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 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.

FIG. 1

Gene map of the ∼5.2-kb insert of pSMB1 containing the katB, ankB, and radA genes. The functions of the gene products are also given. We have previously shown that OxyR, a 34-kDa transactivator, responds to H₂O₂ by activating katB-ankB (39). T, 33-amino-acid inverted repeat that could represent the transcriptional terminator for the katB-ankB operon.

FIG. 2

Alignment of the deduced amino acids from genes coding for bacterial ankyrins PaAnkB (P. aeruginosa; accession no. U59457), PsAnkF (P. syringae; U16026), CvAnkA (C. vinosum; L13419), SlPhlB (S. liquefaciens; P18954), and SvAnkA (S. verticillus; L26954). The 33-amino-acid tandem repeats (underlined) were revealed by using the Ank motif of conserved residues (boldface) as identified by Bennett using the erythrocyte ank repeat consensus sequence (5). Proposed signal sequences are indicated by a double underline. The conserved ank repeat sequences for erythrocyte ankyrin are given below the selected bacterial ALP sequences. RBC, erythrocyte concensus ank repeat sequence.

FIG. 3

Cellular localization of AnkB in P. aeruginosa. (A) Predicted cytoplasmic membrane organization of P. aeruginosa AnkB bacterial ankyrin-like proteins from P. syringae, P. fluorescens, S. liquefaciens, and C. vinosum based upon the positive-inside-rule algorithm developed by von Heijne (57). For the P. aeruginosa AnkB protein, the large number 1 indicates the predicted single MSD. N, N terminus; C, C terminus; LL, loop length; KR, number of lysine and arginine residues; KR Diff, positive charge difference. (B) Schematic diagram of AnkB–β-lactamase and AnkB-PhoA protein fusions in both E. coli and P. aeruginosa PAO1. In both cases, organisms expressing AnkB–β-lactamase were resistant to ampicillin (E. coli) or carbenicillin (P. aeruginosa). Organisms expressing AnkB-PhoA were found to hydrolyze the alkaline phosphatase substrate BCIP in L-agar plates. IM, inner membrane; OM, outer membrane. (C) AP activity in cellular fractions of P. aeruginosa ankB harboring pEX30-ankB::phoA. Bar 1, cytoplasm; bar 2, periplasm; bar 3, cytoplasmic membrane; bar 4, outer membrane.

FIG. 4

Overexpression (A) and circular dichroism analysis (B) of recombinant AnkB proteins. (A) E. coli BL21(λDE3) harboring pET23-ankB-480 was grown aerobically in L broth to mid-logarithmic phase and treated with 1 mM IPTG for 3 h at 37°C. After Ni²⁺-nitrilotriacetic acid purification, purified protein was separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis and the gel was stained with Coomassie blue R-250. Lane 1, molecular mass standard; lane 2, 15 μg of AnkB-480. (B) Circular dichroism spectrum of AnkB-480, using 100 μg ml⁻¹ in 10 mM sodium phosphate (pH 7.0) at 23°C.

FIG. 5

RNase protection assays indicate that katB and ankB comprise an operon and are regulated by OxyR. Riboprobes specific for the katB promoter (katB rp) and for the katB-ankB overlapping region (katB-ankB rp) were used to detect the corresponding transcripts in P. aeruginosa PAO1 or oxyR mutant total RNA isolated during the exponential growth phase in aerobic M9 minimal medium. Paraquat (PQ) was added to final concentrations of 10 and 100 μM 1 h prior to harvest as indicated. Also shown are the digested probes in the absence of any P. aeruginosa RNA as a control. A DNA sequencing reaction was run in parallel and served as a size marker. Numbers on the left are base pairs.

FIG. 6

Ultrastructural analysis of wild-type PAO (A) and PAO ankB mutant (B) bacteria. Bacteria were grown aerobically in L broth to mid-exponential growth phase and treated with 1 mM H₂O₂ for 1 h at 37°C to stimulate transcription of katB-ankB. Organisms were then prepared for TEM examination as described in Materials and Methods. The arrows in panel B point to the larger number of membrane vesicles being produced by the ankB mutant. The width of the cells is ∼800 nm.

FIG. 7

Effect of ankB, katB, and katB ankB on sensitivity to H₂O₂. All bacteria were grown aerobically overnight in M9F medium at 37°C. Fresh prewarmed medium (1 volume of culture in 10-volume flasks) was inoculated with 1/50 of the final culture volume and allowed to reach an OD₆₀₀ of 0.6. Some bacteria were pretreated with a sublethal (1 mM) dose of H₂O₂ for 1 h (shaded bars) relative to control bacteria (open bars). The suspensions were diluted 100-fold in 7 ml of M9F 0.6% top agarose kept at 37°C and poured onto M9F plates. Filter paper disks (7 mm) impregnated with 8.8 M H₂O₂ were placed on the top agar surface. Zones of growth inhibition were measured after a 24-h aerobic incubation at 37°C. Bars: 1, PAO1; 2, ankB; 3, katB; 4, katB ankB; 5, ankB plus pUCP22; 6, ankB plus pankB; 7, katB ankB plus pUCP22; 8, katB ankB plus pankB. Error bars indicate standard errors of the means.

FIG. 8

Absence of AnkB causes a decrease in KatB activity. Bacteria were grown aerobically overnight in L-broth medium at 37°C. Fresh prewarmed medium (1 volume of culture in 10-volume flasks) was inoculated with 1/100 the final culture volume, and the organisms were grown to an OD₆₀₀ of 0.6. Some bacteria (right panel) were then treated with a sublethal (0.35 mM) (11) dose of paraquat for 1 h, and the others (left panel) served as controls. Cell extracts were prepared, and 10 μg was subjected to nondenaturing polyacrylamide gel electrophoresis (5% polyacrylamide). The gels were then stained for catalase activity (58). Lanes: 1, PAO1; 2, ankB; 3, katA; 4, katB; 5, katA ankB.

FIG. 9

Quantitative effect of AnkB on KatB activity. Bacteria were grown aerobically overnight in L-broth medium containing 0.35 mM paraquat at 37°C. Catalase activity and activity gel staining were monitored in cell extracts. Bars: 1, katA plus pUCP22; 2, katA plus pankB; 3, katA ankB plus pUCP22; 4, katA ankB plus pankB. Error bars indicate standard errors of the means. The inset photograph is KatB activity staining of cell extracts in a representative native polyacrylamide gel of each strain. It should be noted that no AhpA activity band (see Fig. 8) could be detected under these conditions.