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Journal of Bacteriology, August 2000, p. 4545-4556, Vol. 182, No. 16
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

AnkB, a Periplasmic Ankyrin-Like Protein in Pseudomonas aeruginosa, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide

Michael L. Howell,1 Eyad Alsabbagh,1 Ju-Fang Ma,1 Urs A. Ochsner,2 Martin G. Klotz,3 Terry J. Beveridge,4 Kenneth M. Blumenthal,1 Eric C. Niederhoffer,5 Randall E. Morris,6 David Needham,7 Gary E. Dean,1 Maqsood A. Wani,1 and Daniel J. Hassett1,*

Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-05241; Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 802622; Department of Biology and Center for Genetics and Molecular Medicine, University of Louisville, Louisville, Kentucky 402923; Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W14; Department of Medical Biochemistry, Southern Illinois University College of Medicine, Carbondale, Illinois 62901-44135; Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-05216; and Department of Materials Science and Mechanical Engineering, Duke University, Durham, North Carolina 277087

Received 11 February 2000/Accepted 19 May 2000

In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is ~65% alpha -helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H2O2, and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H2O2, phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H2O2 detoxification.

* 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, August 2000, p. 4545-4556, Vol. 182, No. 16
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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