Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters.
The cloned Pseudomonas aeruginosa fur (ferric uptake regulator) gene was overexpressed in P. aeruginosa by using a T7 expression system, and the Fur protein (PA-Fur) was purified by using a combination of ion-exchange chromatography and metal affinity chromatography. The DNA binding activity of the PA-Fur protein was confirmed by gel mobility shift assays and DNase I footprints of the synthetic DNA fragment GATAAT GATAATCATTATC, representing a perfect "Fur box". In addition, it was shown that PA-Fur is capable of binding to promoter and operator determinants of the tightly iron-regulated Escherichia coli fepA-fes enterobactin gene system. The activity of PA-Fur on the promoters of iron-regulated genes involved in the production of two siderophores, pyochelin and pyoverdin, and in the expression of exotoxin A was investigated. Data indicating that the promoters of the pchR gene, encoding a transcriptional activator for pyochelin synthesis, and of the pvdS gene, encoding a positive regulator for pyoverdin production, are specifically recognized by Fur-Fe(II) are presented, suggesting that PA-Fur represses expression of pchR and pvdS during growth in an iron-replete environment. However, neither the promoter region of the gene encoding exotoxin A (toxA) nor the promoters of the regAB operon, required for toxA expression, interacted with high concentrations of purified PA-Fur. These data indicate that iron regulation of exotoxin A production involves additional factors which may ultimately be under the control of PA-Fur.
Department of Microbiology, University of Colorado Health Sciences Center, Denver 80262, USA.
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D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D001483:Base Sequence; D001692:Biological Transport; D005098:Exotoxins / Q000096:biosynthesis*; D005290:Ferric Compounds / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D017262:Siderophores / Q000096:biosynthesis*
D001426:Bacterial Proteins; D005098:Exotoxins; D005290:Ferric Compounds; D012097:Repressor Proteins; D017262:Siderophores; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron