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.
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA.
M L
ML
Howell
E
E
Alsabbagh
J F
JF
Ma
U A
UA
Ochsner
M G
MG
Klotz
T J
TJ
Beveridge
K M
KM
Blumenthal
E C
EC
Niederhoffer
R E
RE
Morris
D
D
Needham
G E
GE
Dean
M A
MA
Wani
D J
DJ
Hassett
D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D017487:Ankyrins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002374:Catalase / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008969:Molecular Sequence Data; D033903:Periplasmic Proteins; D010957:Plasmids; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
C412465:AnkB protein, Pseudomonas aeruginosa; D017487:Ankyrins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D002374:Catalase