Differential roles of OxyR-controlled antioxidant enzymes alkyl hydroperoxide reductase (AhpCF) and catalase (KatB) in the protection of Pseudomonas aeruginosa against hydrogen peroxide in biofilm vs. planktonic culture

The role of the Pseudomonas aeruginosa OxyR-controlled antioxidants alkyl hydroperoxide reductase CF (AhpCF) and catalase B (KatB) was evaluated in biofilm vs. planktonic culture upon exposure to hydrogen peroxide. AhpCF was found to be critical for survival of biofilm bacteria while KatB was more important for survival of planktonic free-swimming organisms.     

Catalase-peroxidases of Legionella pneumophila: cloning of the katA gene and studies of KatA function

Legionella pneumophila, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for katA was cloned and its function was studied in L. pneumophila and Escherichia coli and compared with prior studies of katB in this laboratory. katA is induced during exponential growth and is the predominant peroxidase in stationary phase. When katA is inactivated, L. pneumophila is more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to katB. Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine. In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when katA is inactivated, while no change from wild type is seen for the katB null. The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic. This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation. Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by L. pneumophila, further studies on the function and regulation of katA in stationary phase may give insights on the mechanisms of infectivity of this pathogen.     

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.     

Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities.

Pseudomonas aeruginosa is considered a strict aerobe that possesses several enzymes important in the disposal of toxic oxygen reduction products including iron- and manganese-cofactored superoxide dismutase and catalase. At present, the nature of the regulation of these enzymes in P. aeruginosa Is not understood. To address these issues, we used two mutants called A4 and C6 which express altered Fur (named for ferric uptake regulation) proteins and constitutively produce the siderophores pyochelin and pyoverdin. Both mutants required a significant lag phase prior to log-phase aerobic growth, but this lag was not as apparent when the organisms were grown under microaerobic conditions. The addition of iron salts to mutant A4 and, to a greater extent, C6 cultures allowed for an increased growth rate under both conditions relative to that of bacteria without added iron. Increased manganese superoxide dismutase (Mn-SOD) and decreased catalase activities were also apparent in the mutants, although the second catalase, KatB, was detected in cell extracts of each fur mutant. Iron deprivation by the addition of the iron chelator 2,2'-dipyridyl to wild-type bacteria produced an increase in Mn-SOD activity and a decrease in total catalase activity, similar to the fur mutant phenotype. Purified wild-type Fur bound more avidly than mutant Fur to a PCR product containing two palindromic 19-bp "iron box" regions controlling expression of an operon containing the sodA gene that encodes Mn-SOD. All mutants were defective in both ferripyochelin- and ferripyoverdin-mediated iron uptake. Two mutants of strain PAO1, defective in pyoverdin but not pyochelin biosynthesis, produced increased Mn-SOD activity. Sensitivity to both the redox-cycling agent paraquat and hydrogen peroxide was greater in each mutant than in the wild-type strain. In summary, the results indicate that mutations in the P. aeruginosa fur locus affect aerobic growth and SOD and catalase activities in P. aeruginosa. We postulate that reduced siderophore-mediated iron uptake, especially that by pyoverdin, may be one possible mechanism contributing to such effect.     

Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents.

Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective alpha/beta-type small, acid-soluble, spore proteins (termed alpha(-)beta(-) spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or alpha(-)beta(-) spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.     

Purification and physical-chemical characterization of the three hydroperoxidases from the symbiotic bacterium Sinorhizobium meliloti

