Regulation of the iron transporter gene fepA is crucial for cell viability Eva Ursula Weiss
FepA is an Escherichia coli (E. coli) membrane protein which transports the iron-bound siderophore ferric enterobactin from the exterior into the periplasm. Thereby it helps to provide the cells with soluble iron. In the study, its gene, fepA, was ligated into pUC8. E. coli cells were then transformed with this construct and tested for presence and orientation of sense or antisense expression. The majority of colonies that have taken up pUC8 with a fepA fragment we re found to contain the gene in antisense orientation. It was concluded that removal of the gene from its regulation leading to its over-expression results in unviable clones possibly due to membrane protein toxicity or iron toxicity.
Iron uptake is crucial for the survival of most organisms (1). It is needed as cofactor in many important metabolic pathways including the electron transport chain (1). Availability of iron is complicated by the fact that at neutral pH the metal forms stable, insoluble complexes with hydride in water (1). In order to access the metal bacteria use siderophores, which when secreted compete with hydroxide to complex iron (1). These chelators are called enterobactin or ferric enterobacting (FeEnt) when bound to iron in Escherichia coli (E. c oli) (2). Uptake into the cells is mediated by FepA in the presence of TonB (2, 3).
FepA is a 81 kDa protein located on the outer membrane of gram negative bacteria like E. coli (2). There it functions as receptor for FeEnt (2). It is very similar to other E. coli membrane proteins with respect to the distribution of charges and hydrophilic and hydrophobic parts on the molecule (2). Its structure is likely to also involve specific domains at the periplasm and exterior side in order to interact with its ligands and other proteins (4). It is thought to interact with TonB and ExbB both being required for function(4). More specifically, the interaction of FepA with the energy-supplying species, TonB, is thought to occur at the N-terminus of FepA (2). The amino-terminal globular domain forming a loop structure is believed to be important for the translocation of FeEnt whereas the loops of the β-barrel domain at the carboxyl terminus, to which the N-domain is associated, recognize the correct ligand (7, 8). The actual first binding of the ligand, on the other hand, was found to be performed by specific aromatic side chains on the protein (8). This causes a minor conformational change in the protein needed to move away the globular domain, which plugs the barrel, enabling FeEnt to be translocated (7). This mechanism of closing and opening the channel makes the transport specific (3). Additionally, FepA is believed to interact with the other “fep” proteins FepB, FepC, FepD and FepE (4). The latter three are likely to form a complex at the inner membrane to allow FeEnt to pro ceed from the periplasm to the cytoplasm whereas FepB is found in the periplasm itself (4). The exact function of these proteins and how they interact with FepA are not known (2).
When bacterial cells are deprived of iron they boost the production of prote ins involved in its uptake whereas the proteins' gene expression is repressed at high cellular iron(II) levels (2, 5). It is thought that a dyad sequence with a A- T doublet motif in the promoter is the site to which the transcriptional repressor Fur would bind to repress expression of the fepA gene when iron levels are high (2, 6). This regulation of fepA was found to be connected to other genes involved with iron by overlaps in promoter sequences (6). Interestingly, iron (II) itself binds to the repressor protein increasing its activity; thus, a high amount of iron(II) results in more repression of the genes involved in FeEnt uptake (5). Although so far only Fur is known to act as repressor in this process, it is possible that more regulatory proteins are involved (6).
For the study of fepA the gene was obtained from pPC104. The plasmid was first described by Coderre and Earhart. It was originally derived from E. coli genomic sequence. Caderre and Earhart have shown that pPC104 also contains the genes entD fepA and fes, in that order, together with the respective promoter. The products of all of these genes are essential for iron uptake in E. coli (9). fepA was moved to pUC8 in order to remove it from its endogenous promoter and
MATERIALS & METHODS
Amplification, isolation and digestion of pPC104
E. coli cells carrying pPC104, which contains the fepA gene, were incubated with chloramphenicol (60 μg/mL, overnight) allowing for replication of the plasmid but genomic DNA. This led to an amplification of pPC104 in the cells. It was subsequently isolated by centrifugation of the cells at 7,000 rpm with a Sorvall GSA rotor and then resuspended in buffer with lysozyme. The cells were lysed using the alkaline lysis approach (5 mg/mL lysozyme, 0.2 N NaOH, 1% SDS and later 2 N acetic acid). Precipitated genomic DNA was removed by centrifugation at 14,000 rpm in an Eppendorf microcentrifuge and filtration of the supernatant. Further purification was achieved by isopropanol precipitation, RNase (0.3 mg/mL) treatment and phenol/chloroform extraction with subsequent ethanol precipitation. The isolated and purified plasmid DNA was cut in a double digest with the restriction enzymes Ssp I (15 U) and Eco 147 I (15 U) in Buffer Y/Tango (Fermentas Inc, Glen Burnie, MD) for 2 hrs at 37°C. The fepA fragment was isolated by agarose gel electrophoresis followed by gel extraction using the QIAEX II kit (QUIAGEN, Valencia, CA).
Preparation of pUC8 for cloning
Pure pUC8 was cut with the restriction enzyme Hinc II (10 U) in Buffer Y/Tango (Fermentas Inc, Glen Burnie, MD) for 2 hrs at 37°C. Since only one restriction site for this enzyme is present on pUC8, the digest led to an opening of the plasmid but no fragmentation resulting in a linear piece of pUC8 with blunt ends. To later prevent self- ligation of the plasmid, it was also treated with Shrimp alkaline phosphatase (SAP) for 1 hr at 37°C, which removed the 5' phosphate groups at the ends, in SAP buffer.
Preparation of competent JM83 E. coli
An aliquot of an overnight JM83 E. coli culture was grown to an OD550 of between 0.4 and 0.6. Then the cells were kept cold to prevent further growth. Competency was induced by pelleting the cells and resuspending them in TSS (1 % tryptone, 0.5 % yeast extract, 1 % NaCl, 10 % PEG, 30 % glycero l, 0.05 M MgSO4, pH 6.5). The level of achieved competency was determined by heat-shock transforming 100 μL of competent cells with 15 ng, 1.5 ng and 0.15 ng of pure pUC8, adding 900 μL LB medium, plating 100 μL of each sample on an agar plate and counting the number of colonies after an overnight incubation.
Ligation of pUC8 and fepA fragment
Linearized pUC8 and isolated fepA- containing pPC104 fragment both had blunt and therefore compatible ends. They were ligated together using T4 ligase (1.5 U) in the respective ligase buffer (Fermentas Inc, Glen Burnie, MD). A two- molar excess of insert compared to vector were used (60 fmoles of fepA insert and 30 fmoles of pUC8) to increase the likelihood of a reaction between the two. The ligation was allowed to incubate overnight at 15°C.
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