Molecular characterization of the Fur protein of Listeria monocytogenes.
Iron is essential for the survival of almost all organisms, although excess iron can result in the generation of free radicals which are toxic to cells. To avoid the toxic effects of free radicals, the concentration of intracellular iron is generally regulated by the ferric uptake regulator Fur in bacteria. The 150 aa fur ORF from Listeria monocytogenes was cloned into pRSETa, and the His-tagged fusion protein was purified by nickel affinity column chromatography. DNA binding activity of this protein was studied by an electrophoretic mobility shift assay using the end-labelled promoters P(fhuDC) and P(fur). The results showed a decrease in migration for both promoter DNAs in the presence of the Fur protein, and the change in migration was competitively inhibited with an excess of the same unlabelled promoters. No shift in migration was observed when a similar assay was performed using non-specific end-labelled DNA. The assay showed that binding of Fur to P(fur) or P(fhuDC) was independent of iron or manganese ions, and was not inhibited in the presence of 2 mM EDTA. Inductively coupled plasma MS of the Fur protein showed no iron or manganese, but 0.48 mole zinc per mole protein was detected. A DNase I protection assay revealed that Fur specifically bound to and protected a 19 bp consensus Fur box sequence located in the promoters of fur and fhuDC. There was no requirement for iron or manganese in this assay also. However, Northern blot analysis showed an increase in fur transcription under iron-restricted compared to high-level conditions. Thus, the study suggests that under in vitro conditions, the affinity of the Fur protein for the 19 bp Fur box sequence does not require iron, but iron availability regulates fur transcription in vivo. Thus, the regulation by Fur in this intracellular pathogen may be dependent on either the structure of the DNA binding domain or other intracellular factors yet to be identified.
Ledala
Nagender
N
Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA.
Pearson
Stacy L
SL
Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA.
Wilkinson
Brian J
BJ
Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA.
Jayaswal
R K
RK
Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA.
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015152:Blotting, Northern; D016384:Consensus Sequence; D003850:Deoxyribonuclease I / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D007501:Iron / Q000378:metabolism; D008089:Listeria monocytogenes / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron; D003850:Deoxyribonuclease I