Three genes encoding heme hydroperoxidases (katA, katB, and katC) have been identified in the soil bacterium Sinorhizobium meliloti. The recombinant proteins were overexpressed in Escherichia coli and purified in order to achieve a spectral and kinetic characterization. The three proteins contain heme b with high-spin Fe(III). KatB is an acidic bifunctional homodimeric catalase-peroxidase exhibiting both catalase (k(cat) = 2400 s(-1)) and peroxidase activity and having a high affinity for hydrogen peroxide (apparent K(M) = 1.6 mM). KatA and KatC are acidic monofunctional homotetrameric catalases. Although different in size (KatA is a small subunit catalase while KatC is a large subunit catalase) both enzymes exhibit the same heme type and a similar affinity for H(2)O(2) (apparent K(M) values of 160 and 150 mM). However, the turnover rate of KatA (k(cat) = 279000 s(-1)) exceeds that of KatC (k(cat) = 3100 s(-1)) significantly. The kinetic parameters are in good agreement with the physiological role of these heme proteins. KatB is the housekeeping hydroperoxidase exhibiting the highest affinity for hydrogen peroxide, while KatA has the lowest H(2)O(2) affinity but the highest k(cat)/K(M) value (1.75 x 10(6) M(-1) s(-1)), in agreement with the hydrogen peroxide inducibility of the encoding gene. Moreover, the lower catalytic efficiency of KatC (2.1 x 10(4) M(-1) s(-1)) appears to be enough for growing in the stationary phase and/or under heat or salt stress (conditions that are known to favor katC expression).     

Oxidative stress response in an anaerobe, Bacteroides fragilis: a role for catalase in protection against hydrogen peroxide.

Survival of Bacteroides fragilis in the presence of oxygen was dependent on the ability of bacteria to synthesize new proteins, as determined by the inhibition of protein synthesis after oxygen exposure. The B. fragilis protein profile was significantly altered after either a shift from anaerobic to aerobic conditions with or without paraquat or the addition of exogenous hydrogen peroxide. As determined by autoradiography after two-dimensional gel electrophoresis, approximately 28 newly synthesized proteins were detected in response to oxidative conditions. These proteins were found to have a broad range of pI values (from 5.1 to 7.2) and molecular weights (from 12,000 to 79,000). The hydrogen peroxide- and paraquat-inducible responses were similar but not identical to that induced by oxygen as seen by two-dimensional gel protein profile. Eleven of the oxidative response proteins were closely related, with pI values and molecular weights from 5.1 to 5.8 and from 17,000 to 23,000, respectively. As a first step to understanding the resistance to oxygen, a catalase-deficient mutant was constructed by allelic gene exchange. The katB mutant was found to be more sensitive to the lethal effects of hydrogen peroxide than was the parent strain when the ferrous iron chelator bipyridyl was added to culture media. This suggests that the presence of ferrous iron in anaerobic culture media exacerbates the toxicity of hydrogen peroxide and that the presence of a functional catalase is important for survival in the presence of hydrogen peroxide. Further, the treatment of cultures with a sublethal concentration of hydrogen peroxide was necessary to induce resistance to higher concentrations of hydrogen peroxide in the parent strain, suggesting that this was an inducible response. This was confirmed when the bacterial culture, treated with chloramphenicol before the cells were exposed to a sublethal concentration of peroxide, completely lost viability. In contrast, cell viability was greatly preserved when protein synthesis inhibition occurred after peroxide induction. Complementation of catalase activity in the mutant restored the ability of the mutant strain to survive in the presence of hydrogen peroxide, showing that the catalase (KatB) may play a role in oxidative stress resistance in aerotolerant anaerobic bacteria.     

The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe.

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.     

Identification of a non-haem catalase in Salmonella and its regulation by RpoS (sigmaS)

We report the identification and functional analysis of katN, a gene encoding a non-haem catalase of Salmonella enterica serotype Typhimurium. katN, which is not present in Escherichia coli, is located between the yciGFE and yciD E. coli homologues in the Salmonella genome. Its predicted protein product has a molecular weight of 31 826 Da and is similar to the Mn-catalases of Lactobacillus plantarum and Thermus spp. Its product, KatN, was visualized as a 37 kDa protein in E. coli maxicells. A KatN recombinant protein, containing six histidine residues at its C-terminus, was purified, and its catalase activity was observed on a non-denaturing polyacrylamide gel. KatN was also visualized by catalase activity gel staining of bacterial cell extracts. Its expression was shown to be regulated by growth phase and rpoS. Northern blotting indicated that kat forms an operon with the upstream yciGFE genes. A putative rpoS-regulated promoter was identified upstream of yciG. Southern blotting revealed that katN is conserved within Salmonella serovars. katN homologues were found in Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae and Serratia marcescens. A katN mutation did not appear to affect the hydrogen peroxide (H2O2) response of Salmonella. However, the expression of katN increased the H2O2 resistance of unadapted cells in the exponential phase and of rpoS mutants in stationary phase. Thus, KatN may contribute to hydrogen peroxide resistance in Salmonella in certain environmental conditions.     

