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

Abstract

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 ϳ65% ␣-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.

Extensive genomic diversity of closely related Wolbachia strains

“…In eukaryotes, they can be involved in many functions, including cell cycle regulation and cytoskeleton interactions (Voronin & Kiseleva, 2007;Wu et al, 2004), making them candidates for mediating Wolbachia-host interaction and host reproductive phenotypes (Foster et al, 2005;Iturbe-Ormaetxe et al, 2005a). In other symbiotic relationships, ankyrin repeat genes have been shown to bind to host chromatin in A. phagocytophilum (Caturegli et al, 2000;Park et al, 2004), have a role in hydrogen peroxide resistance of Pseudomonas aeruginosa (Howell et al, 2000) and be key in establishing a plantbacterium relationship between Lotus japonicus and Mesorhizobium loti (Kumagai et al, 2007).…”

A Protease-Resistant Catalase, KatA, Released upon Cell Lysis during Stationary Phase Is Essential for Aerobic Survival of a Pseudomonas aeruginosa oxyR Mutant at Low Cell Densities

“…We have recently shown that KatB is required for optimal H 2 O 2 resistance and that KatB activity is increased ϳ50-fold in its presence (4,13). Because KatB can be detected in the cytoplasmic membrane and periplasmic fractions (13), it could act to protect H 2 O 2 -sensitive respiratory chain components. Optimal KatB-mediated protection also requires AnkB, a cytoplasmic membrane-periplasmic ankyrin-like protein that we believe anchors KatB to the inner membrane and/or stabilizes it is the periplasmic space (13).…”

“…One likely reason for the dramatic H 2 O 2 sensitivity of the oxyR mutant is that each OxyR-regulated gene product contributes some measure of protection against it (17). We have recently shown that KatB is required for optimal H 2 O 2 resistance and that KatB activity is increased ϳ50-fold in its presence (4,13). Because KatB can be detected in the cytoplasmic membrane and periplasmic fractions (13), it could act to protect H 2 O 2 -sensitive respiratory chain components.…”

“…Because KatB can be detected in the cytoplasmic membrane and periplasmic fractions (13), it could act to protect H 2 O 2 -sensitive respiratory chain components. Optimal KatB-mediated protection also requires AnkB, a cytoplasmic membrane-periplasmic ankyrin-like protein that we believe anchors KatB to the inner membrane and/or stabilizes it is the periplasmic space (13). Although AhpB and AhpCF are predicted to encode proteins that are important in the detoxification of organic hydroperoxides, both AhpB and AhpCF appear to be important for some resistance to H 2 O 2 when they are overexpressed in an oxyR mutant (Table 3 (15a), and E. coli oxyR mutants require catalase for normal aerobic growth.…”

The Genome of the Obligately Intracellular Bacterium Ehrlichia canis Reveals Themes of Complex Membrane Structure and Immune Evasion Strategies

“…Although motility has not been demonstrated with Ehrlichia spp., Ecaj_0062 exhibits similarity (E ϭ 5 ϫ 10 Ϫ5 ) to RickA proteins, suggesting that a mechanism of interacting with the cytoskeleton exists in this pathogen. Furthermore, it has been shown in Pseudomonas aeruginosa that AnkB, an ankyrin repeat-containing protein, is essential for optimal activity of periplasmic catalase (28). It is therefore reasonable to propose that these ankyrin-containing proteins may play a significant role in the adaptation of the pathogen to the host cell, mediating a series of interactions or reprogramming of the host cell functions.…”