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Journal of Bacteriology, November 2001, p. 6394-6403, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6394-6403.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Homologues of Neisserial Heme Oxygenase in
Gram-Negative Bacteria: Degradation of Heme by the Product of the
pigA Gene of Pseudomonas aeruginosa
Melanie
Ratliff,1
Wenming
Zhu,1
Rahul
Deshmukh,2
Angela
Wilks,2 and
Igor
Stojiljkovic1,*
Department of Microbiology and Immunology,
Emory School of Medicine, Atlanta, Georgia
30322,1 and Department of Pharmaceutical
Sciences, School of Pharmacy, University of Maryland, Baltimore,
Maryland 212012
Received 18 June 2001/Accepted 3 August 2001
The oxidative cleavage of heme to release iron is a mechanism by
which some bacterial pathogens can utilize heme as an iron source. The
pigA gene of Pseudomonas aeruginosa is shown to
encode a heme oxygenase protein, which was identified in the genome
sequence by its significant homology (37%) with HemO of
Neisseria meningitidis. When the gene encoding the
neisserial heme oxygenase, hemO, was replaced with
pigA, we demonstrated that pigA could
functionally replace hemO and allow for heme utilization by
neisseriae. Furthermore, when pigA was disrupted by
cassette mutagenesis in P. aeruginosa, heme utilization was
defective in iron-poor media supplemented with heme. This defect could
be restored both by the addition of exogenous FeSO4,
indicating that the mutant did not have a defect in iron metabolism,
and by in trans complementation with pigA from
a plasmid with an inducible promoter. The PigA protein was purified by
ion-exchange chromotography. The UV-visible spectrum of PigA
reconstituted with heme showed characteristics previously reported for
other bacterial and mammalian heme oxygenases. The heme-PigA complex
could be converted to ferric biliverdin in the presence of ascorbate,
demonstrating the need for an exogenous reductant. Acidification and
high-performance liquid chromatography analysis of the ascorbate
reduction products identified a major product of biliverdin IX-
.
This differs from the previously characterized heme oxygenases in which
biliverdin IX-
is the typical product. We conclude that PigA is a
heme oxygenase and may represent a class of these enzymes with novel regiospecificity.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Emory School of Medicine, 3001 Rollins
Research Center, Atlanta, GA 30322. Phone: (404) 727-1322. Fax: (404)
727-3659. E-mail: stojiljk{at}microbio.emory.edu.
Journal of Bacteriology, November 2001, p. 6394-6403, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6394-6403.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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