Catalase activity and the survival of Pseudomonas putida, a root colonizer, upon treatment with peracetic acid

Peracetic acid is used as a sterilant in several industrial settings. Cells of a plant-colonizing bacterium, Pseudomonas putida in liquid suspension, were more sensitive to killing by peracetic acid when they lacked a major catalase activity, catalase A. Low doses of peracetic acid induced promoter activity of the gene encoding catalase A and increased total catalase specific activity in cell extracts. Microbes present in native agricultural soils rapidly degraded the active oxygen species present in peracetic acid. The simultaneous release of oxygen was consistent with a role for catalase in degrading the hydrogen peroxide that is part of the peracetic acid-equilibrium mixture. Amendment of sterilized soils with wild-type P. putida restored the rate of degradation of peracetic acid to a higher level than was observed in the soils amended with the catalase A-deficient mutant. The association of the bacteria with the plant roots resulted in protection of the wild-type as well as the catalase-deficient mutant from killing by peracetic acid. No differential recovery of the wild-type and catalase A mutant of P. putida was observed from roots after the growth matrix containing the plants was flushed with peracetic acid.     

The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product.

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.     

Effect of a waaL mutation on lipopolysaccharide composition, oxidative stress survival, and virulence in Erwinia amylovora

Erwinia amylovora , the causal agent of fire blight, is an enterobacterial pathogen of Rosaceous plants including apple and pear. We have been studying the response of E. amylovora to oxidative stress because, during infection, the bacterium elicits an oxidative burst response in host plants. During the screening of a transposon mutant library for hydrogen peroxide sensitivity, we identified a mutant carrying an insertion in waaL , a gene involved in lipopolysaccharide biosynthesis, that was more sensitive to hydrogen peroxide than the parental wild-type strain. We also confirmed that a waaL mutant of Pseudomonas aeruginosa exhibited an increased sensitivity to hydrogen peroxide compared with the wild-type strain. The E. amylovora waaL mutant was also reduced in virulence, showed a decrease in twitching motility, and was more sensitive to polymyxin B than the wild type. Each of these phenotypes was complemented by the cloned waaL gene. Our results highlight the importance of the lipopolysaccharide layer to virulence in E. amylovora and the unexpected finding of an additional function of lipopolysaccharide in protection from oxidative stress in E. amylovora and P. aeruginosa .     

Pseudomonas syringae Catalases Are Collectively Required for Plant Pathogenesis

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 must detoxify plant-produced hydrogen peroxide (H(2)O(2)) in order to survive in its host plant. Candidate enzymes for this detoxification include the monofunctional catalases KatB and KatE and the bifunctional catalase-peroxidase KatG of DC3000. This study shows that KatG is the major housekeeping catalase of DC3000 and provides protection against menadione-generated endogenous H(2)O(2). In contrast, KatB rapidly and substantially accumulates in response to exogenous H(2)O(2). Furthermore, KatB and KatG have nonredundant roles in detoxifying exogenous H(2)O(2) and are required for full virulence of DC3000 in Arabidopsis thaliana. Therefore, the nonredundant ability of KatB and KatG to detoxify plant-produced H(2)O(2) is essential for the bacteria to survive in plants. Indeed, a DC3000 catalase triple mutant is severely compromised in its ability to grow in planta, and its growth can be partially rescued by the expression of katB, katE, or katG. Interestingly, our data demonstrate that although KatB and KatG are the major catalases involved in the virulence of DC3000, KatE can also provide some protection in planta. Thus, our results indicate that these catalases are virulence factors for DC3000 and are collectively required for pathogenesis.