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Journal of Bacteriology, August 2000, p. 4545-4556, Vol. 182, No. 16
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%
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
-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.
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