (iv) LB broth containing 40 μM FeSO4, 12.5 μg/ml tetracycline and 50 μg/ml kanamycin, 0.2 % glucose. Diluted cultures were incubated at 30 °C for 16 h with agitation. Cell densities were measured as A600 values. Growth experiments were performed in triplicate.
Expression constructs were transformed into competent E. coli BW25113 cells. Single colony picks of transformants were used to inoculate LB broth supplemented with appropriate antibiotics. Overnight cultures were diluted 1:100 in LB plus antibiotics and then grown at 30 °C until they reached an A600 of 1.0. Cultures were centrifuged for 1 min at 21,000 × g and cell pellets were resuspended in 1× modified M9 medium and then diluted to an equivalent cell density (A600 = 1.00). Cultures were prepared by 1:1000 dilution into iron-depleted medium (1× modified M9 medium plus 100 μM 2,2′-dipyridyl and appropriate antibiotics) and/or iron-rich medium (LB broth containing 40 μM FeSO4, 0.2 % glucose and appropriate antibiotics), followed by incubation at 30 °C for 16 h with agitation. Cells (100 mg wet weight) from overnight cultures were pelleted by centrifugation at 3000 × g at 4 °C for 30 min and then resuspended in Lysis Buffer (50 mM Tris (pH 8.0), 1 % n-octyl-B-D-thioglucopyranoside, 3 μg/ml DNase I, 3 μg/ml RNase A, 30 μg/ml lysozyme, 1 mM DTT, 1× Protease Inhibitor Cocktail). Whole-cell lysates were incubated on a nutating mixer for 30 min at room temperature and then centrifuged for 5 min at 21,000 × g. Supernatants were recovered for Western blots. Aliquots of cleared cell lysates were separated on 10 % SDS-polyacrylamide gels. Following gel electrophoresis, separated proteins were transferred onto a PVDF membrane using a Mini-Trans Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories). The membrane was blocked for 1 h at room temperature using 5 % skim milk powder in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.2 % Tween 20). Blocked membranes were incubated with one of the following primary antibodies for 1 h at room temperature or at 4 °C overnight: (i) mouse monoclonal anti-FLAG antibody (1:1000 dilution; Thermo Fisher Scientific), (ii) mouse monoclonal anti-HA antibody (1:1000 dilution; Pierce), (iii) mouse monoclonal anti-GAPDH antibody (1:10,000; Thermo Fisher Scientific). Goat anti-mouse conjugated with horseradish peroxidase (HRP) (1: 10,000–1:20,000 dilution; Santa Cruz Biotechnology) was used as a secondary antibody. HRP activity was visualized using a SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
Construction of pFCF1, pFCF2 and pFBH1
Assessment of iron-responsive promoter regions
Iron-controlled protein expression
To determine that the fepB/entC Fur promoter region was iron-responsive, we grew E. coli BW25113 cells transformed with pFCF1-entE under iron-rich conditions using LB medium supplemented with FeSO4 as well as under iron-restricted conditions using modified M9 medium supplemented with 2,2′-dipyridyl. Cells were recovered from respective overnight cultures and equivalent amounts of proteins from whole-cell lysates were separated by SDS-PAGE. Western blot analysis using an anti-FLAG antibody revealed that FLAG-EntE was only detected in cells grown in iron-depleted medium, whereas no signal was observed from transformants grown in iron-rich medium (Fig. 5c, upper panel). As a control, proteins from identical lysate loadings were probed with an anti-GAPDH antibody. Comparable GAPDH signals were observed in lysates from cells grown in both iron-depleted and iron-rich media (Fig. 5c, lower panel).
Iron-controlled bidirectional expression from pFCF2
We designed and constructed three vectors (pFCF1, pFCF2, and pFBH1) for iron-controlled protein expression from low-copy-number vectors in E. coli. For pFCF1 and pFCF2, the plasmid pACYC184  (origin of replication: p15A; copy number ~ 15) was used as a backbone, whereas for pFBH1 the plasmid pBR322  (origin of replication: pMB1; copy number ~ 20) was used. These low-copy-number plasmids were chosen as backbones in order to avoid protein overexpression found in systems employing high-copy-number plasmids, such as the pBAD series of expression vectors that contain the pUC origin of replication (copy number ~ 500–700 ) or the pTZ-derived expression vectors . Furthermore, since pFCF1/pFCF2 and pFBH1 have different antibiotic resistance markers along with compatible origins of replication, expression of multiple epitope-tagged proteins from a single co-transformant is possible. For all vectors, gBlock® fragments containing: (i) a wild-type E. coli Fur-controlled bidirectional promoter region, (ii) sequences encoding epitope tags (FLAG or HA), (iii) sequence encoding the TEV protease cleavage site and (iv) a BACTH-compatible MCS, were designed, synthesized and inserted into respective plasmid backbones (pACYC184 or pBR322).
For iron-controlled expression of epitope-tagged proteins, we used the bidirectional promoter region in the intercistronic space between the fepB and entC genes in the E. coli chromosome (Fig. 1a) [18, 19]. This region, which contains all nucleotides between the fepB and entC start codons (624511–624884 in E. coli K12 MG1655 (NCBI Reference Number: NC_000913.3)), has two Fur box sequences. Fur box 1 controls expression of fepB in the anti-clockwise direction whereas Fur box 2 controls expression of the operon containing entC, entE, entB, entA, and entH genes in the clockwise direction. While Fur box 1 overlaps with its cognate −10/−35 sequences, Fur box 2 occurs downstream of its cognate +1/−10/−35 sequences [18, 19].
