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Microbiology 146 (2000), 185-198
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Microbiology (2000), 146, 185-198.
© 2000 Society for General Microbiology


Genetics and Molecular Biology

Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa

Urs A. Ochsner1, Zaiga Johnson1 and Michael L. Vasil1

Department of Microbiology, University of Colorado Health Sciences Center, 4200 E Ninth Avenue, Campus Box B175, Denver, CO 80262, USA1

Author for correspondence: Urs Ochsner. Tel: +1 303 315 5093. Fax: +1 303 315 6785. e-mail: urs.ochsner{at}UCHSC.edu

A gene cluster similar to haem iron uptake loci of bacterial pathogens was identified in Pseudomonas aeruginosa. This phu locus (‘Pseudomonas haem uptake’) consisted of the phuR receptor gene and the phuSTUVW operon encoding a typical ABC transporter. Expression of phuR and phuSTUVW from mapped transcriptional-start sites occurred under iron-restricted growth conditions and was directly controlled by the Fur protein. Binding of Fur was demonstrated by DNase footprinting of two adjacent ‘Fur boxes’ that overlapped both the phuR and phuSTUVW promoters. Two tandem repeats of 154 bp were identified downstream of the phuSTUVW operon, each of which contained a strong Fur-dependent promoter driving expression of iron-regulated RNAs antisense to phuSTUVW. Mutant strains with deletions in phuR and phuSTUV showed greatly reduced growth with either haem or haemoglobin as the only iron source: the defects were complemented by plasmids harbouring the phuR or the phuSTUV genes, respectively. Deletions of phuW or of the tandem repeats had only minor effects on haem utilization. The remaining haem and haemoglobin uptake still observed in the {Delta}phuR or {Delta}phuSTUV deletion mutants was due to a second haem-acquisition system, has, which was also under the direct control of Fur. This second haem-receptor gene, hasR, was identified upstream of and in an operon with hasA, encoding a haem-binding extracellular protein. A {Delta}hasR mutant also exhibited decreased utilization of haem and haemoglobin, and a {Delta}phuR {Delta}hasR double mutant was virtually unable to take up either compound. Both the PhuR and HasR proteins were detected in the outer-membrane fraction of P. aeruginosa grown in low-iron media. Taken together, the evidence suggests that the phu and has loci encode two distinct systems required for the acquisition of haem and haemoglobin in P. aeruginosa.

Keywords: Pseudomonas, iron transport, haem receptor, ferric uptake regulator

Abbreviations: ABC, ATP-binding cassette; EDDHA, ethylenediamine di(o-hydroxyphenylacetic acid)

The GenBank accession numbers for the sequences reported in this paper are AF055999, AF127222, and AF127223.




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