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 prote in 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 repe ats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible ca talase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pi of 5.5 that po ssesses 22 primarily hydrophobic amino acids at residues 3 to 25, predictin g 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 me mbrane 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 ana lyses indicated that katB, and ankB are part of a small operon whose transc ription is induced dramatically by H2O2, and controlled by the global trans activator OxyR. Interestingly, unlike the spherical nature of ankyrin-defic ient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicl es. The mutant also exhibited a fourfold reduction in KatB activity and inc reased 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 cytop lasmic membrane, thus providing a protective lattice work for optimal H2O2 detoxification.