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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 alpha  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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