Function and stationary-phase induction of periplasmic copper-zinc superoxide dismutase and catalase/peroxidase in Caulobacter crescentus.

Although cytosolic superoxide dismutases (SODs) are widely distributed among bacteria, only a small number of species contain a periplasmic SOD. One of these is Caulobacter crescentus, which has a copper-zinc SOD (CuZnSOD) in the periplasm and an iron SOD (FeSOD) in the cytosol. The function of periplasmic CuZnSOD was studied by characterizing a mutant of C. crescentus with an insertionally inactivated CuZnSOD gene. Wild-type and mutant strains showed identical tolerance to intracellular superoxide. However, in response to extracellular superoxide, the presence of periplasmic CuZnSOD increased survival by as much as 20-fold. This is the first demonstration that periplasmic SOD defends against external superoxide of environmental origin. This result has implications for those bacterial pathogens that contain a CuZnSOD. C. crescentus was shown to contain a single catalase/peroxidase which, like Escherichia coli KatG catalase/peroxidase, is present in both the periplasmic and cytoplasmic fractions. The growth stage dependence of C. crescentus catalase/peroxidase and SOD activity was studied. Although FeSOD activity was identical in exponential- and stationary-phase cultures, CuZnSOD was induced nearly 4-fold in stationary phase and the catalase/peroxidase was induced nearly 100-fold. Induction of antioxidant enzymes in the periplasm of C. crescentus appears to be an important attribute of the stationary-phase response and may be a useful tool for studying its regulation.     

Legionella pneumophila Catalase-Peroxidases: Cloning of the katB Gene and Studies of KatB Function

Legionella pneumophila, the causative organism of Legionnaires’ pneumonia, is spread by aerosolization from man-made reservoirs, e.g., water cooling towers and air conditioning ducts, whose nutrient-poor conditions are conducive to entrance into stationary phase. Exposure to starvation conditions is known to induce several virulence traits in L. pneumophila. Since catalase-peroxidases have been extremely useful markers of the stationary-phase response in many bacterial species and may be an avenue for identifying virulence genes in L. pneumophila, an investigation of these enzymes was initiated. L. pneumophila was shown to contain two bifunctional catalase-peroxidases and to lack monofunctional catalase and peroxidase. The gene encoding the KatB catalase-peroxidase was cloned and sequenced, and lacZ fusion and null mutant strains were constructed. Null mutants in katB are delayed in the infection and lysis of cultured macrophage-like cell lines. KatB is similar to the KatG catalase-peroxidase of Escherichia coli in its 20-fold induction during exponential growth and in playing a role in resistance to hydrogen peroxide. Analysis of the changes in katB expression and in the total catalase and peroxidase activity during growth indicates that the 8- to 10-fold induction of peroxidase activity that occurs in stationary phase is attributable to KatA, the second L. pneumophila catalase-peroxidase.     

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.     

A peroxide/ascorbate-inducible catalase from Haemophilus influenzae is homologous to the Escherichia coli katE gene product.

Bacterial catalases are induced by exposure to peroxide (e.g., Escherichia coli katG) or entry into stationary phase (e.g., E. coli katE). To study regulatory systems in Haemophilus influenzae, we complemented an E. coli rpoS mutant, which is unable to induce katE in stationary phase, with a plasmid library of H. influenzae Rd- chromosomal DNA. Nineteen complementing clones with a catalase-positive phenotype were obtained and characterized after screening about 10(5) transformants. All carried the same structural gene for an H. influenzae catalase. The DNA sequence of this gene, called hktE, encodes a 508-amino-acid polypeptide with strong homology to eukaryotic catalases and E. coli katE. However, hktE is regulated like E. coli katG, with catalase activity increasing 10-fold and hktE mRNA levels increasing 4-fold upon exposure to ascorbic acid, which serves to generate hydrogen peroxide. Mutations in the known global regulatory genes of H. influenzae--crp, cya, and sxy--do not affect the inducibility of hktE. The hktE gene maps to a 225-kb segment of the H. influenzae chromosome in a region encoding resistance to spectinomycin.     

Role of glutathione in regulation of hydroperoxidase I in growing Escherichia coli.

