Analysis of growth phase regulated KatA and CatE and their physiological roles in determining hydrogen peroxide resistance in Agrobacterium tumefaciens

Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H2O2 resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H2O2 resistance. However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H2O2 toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H2O2 resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.     

Cj1386 is an ankyrin-containing protein involved in heme trafficking to catalase in Campylobacter jejuni

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.     

Role of a bacterial organic hydroperoxide detoxification system in preventing catalase inactivation

In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H. pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase. H. pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H2O2. However, it inactivated is by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation.     

Unusual properties of catalase A (KatA) of Pseudomonas aeruginosa PA14 are associated with its biofilm peroxide resistance

Pseudomonas aeruginosa is a ubiquitous environmental bacterium whose major catalase (KatA) is highly stable, extracellularly present, and required for full virulence as well as for peroxide resistance in planktonic and biofilm states. Here, we dismantled the function of P. aeruginosa KatA (KatA(Pa)) by comparing its properties with those of two evolutionarily related (clade 3 monofunctional) catalases from Bacillus subtilis (KatA(Bs)) and Streptomyces coelicolor (CatA(Sc)). We switched the coding region for KatA(Pa) with those for KatA(Bs) and CatA(Sc), expressed the catalases under the potential katA-regulatory elements in a P. aeruginosa PA14 katA mutant, and verified their comparable protein levels by Western blot analysis. The activities of KatA(Bs) and CatA(Sc), however, were less than 40% of the KatA(Pa) activity, suggestive of the difference in intrinsic catalatic activity or efficiency for posttranslational activity modulation in P. aeruginosa. Furthermore, KatA(Bs) and CatA(Sc) were relatively susceptible to proteinase K, whereas KatA(Pa) was highly stable upon proteinase K treatment. As well, KatA(Bs) and CatA(Sc) were undetectable in the extracellular milieu. Nevertheless, katA(Bs) and catA(Sc) fully rescued the peroxide sensitivity and osmosensitivity of the katA mutant, respectively. Both catalase genes rescued the attenuated virulence of the katA mutant in mouse acute infection and Drosophila melanogaster models. However, the peroxide susceptibility of the katA mutant in a biofilm growth state was rescued by neither katA(Bs) nor catA(Sc). Based on these results, we propose that the P. aeruginosa KatA is highly stable compared to the two major catalases from gram-positive bacteria and that its unique properties involving metastability and extracellular presence may contribute to the peroxide resistance of P. aeruginosa biofilm and presumably to chronic infections.     

Characterization of the OxyR regulon of Neisseria gonorrhoeae

OxyR regulates the expression of the majority of H(2)O(2) responses in Gram-negative organisms. In a previous study we reported the OxyR-dependent derepression of catalase expression in the human pathogen Neisseria gonorrhoeae. In the present study we used microarray expression profiling of N. gonorrhoeae wild-type strain 1291 and an oxyR mutant strain to define the OxyR regulon. In addition to katA (encoding catalase), only one other locus displayed a greater than two-fold difference in expression in the wild type : oxyR comparison. This locus encodes an operon of two genes, a putative peroxiredoxin/glutaredoxin (Prx) and a putative glutathione oxidoreductase (Gor). Mutant strains were constructed in which each of these genes was inactivated. A previous biochemical study in Neisseria meningitidis had confirmed function of the glutaredoxin/peroxiredoxin. Assay of the wild-type 1291 cell free extract confirmed Gor activity, which was lost in the gor mutant strain. Phenotypic analysis of the prx mutant strain in H(2)O(2) killing assays revealed increased resistance, presumably due to upregulation of alternative defence mechanisms. The oxyR, prx and gor mutant strains were deficient in biofilm formation, and the oxyR and prx strains had decreased survival in cervical epithelial cells, indicating a key role for the OxyR regulon in these processes.     

