Dataset Information

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.

SUBMITTER: Howell ML

PROVIDER: S-EPMC94626 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide.

Howell M L ML Alsabbagh E E Ma J F JF Ochsner U A UA Klotz M G MG Beveridge T J TJ Blumenthal K M KM Niederhoffer E C EC Morris R E RE Needham D D Dean G E GE Wani M A MA Hassett D J DJ

Journal of bacteriology 20000801 16

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 re ...[more]

PMID: 10913088

Similar Datasets

Project description:Pseudomonas aeruginosa is an obligate aerobe that is virtually ubiquitous in the environment. During aerobic respiration, the metabolism of dioxygen can lead to the production of reactive oxygen intermediates, one of which includes hydrogen peroxide. To counteract the potentially toxic effects of this compound, P. aeruginosa possesses two heme-containing catalases which detoxify hydrogen peroxide. In this study, we have cloned katB, encoding one catalase gene of P. aeruginosa. The gene was cloned on a 5.4-kb EcoRI fragment and is composed of 1,539 bp, encoding 513 amino acids. The amino acid sequence of the P. aeruginosa katB was approximately 65% identical to that of a catalase from a related species, Pseudomonas syringae. The katB gene was mapped to the 71- to 75-min region of the P. aeruginosa chromosome, the identical region which harbors both sodA and sodB genes encoding both manganese and iron superoxide dismutases. When cloned into a catalase-deficient mutant of Escherichia coli (UM255), the recombinant P. aeruginosa KatB was expressed (229 U/mg) and afforded this strain resistance to hydrogen peroxide nearly equivalent to that of the wild-type E. coli strain (HB101). The KatB protein was purified to homogeneity and determined to be a tetramer of approximately 228 kDa, which was in good agreement with the predicted protein size derived from the translated katB gene. Interestingly, KatB was not produced during the normal P. aeruginosa growth cycle, and catalase activity was greater in nonmucoid than in mucoid, alginate-producing organisms. When exposed to hydrogen peroxide and, to a greater extent, paraquat, total catalase activity was elevated 7- to 16-fold, respectively. In addition, an increase in KatB activity caused a marked increase in resistance to hydrogen peroxide. KatB was localized to the cytoplasm, while KatA, the "housekeeping" enzyme, was detected in both cytoplasmic and periplasmic extracts. A P. aeruginosa katB mutant demonstrated 50% greater sensitivity to hydrogen peroxide than wild-type bacteria, suggesting that KatB is essential for optimal resistance of P. aeroginosa to exogenous hydrogen peroxide.

Project description:The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H(2)O(2)) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H(2)O(2). Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H(2)O(2), whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H(2)O(2). The katB mutant, lacking the H(2)O(2)-inducible catalase KatB, was similar to the wild-type strain with respect to H(2)O(2) resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H(2)O(2), while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H(2)O(2). Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H(2)O(2), suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H(2)O(2), while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of beta-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H(2)O(2), particularly at high H(2)O(2) concentrations; KatB is induced in both planktonic and biofilm cells in response to H(2)O(2) insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H(2)O(2) are sublethal.

Project description:Pseudomonas aeruginosa possesses an extensive armament of genes involved in oxidative stress defense, including katB-ankB, ahpB, and ahpC-ahpF. Transcription of these genes was regulated in response to H(2)O(2), paraquat, or organic peroxides. Expression of katB-lacZ and the observed KatB catalase levels in P. aeruginosa PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds. Also, ahpB-lacZ and ahpC-lacZ expression was 90- and 3-fold higher, respectively, upon exposure to paraquat. The dose- and time-response curves revealed that 1 microM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure. Expression of these genes was not observed in a DeltaoxyR mutant, indicating that OxyR was essential for this response. The transcriptional start sites of katB-ankB, ahpB, and ahpC-ahpF were mapped, putative OxyR-binding sites were identified upstream of the -35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated. The oxyR mutant was hypersusceptible to oxidative stress-generating agents, including H(2)O(2) and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type. The oxyR phenotype was fully complemented by a plasmid containing the oxyR gene, while any of the katB, ahpB, or ahpCF genes alone resulted in only marginal complementation. Increased katB-lacZ expression and higher KatB catalase levels were detected in a DeltaahpCF background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF. In P. aeruginosa, oxyR is located upstream of recG, encoding a putative DNA repair enzyme. oxyR-lacZ and recG-lacZ reporter activities and oxyR-recG mRNA analysis showed that oxyR and recG are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of oxyR. Mutants affected in recG but not oxyR were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation. In conclusion, we present evidence that the oxyR-recG locus is essential for oxidative stress defense and for DNA repair.

Project description:Pseudomonas stutzeri A1501 is a versatile nitrogen-fixing bacterium capable of living in diverse environments and coping with various oxidative stresses. NfiS, a regulatory noncoding RNA (ncRNA) involved in the control of nitrogen fixation in A1501, was previously shown to be required for optimal resistance to H2O2; however, the precise role of NfiS and the target genes involved in the oxidative stress response is entirely unknown. In this work, we systematically investigated the NfiS-based mechanisms underlying the response of this bacterium to H2O2 at the cellular and molecular levels. A mutant strain carrying a deletion of nfiS showed significant downregulation of oxidative stress response genes, especially katB, a catalase gene, and oxyR, an essential regulator for transcription of catalase genes. Secondary structure prediction revealed two binding sites in NfiS for katB mRNA. Complementation experiments using truncated nfiS genes showed that each of two sites is functional, but not sufficient, for NfiS-mediated regulation of oxidative stress resistance and nitrogenase activities. Microscale thermophoresis assays further indicated direct base pairing between katB mRNA and NfiS at both sites 1 and 2, thus enhancing the half-life of the transcript. We also demonstrated that katB expression is dependent on OxyR and that both OxyR and KatB are essential for optimal oxidative stress resistance and nitrogenase activities. H2O2 at low concentrations was detoxified by KatB, leaving O2 as a by-product to support nitrogen fixation under O2-insufficient conditions. Moreover, our data suggest that the direct interaction between NfiS and katB mRNA is a conserved and widespread mechanism among P. stutzeri strains.IMPORTANCE Protection against oxygen damage is crucial for survival of nitrogen-fixing bacteria due to the extreme oxygen sensitivity of nitrogenase. This work exemplifies how the small ncRNA NfiS coordinates oxidative stress response and nitrogen fixation via base pairing with katB mRNA and nifK mRNA. Hence, NfiS acts as a molecular link to coordinate the expression of genes involved in oxidative stress response and nitrogen fixation. Our study provides the first insight into the biological functions of NfiS in oxidative stress regulation and adds a new regulation level to the mechanisms that contribute to the oxygen protection of the MoFe nitrogenase.

Dataset Information

AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide.

Publications

AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets