Molecular characterization of the ferric-uptake regulator, fur, from Staphylococcus aureus.
Iron is an essential nutrient for the survival and pathogenesis of bacteria, but relatively little is known regarding its transport and regulation in staphylococci. Based on the known sequences of ferric-uptake regulatory (fur) genes from several Gram-positive and Gram-negative bacteria, a fragment containing the fur homologue was cloned from a genomic library of Staphylococcus aureus RN450. Nucleotide sequence analysis of this fragment revealed the presence of a 447 bp ORF that encodes a putative 149 aa polypeptide with an apparent molecular mass of 17 kDa. A putative ferrichrome-uptake (fhu) operon, containing the conserved Fur-binding sequences (Fur box) in the promoter region, was also cloned from the same S. aureus library. To characterize the impact of Fur on the fhu operon, fur was cloned, overexpressed as a His-tagged protein and purified by Ni2+-affinity column chromatography. The recombinant protein was digested with enterokinase to remove the His tag. Electrophoretic mobility-shift assays indicated that Fur binds to the promoter region of the fhu operon in the presence of divalent cations. Fur also interacted with the promoter region of the recently reported sir operon that has been proposed to constitute a siderophore-transport system in S. aureus. The DNase I-protection assay revealed that Fur specifically binds to the Fur box located in the promoter region of the fhu operon. The primer-extension reaction indicated that the transcription-start site of the fhu operon was located inside the Fur box. S. aureus fur partially complemented a fur- mutation in Bacillus subtilis. The data suggest that Fur regulates iron-transport processes in S. aureus.
Xiong
Anming
A
Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA1.
Singh
Vineet K
VK
Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA1.
Cabrera
Guillermo
G
Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA1.
Jayaswal
Radheshyam K
RK
Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA1.
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003851:Deoxyribonucleases / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism*; D005291:Ferrichrome / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D017136:Ion Transport; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C087175:Emm22 protein,Streptococcus pyogenes; D005290:Ferric Compounds; D008565:Membrane Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D005291:Ferrichrome; D003851:Deoxyribonucleases