Derepression of the Bacillus subtilis PerR peroxide stress response leads to iron deficiency

The Bacillus subtilis PerR repressor regulates the adaptive response to peroxide stress. The PerR regulon includes the major vegetative catalase (katA), an iron storage protein (mrgA), an alkylhydroperoxide reductase (ahpCF), a zinc uptake system (zosA), heme biosynthesis enzymes (hemAXCDBL), the iron uptake repressor (fur), and perR itself. A perR null strain is resistant to hydrogen peroxide, accumulates a porphyrin-like compound, and grows very slowly. The poor growth of the perR mutant can be largely accounted for by the elevated expression of two proteins: the KatA catalase and Fur. Genetic studies support a model in which poor growth of the perR null mutant is due to elevated repression of iron uptake by Fur, exacerbated by heme sequestration by the abundant catalase protein. Analysis of the altered-function allele perR991 further supports a link between PerR and iron homeostasis. Strains containing perR991 are peroxide resistant but grow nearly as well as the wild type. Unlike a perR null allele, the perR991 allele (F51S) derepresses KatA, but not Fur, which likely accounts for its comparatively rapid growth.     

Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid

The parental strain (A⁺T⁺) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A⁺T^–), catalase A (A^–T⁺) or both catalases (A^–T^–), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two oleic acid-grown A⁺-strains (A⁺T⁺ and A⁺T^–) high catalase activities were found; catalase activity invariably remained low in the A^–T⁺ strain and was never detected in the A^–T^– strain. The levels of -oxidation enzymes in oleic acid-grown cells of the parental and all mutant strains were not significantly different. However, cytochrome C peroxidase activity had increased 8-fold in oleic acid grown A^– strains (A^–T⁺ and A^–T^–) compared to parental strain cells. The degree of peroxisomal proliferation was comparable among the different strains. Catalase A was shown to be located in peroxisomes. Catalase T is most probably cytosolic in nature and/or present in the periplasmic space.     

Stable accumulation of sigma54 in Helicobacter pylori requires the novel protein HP0958

Several flagellar genes in Helicobacter pylori are dependent on sigma(54) (RpoN) for their expression. These genes encode components of the basal body, the hook protein, and a minor flagellin, FlaB. A protein-protein interaction map for H. pylori constructed from a high-throughput screen of a yeast two-hybrid assay (http://pim.hybrigenics.com/pimriderext/common/) revealed interactions between sigma(54) and the conserved hypothetical protein HP0958. To see if HP0958 influences sigma(54) function, the corresponding gene was disrupted with a kanamycin resistance gene (aphA3) in H. pylori ATCC 43504 and the resulting mutant was analyzed. The hp0958:aphA3 mutant was nonmotile and failed to produce flagella. Introduction of a functional copy of hp0958 into the genome of the hp0958:aphA3 mutant restored flagellar biogenesis and motility. The hp0958:aphA3 mutant was deficient in expressing two sigma(54)-dependent reporter genes, flaB'-'xylE and hp1120'-'xylE. Levels of sigma(54) in the hp0958 mutant were substantially lower than those in the parental strain, suggesting that the failure of the mutant to express the genes in the RpoN regulon and produce flagella was due to reduced sigma(54) levels. Expressing sigma(54) at high levels by putting rpoN under the control of the ureA promoter restored flagellar biogenesis and motility in the hp0958:aphA3 mutant. Turnover of sigma(54) was more rapid in the hp0958:aphA3 mutant than it was in the wild-type strain, suggesting that HP0958 supports wild-type sigma(54) levels in H. pylori by protecting it from proteolysis.     

Campylobacter jejuni Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.     

Duodenal ulcer-related antigens from Helicobacter pylori: immunoproteome and protein microarray approaches

