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Journal of Bacteriology, June 1999, p. 3730-3742, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Bacterioferritin A Modulates Catalase A (KatA)
Activity and Resistance to Hydrogen Peroxide in Pseudomonas
aeruginosa
Ju-Fang
Ma,1
Urs
A.
Ochsner,2
Martin G.
Klotz,3,4
Vagira K.
Nanayakkara,1,5
Michael L.
Howell,1
Zaiga
Johnson,2
James E.
Posey,6
Michael L.
Vasil,2
John J.
Monaco,1,5 and
Daniel J.
Hassett1,*
Department of Molecular Genetics,
Biochemistry and Microbiology1 and
Howard Hughes Medical Institute,5
University of Cincinnati College of Medicine, Cincinnati, Ohio
45267-0524; Department of Microbiology and Immunology,
University of Colorado Health Sciences Center, Denver, Colorado
802622; Department of
Biology3 and Center for Genetics and
Molecular Medicine,4 University of
Louisville, Louisville, Kentucky 40292; and Department of
Microbiology, University of Georgia, Athens, Georgia
30609-40666
Received 16 September 1998/Accepted 8 April 1999
We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas
aeruginosa harboring the rpoA, rplQ,
katA, and bfrA genes. These loci are predicted
to encode, respectively, (i) the
subunit of RNA polymerase; (ii)
the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one
of two iron storage proteins called bacterioferritin A (BfrA;
cytochrome b1 or b557).
Our goal was to determine the contributions of KatA and BfrA to the
resistance of P. aeruginosa to hydrogen peroxide
(H2O2). When provided on a multicopy plasmid,
the P. aeruginosa katA gene complemented a
catalase-deficient strain of Escherichia coli. The
katA gene was found to contain two translational start
codons encoding a heteromultimer of ~160 to 170 kDa and having an
apparent Km for H2O2 of
44.7 mM. Isogenic katA and bfrA mutants were
hypersusceptible to H2O2, while a katA
bfrA double mutant demonstrated the greatest sensitivity. The
katA and katA bfrA mutants possessed no
detectable catalase activity. Interestingly, a bfrA mutant
expressed only ~47% the KatA activity of wild-type organisms,
despite possessing wild-type katA transcription and
translation. Plasmids harboring bfrA genes encoding BfrA
altered at critical amino acids essential for ferroxidase activity
could not restore wild-type catalase activity in the bfrA
mutant. RNase protection assays revealed that katA and
bfrA are on different transcripts, the levels of which are
increased by both iron and H2O2. Mass
spectrometry analysis of whole cells revealed no significant difference
in total cellular iron levels in the bfrA,
katA, and katA bfrA mutants relative to
wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against
H2O2.
*
Corresponding author. Mailing address: Department of
Molecular Genetics, Biochemistry and Microbiology, University of
Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH
45267-0524. Phone: (513) 558-1154. Fax: (513) 558-8474. E-mail:
Daniel.Hassett{at}UC.Edu.
Journal of Bacteriology, June 1999, p. 3730-3742, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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