A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli.
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.
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St., W., Montreal, QC, H4B 1R6, Canada.
Paknoosh
P
Pakarian
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St., W., Montreal, QC, H4B 1R6, Canada. peter.pawelek@concordia.ca.|Groupe de Recherche Axé sur la Structure des Protéines (GRASP), Montreal, Canada. peter.pawelek@concordia.ca.
Peter D
PD
Pawelek
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D007501:Iron / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron
Bacterial two-hybrid; Bidirectional promoter; CAS assay; Ferric uptake regulator; Protein expression