Helicobacter pylori is an important risk factor of duodenal ulcer (DU). Although many virulence factors of H. pylori have been identified, few have been reported to show an association with the pathogenesis of DU. The aims of this study were to identify H. pylori antigens showing a high seropositivity in DU and to develop a platform for rapid and easy diagnosis for DU. Because DU and gastric cancer (GC) are considered clinical divergent gastroduodenal diseases, we compared two-dimensional immunoblots of an acid-glycine extract of an H. pylori strain from a patient with DU probed with serum samples from 10 patients with DU and 10 with GC to identify DU-related antigens. Of the 11 proteins that were strongly recognized by serum IgG from DU patients, translation elongation factor EF-G (FusA), catalase (KatA), and urease alpha subunit (UreA) were identified as DU-related antigens, showing a higher seropositivity in DU samples (n = 124) than in GC samples (n = 95) (FusA, 70.2 versus 45.3%; KatA, 50.8 versus 41.1%; UreA, 44.4 versus 27.4%). In addition, we found that the use of multiple antigens improved the discrimination between patients with DU and those with GC as the odds ratios increased from 1.82 (95% confidence interval (CI), 0.79-4.21; p = 0.1607) for seropositivity for FusA, KatA, or UreA alone to 4.95 (95% CI, 2.05-12.0; p = 0.0004) for two of the three antigens and to 5.71 (95% CI, 1.86-17.6; p = 0.0024) for all three antigens. Moreover a protein array containing the three DU-related antigens was developed to test the idea of using multiple biomarkers in diagnosis. We conclude that FusA, KatA, and UreA are DU-related antigens of H. pylori, and the combination of these on a protein array provided a rapid and convenient method for detecting serum antibody patterns of DU patients.     

Recombinant Herpesvirus Glycoprotein G Improves the Protective Immune Response to Helicobacter pylori Vaccination in a Mouse Model of Disease

Alphaherpesviruses, which have co-evolved with their hosts for more than 200 million years, evade and subvert host immune responses, in part, by expression of immuno-modulatory molecules. Alphaherpesviruses express a single, broadly conserved chemokine decoy receptor, glycoprotein G (gG), which can bind multiple chemokine classes from multiple species, including human and mouse. Previously, we demonstrated that infection of chickens with an infectious laryngotracheitis virus (ILTV) mutant deficient in gG resulted in altered host immune responses compared to infection with wild-type virus. The ability of gG to disrupt the chemokine network has the potential to be used therapeutically. Here we investigated whether gG from ILTV or equine herpesvirus 1 (EHV-1) could modulate the protective immune response induced by the Helicobacter pylori vaccine antigen, catalase (KatA). Subcutaneous immunisation of mice with KatA together with EHV-1 gG, but not ILTV gG, induced significantly higher anti-KatA IgG than KatA alone. Importantly, subcutaneous or intranasal immunisation with KatA and EHV-1 gG both resulted in significantly lower colonization levels of H. pylori colonization following challenge, compared to mice vaccinated with KatA alone. Indeed, the lowest colonization levels were observed in mice vaccinated with KatA and EHV-1 gG, subcutaneously. In contrast, formulations containing ILTV gG did not affect H. pylori colonisation levels. The difference in efficacy between EHV-1 gG and ILTV gG may reflect the different spectrum of chemokines bound by the two proteins. Together, these data indicate that the immuno-modulatory properties of viral gGs could be harnessed for improving immune responses to vaccine antigens. Future studies should focus on the mechanism of action and whether gG may have other therapeutic applications.     

Cloning and characterization of the katA gene of Rhizobium meliloti encoding a hydrogen peroxide-inducible catalase.

To investigate the involvement of bacterial catalases of the symbiotic gram-negative bacterium Rhizobium meliloti in the development of Medicago-Rhizobium functional nodules, we cloned a putative kat gene by screening a cosmid library with a catalase-specific DNA probe amplified by PCR from the R. meliloti genome. Nucleotide sequence analysis of a 1.8-kb DNA fragment revealed an open reading frame, called katA, encoding a peptide of 562 amino acid residues with a calculated molecular mass of 62.9 kDa. The predicted amino acid sequence showed a high homology with the primary structure of monofunctional catalases from eucaryotes and procaryotes. The katA gene was localized on the chromosome, and the katA gene product was essentially found in the periplasmic space. A katA::Tn5 mutant was obtained and showed a drastic sensitivity to hydrogen peroxide, indicating an essential protective role of KatA. However, neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that KatA is not essential for nodulation and establishment of nitrogen fixation. Exposure to a sublethal concentration of H2O2 enhanced KatA activity (100-fold) and also increased survival to subsequent H2O2 exposure at higher concentrations. No protection is observed in katA::Tn5, indicating that KatA is the major component of an adaptive response.     

