AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide.
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 approximately 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 H(2)O(2), 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 H(2)O(2), 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 H(2)O(2) detoxification.
Howell, ML; Alsabbagh, E; Ma, JF; Ochsner, UA; Klotz, MG; Beveridge, TJ; Blumenthal, KM; Niederhoffer, EC; Morris, RE; Needham, D; Dean, GE; Wani, MA; Hassett, DJ
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