The Role of the Cytoplasmic Heme-binding Protein (PhuS) of Pseudomonas aeruginosa in Intracellular Heme Trafficking and Iron Homeostasis*
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1140
- 1 To whom correspondence should be addressed: Dept. of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, HSF II, 20 Penn St., Baltimore, MD 21201-1140. Tel.: 410-706-2537; Fax: 410-706-5017; E-mail: awilks{at}rx.umaryland.edu.
Abstract
The cytoplasmic heme-binding protein PhuS, encoded within the Fur-regulated Pseudomonas heme utilization (phu) operon, has previously been shown to traffic heme to the iron-regulated heme oxygenase (HO). We further investigate the role of PhuS in heme trafficking to HO on disruption of the phuS and hemO genes in a Pseudomonas aeruginosa siderophore-deficient and wild-type background. Previous studies have shown that deletion of hemO prevents the cells from utilizing heme as the sole source of iron. However, disruption of phuS alone resulted in a slow growth phenotype, consistent with its role as a heme-trafficking protein to HO. Furthermore, in contrast to the hemO and hemO/phuS deletion strains, the phuS knockout prematurely produced pyocyanin in the presence of heme. Western blot analysis of PhuS protein levels in the wild-type strain showed that Fur-regulation of the phu operon could be derepressed in the presence of heme. In addition the premature onset of pyocyanin production requires both heme and a functional HO. Suppression of the phenotype on increasing the external heme concentration suggested that the decreased heme-flux through HO results in premature production of pyocyanin. The premature production of pyocyanin was not due to lower intracellular iron levels as a result of decreased heme flux through HO. However, transcriptional analysis of the phuS mutants indicates that the cells are sensing iron deprivation. The present data suggest that PhuS has a dual function in trafficking heme to HO, and in directly or indirectly sensing and maintaining iron and heme homeostasis.
Footnotes
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↵2 The abbreviations used are: HO, heme oxygenase; QS, quinolone signal; PQS, Pseudomonas quinolone signal; Phu, Pseudomonas heme utilization; ABC, ATP binding cassette; Fur, ferric uptake regulator; PIA, Pseudomonas isolation agar; FDR, false discovery rate; RND, root nodule cell division.
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↵3 A. P. Kaur and A. Wilks, unpublished data.
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↵* This work was supported, in whole or in part, by National Institutes of Health Grant AI-55912 (to A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1, Figs. S1 and S2, and references.
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- Received August 6, 2008.
- Revision received November 5, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