The role of cytoplasmic catalase in dehydration tolerance of Saccharomyces cerevisiae

In this study, we investigated the role played by cytoplasmic catalase (Ctt1) in resistance against water loss using the yeast Saccharomyces cerevisiae as eukaryotic cell model. Comparing a mutant possessing a specific lesion in CTT1 with its parental strain, it was observed that both control and ctt1 strains exhibited increased levels of lipid peroxidation after dehydration, suggesting that catalase does not protect membranes during drying. Although the ctt1 strain has only 1 catalase isoform (peroxisomal catalase), the mutant showed the same levels of total catalase activity as the control strain. Furthermore, in cells deficient in Ctt1, the reduced glutathione:oxidized glutathione ratio (GSH:GSSG) of dry cells was higher than that of the control strain, indicating a compensatory mechanism of defense in response to dehydration. Even so, desiccation tolerance of the ctt1 strain was significantly lower than in the control strain. Using a fluorescent probe sensitive to oxidation, we observed that cells of the ctt1 strain showed levels of intracellular oxidation 70% higher than those of control strain, suggesting that Ctt1 plays a role in the maintenance of the intracellular redox balance during dehydration and, therefore, in tolerance against a water stress.     

The Periplasmic, Group III Catalase of Vibrio fischeri Is Required for Normal Symbiotic Competence and Is Induced Both by Oxidative Stress and by Approach to Stationary Phase

The catalase gene, katA, of the sepiolid squid symbiont Vibrio fischeri has been cloned and sequenced. The predicted amino acid sequence of KatA has a high degree of similarity to the recently defined group III catalases, including those found in Haemophilus influenzae, Bacteroides fragilis, and Proteus mirabilis. Upstream of the predicted start codon of katA is a sequence that closely matches the consensus sequence for promoters regulated in Escherichia coli by the alternative sigma factor encoded by rpoS. Further, the level of expression of the cloned katA gene in an E. coli rpoS mutant is much lower than in wild-type E. coli. Catalase activity is induced three- to fourfold both as growing V. fischeri cells approach stationary phase and upon the addition of a small amount of hydrogen peroxide during logarithmic growth. The catalase activity was localized in the periplasm of wild-type V. fischeri cells, where its role could be to detoxify hydrogen peroxide coming from the external environment. No significant catalase activity could be detected in a katA null mutant strain, demonstrating that KatA is the predominately expressed catalase in V. fischeri and indicating that V. fischeri carries only a single catalase gene. The catalase mutant was defective in its ability to competitively colonize the light organs of juvenile squids in coinoculation experiments with the parent strain, suggesting that the catalase enzyme plays an important role in the symbiosis between V. fischeri and its squid host.     

Expression analysis and characterization of the mutant of a growth-phase- and starvation-regulated monofunctional catalase gene from Xanthomonas campestris pv. phaseoli

Analysis of the Xanthomonas campestris pv. phaseoli (Xp) catalase profile using an activity gel revealed at least two distinct monofunctional catalase isozymes denoted Kat1 and Kat2. Kat1 was expressed throughout growth, whereas Kat2 was expressed only during the stationary phase of growth. The nucleotide sequence of a previously isolated monofunctional catalase gene, Xp katE, was determined. The deduced amino acid sequence of Xp KatE showed a high percentage identity to an atypical group of monofunctional catalases that includes the well-characterized E. coli katE. Expression of Xp katE was growth phase-dependent but was not inducible by oxidants. In addition, growth of Xp in a carbon-starvation medium induced expression of the gene. An Xp katE mutant was constructed, and analysis of its catalase enzyme pattern showed that Xp katE coded for the Kat2 isozyme. Xp katE mutant had resistance levels similar to the parental strain against peroxide and superoxide killing at both exponential and stationary phases of growth. Interestingly, the level of total catalase activity in the mutant was similar to that of the parental strain even in stationary phase. These results suggest the existence of a novel compensatory mechanism for the activity of Xp catalase isozymes.