To examine role of glutathione in regulation of catalases in growing Escherichia coli, katG::lacZ and katE::lacZ fusions were transformed into a glutathione-deficient Escherichia coli strain and wild-type parent. In the absence of H2O2 and in the presence of the low H2O2 concentrations (0.1-3 mM), the gshA mutation stimulated katG::lacZ expression and the total catalase activity in exponential phase. In the absence of H2O2, the mutation in gshA also stimulated katE::lacZ expression. At higher H2O2 concentrations, the gshA mutation suppressed katG::lacZ expression and catalase activity. In stationary and mid-exponential phases, the intracellular concentrations of H2O2 in the gshA mutant were markedly increased compared to those in the wild type. These results suggest that glutathione may be involved in regulation of catalases.     

Growth of Escherichia coli in iron-enriched medium increases HPI catalase activity

Escherichia coli has two catalases, HPI and HPII. HPI is induced during logarithmic growth in response to low concentrations of hydrogen peroxide. This induction is OxyR-dependent. On the other hand, HPII is not peroxide-inducible but is induced in entry to the stationary phase. We demonstrate here that E. coli displayed higher HPI catalase activity when compared to the cultures that were grown in a normal medium, if grown in a medium supplemented with iron-citrate. Iron supplementation had no effect on HPII catalase. This increase of HPI activity was OxyR-independent and not observed in a Deltafur mutant. The physiological significance of the increase of HPI activity is unclear, but it appears that the katG gene that codes for HPI catalase is among the genes that are regulated by Fur.     

Site-directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare , like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H₂O₂ and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H₂O₂ and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.     

Genetic mapping of katG, a locus that affects synthesis of the bifunctional catalase-peroxidase hydroperoxidase I in Escherichia coli.

A locus unlinked to either katE or katF that affected catalase levels in Escherichia coli was identified and localized between metB and ppc at 89.2 min on the genome. The locus was named katG. Mutations in katG which prevented the formation of both isoenzyme forms of the bifunctional catalase-peroxidase HPI were created both by nitrosoguanidine and by transposon Tn10 insertions. All katG+ recombinants and transductants contained both HPI isoenzymes. Despite the common feature of little or no catalase activity in four of the catalase-deficient strains, subtle differences in the phenotypes of each strain resulted from the different katG mutations. All three mutants caused by nitrosoguanidine produced a protein with little or no catalase activity but with the same subunit molecular weight and with similar antigenic properties to HPI, implying the presence of missense mutations rather than nonsense mutations in each strain. Indeed one mutant produced an HPI-like protein that retained peroxidase activity, whereas the HPI-like protein in a second mutant exhibited no catalase or peroxidase activity. The third mutant responded to ascorbate induction with the synthesis of near normal catalase levels, suggesting a regulatory defect. The Tn10 insertion mutant produced no catalase and no protein that was antigenically similar to HPI.     

Cloning, expression, and characterization of the katG gene, encoding catalase-peroxidase, from the polycyclic aromatic hydrocarbon-degrading bacterium Mycobacterium sp. strain PYR-1

A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.     

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.     

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.     

Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus

DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.     

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met-Tyr-Trp cross-linked adduct

Mycobacterium tuberculosis catalase-peroxidase (Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous-CO complex of KatG(Y229F) suggest a non-linear Fe-CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met-Tyr-Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.     

Homology among bacterial catalase genes

Catalase activities in crude extracts of exponential and stationary phase cultures of various bacteria were visualized following gel electrophoresis for comparison with the enzymes from Escherichia coli. Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Klebsiella pneumoniae, and Salmonella typhimurium exhibited patterns of catalase activity similar to E. coli, including bifunctional HPI-like bands and a monofunctional HPII-like band. Proteus mirabilis, Erwinia carotovora, and Serratia marcescens contained a single band of monofunctional catalase with a mobility intermediate between the HPI-like and HPII-like bands. The cloned genes for catalases HPI (katG) and HPII (katE) from E. coli were used as probes in Southern hybridization analyses for homologous sequences in genomic DNA of the same bacteria. katG was found to hybridize with fragments from C. freudii, Ent. aerogenes, Sal. typhimurium, and K. pneumoniae but not at all with Ed. tarda, P. mirabilis, S. marcesens, or Er. carotovora. katE hybridized with C. freundii and K. pneumoniae DNAs and not with the other bacterial DNAs.