In order to test the functionality of pFCF1, pFCF2 and pFBH1, derivative constructs containing ORFs (entA, entE, and entB) under the control of Fur box 2 were prepared. Chromosomal expression of these ORFs in E. coli is under Fur control, and they were therefore logical candidates for testing the plasmid-borne bidirectional promoter region in pFCF1/2 and pFBH1. CAS assays and growth studies (Fig. 4) confirmed that the derivative constructs were able to complement respective knockout phenotypes upon iron restriction due to Fur derepression. Western blotting demonstrated that the bidirectional promoter region was controlling expression of epitope-tagged proteins FLAG-EntA and HA-EntB from pFCF1 and pFBH1, respectively. Also, expression of FLAG-T25, a FLAG-tagged B. pertussis polypeptide that is typically not under control of E. coli Fur, was detected. Taken together, these outcomes demonstrate that the fepB/entC promoter region (Fig. 1a) is functional in pFCF1 and pFBH1. Experiments using iron-depleted and iron-rich media showed that expression of FLAG-EntE from pFCF1-entE occurred in an iron-controlled manner. This is consistent with recent reports demonstrating that elevated expression of genes under Fur control occurred under iron-restricted conditions [10, 31].
Although unidirectional protein expression controlled by Fur box 2 was observed in pFCF1 and pFBH1, the bidirectionality of the fepB/entC promoter region had to be tested. We designed pFCF2 such that expression of the KanR gene would be controlled by Fur box 1 while Fur box 2 would simultaneously control expression of an ORF subcloned into the MCS in the other direction. A pFCF2-entA transformant grown under iron-restricted conditions was expected to exhibit complementation of the entA − phenotype with concomitant kanamycin resistance. Using iron-depleted CAS agar plates supplemented with tetracycline and kanamycin, the pFCF2-entA transformant complemented the entA − phenotype and produced a CAS halo while being resistant to kanamycin (Fig. 4a, lower left panel). This demonstrated that the KanR gene in pFCF2-entA was under the control of Fur box 1 resulting in iron-regulated expression. Furthermore, growth studies revealed that the pFCF2-entA transformant grew more poorly in iron-rich medium supplemented with kanamycin compared to growth in iron-depleted medium plus kanamycin. This suggests that under iron-replete conditions, Fur-controlled expression of the KanR gene is repressed relative to that of the same transformant grown under iron-depleted conditions. Taken together our results demonstrate that bidirectional iron-controlled in trans protein expression from pFCF2-entA occurs in iron-starved E. coli transformants. Protein expression controlled by Fur box 1 in pFCF2 is currently restricted to the KanR gene. By replacement of this gene with an additional MCS, bidirectional expression of any two epitope-tagged proteins can be achieved in a single transformant. The compatibility of pFCF- and pFBH-derived constructs would further allow for expression of up to four epitope-tagged proteins in a single co-transformant.
Constructs containing engineered Fur box sequences for unidirectional expression of toxic genes in E. coli have previously been reported . To facilitate bidirectional expression of epitope-tagged proteins under iron control, we have designed and constructed three novel low-copy-number vectors derived from pACYC184 and pBR322. These vectors contain the wild-type intercistronic region found between the fepB and entC genes in the E. coli chromosome that can be used for bidirectional expression. As we have demonstrated, when inserted into low-copy-number plasmid vectors, this region can control simultaneous expression of two proteins in a single transformant. Since the pFCF1/2 and pFBH1 have compatible origins of replication and different antibiotic resistance gene markers, they can also be useful for co-transformation.
Our current understanding of Fur regulation indicates that there are approximately 80 E. coli genes under the control of Fur . By transferring ORFs naturally found in Fur regulons, the vectors reported here can be used for a wide variety of experiments such as the study of in trans complementation of knockout phenotypes, effects of iron-controlled protein expression on cellular processes (e.g., oxidative stress response, TCA cycle, etc.), as well as studies on proteins that are involved in Fur-controlled virulence mechanisms (e.g., Type 3 secretion system) . Furthermore, genes not typically under Fur control can be expressed in an iron-controlled manner. Since the MCS in pFCF1 and pFBH1 is compatible with the BACTH system, ORFs encoding interacting partners detected by BACTH could be easily subcloned into the vectors reported here. Such constructs could be used for follow-up studies such as co-immunoprecipitation experiments using appropriate antibodies directed against vector-encoded epitope-tagged proteins.
We acknowledge NBRP-E. coli at NIG (Japan) for providing KEIO strains.
This work was supported by Discovery Grant 341983 from the Natural Sciences and Engineering Research Council of Canada to P.D.P. Financial support for P. Pakarian was provided by an FQRNT PBEEE/MELS Scholarship and GRASP.
Availability of data and materials
The data generated for this study are included in this article and its additional files.
PDP participated in experimental design and directed the research. PP conceived of the study and performed all experimental work. PDP and PP participated in drafting the manuscript. Both PDP and PP approved of the manuscript in its final form.
The authors declare that they have no competing interests.
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- 6.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.Google Scholar
- 12.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.CrossRefGoogle Scholar
- 13.Bagg A, Neilands JB. Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiol Rev. 1987;51:509–18.Google Scholar
- 18.Elkins MF, Earhart CF. Nucleotide sequence and regulation of the Escherichia coli gene for ferrienterobactin transport protein FepB. J Bacteriol. 1989;171:5443–51.Google Scholar
- 20.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.Google Scholar
- 27.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.Google Scholar
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