A Mechanism of Resistance to Hydrogen Peroxide in Vibrio rumoiensis S-1

A possible mechanism of resistance to hydrogen peroxide (H₂O₂) in Vibrio rumoiensis, isolated from the H₂O₂-rich drain pool of a fish processing plant, was examined. When V. rumoiensis cells were inoculated into medium containing either 5 mM or no H₂O₂, they grew in similar manners. A spontaneous mutant strain, S-4, derived from V. rumoiensis and lacking catalase activity did not grow at all in the presence of 5 mM H₂O₂. These results suggest that catalase is inevitably involved in the resistance and survival of V. rumoiensis in the presence of H₂O₂. Catalase activity was constitutively present in V. rumoiensis cells grown in the absence of H₂O₂, and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of Escherichia coli. The presence of the HP II-type catalase in V. rumoiensis cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism. A notable difference between V. rumoiensis and E. coli is that catalase is accumulated at very high levels (~2% of the total soluble proteins) in V. rumoiensis, in contrast to the case for E. coli. When V. rumoiensis cells which had been exposed to 5 mM H₂O₂ were centrifuged, most intracellular proteins, including catalase, were recovered in the medium. On the other hand, when V. rumoiensis cells were grown on plates containing various concentrations of H₂O₂, individual cells had a colony-forming ability inferior to those of E. coli, Bacillus subtilis, and Vibrio parahaemolyticus. Thus, it is suggested that when V. rumoiensis cells are exposed to high concentrations of H₂O₂, most cells will immediately be broken by H₂O₂. In addition, the cells which have had little or no damage will start to grow in a medium where almost all H₂O₂ has been decomposed by the catalase released from broken cells.     

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H₂O₂) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (P_n) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.     

Ultrastructural Localization of Hydrogen Peroxide Production in Ligninolytic Phanerochaete chrysosporium Cells

Previous studies have shown that the hydroxyl radical derived from hydrogen peroxide (H₂O₂) is involved in lignin degradation by Phanerochaete chrysosporium. In the present study, the ultrastructural sites of H₂O₂ production in ligninolytic cells of P. chrysosporium were demonstrated by cytochemically staining cells with 3,3′-diaminobenzidine (DAB). Hydrogen peroxide production, as evidenced by the presence of oxidized DAB deposits, appeared to be localized in the periplasmic space of cells from ligninolytic cultures grown for 14 days in nitrogen-limited medium. When identical cells were treated with DAB in the presence of aminotriazole, periplasmic deposits of oxidized DAB were not observed, suggesting that the deposits resulted from the H₂O₂-dependent peroxidatic oxidation of DAB by catalase. Cells from cultures grown for 3 or 6 days in nitrogen-limited medium or for 14 days in nitrogen-sufficient medium had little ligninolytic activity and low specific activity for H₂O₂ production and did not contain periplasmic oxidized DAB deposits. The results suggest that in cultures grown in nitrogen-limited medium, there is a positive correlation between the occurrence of oxidized DAB deposits, the specific activity for H₂O₂ production in cell extracts, and ligninolytic activity.     

alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (?uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ?uspA E. coli strain. The IPTG induced expression of a 56.6 kDa GST fusion protein was visualized on SDS–PAGE and attested by immunoblotting. E. coli ?uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl₂, H₂O₂, UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl₂ demonstrated about 22.6–51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.     

Heterologous extracellular expression and initial characterization of the peroxisomal catalase from the methylotrophic yeast Hansenula polymorpha in Pichia pastoris

Catalase is well known to eliminate H₂O₂ in cells and reduces the toxicity of peroxide compounds. A catalase gene HpCAT1 of methylotrophic yeast Hansenula polymorpha without the part coding the native signal peptide was cloned into expression vector pYM3165 and then integrated into genome of Pichia pastoris GS115 by electroporation. The result of the enzyme activity assay and SDS-PAGE demonstrated that the recombinant protein (HpCAT1) of H. polymorpha was extracellularly expressed in P. pastoris. The expressed catalase was recovered from the culture supernatant of P. pastoris GS115 and purified by (NH₄)₂SO₄ fractionation and Ni-NTA affinity chromatography. The main biochemical properties of the recombinant protein HpCAT1, such as thermodependence and thermostability, pH optimum and pH stability, as well as the effect of metal ions and chemicals, were characterized. With H₂O₂ as the substrate, HpCAT1 displayed pH and temperature optima of ～2.6 and 45°C, respectively. The recombinant HpCAT1 activity was inhibited by 1 mM Hg²⁺ and Cu²⁺, but was highly enhanced by 1.0 mM Fe²⁺.     

Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H₂O₂) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H₂O₂ compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.     

Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)₆ tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.     

Superoxide radicals and phagocytosis

Escherichia coli B, grown in iron-rich media, were more resistant toward the aerobic bactericidal action of the formed elements of blood than were comparable iron-deficient cells. The iron replete cells contained 2.5 times more ferrisuperoxide dismutase, 12 times more peroxidase, and 1.5 times more catalase than did the iron-deficient cells. The iron-deficient cells were more susceptible to exogenous O₂^? and to H₂O₂ than were iron-replete cells. Cyanide permitted a differentiation between ferrisuperoxide dismutase and catalase or peroxidase since it inhibited the latter peroxide-consuming enzymes but had no effect on the superoxide-utilizing enzyme. In the presence of 2 mm cyanide, the iron-replete E. coli were much more resistant toward phagocytic kill than were the iron-deficient cells even though this level of cyanide completely inhibited catalase and peroxidase. It can be concluded that a large part of the enhanced resistance toward phagocytic kill, exhibited by iron-replete E. coli B, was due to their increased content of the periplasmic ferrisuperoxide dismutase. It follows that O₂^? is probably an important agent in the killing of phagocytized E. coli B.     

How Escherichia coli Tolerates Profuse Hydrogen Peroxide Formation by a Catabolic Pathway

When Escherichia coli grows on conventional substrates, it continuously generates 10 to 15 μM/s intracellular H₂O₂ through the accidental autoxidation of redox enzymes. Dosimetric analyses indicate that scavenging enzymes barely keep this H₂O₂ below toxic levels. Therefore, it seemed potentially problematic that E. coli can synthesize a catabolic phenylethylamine oxidase that stoichiometrically generates H₂O₂. This study was undertaken to understand how E. coli tolerates the oxidative stress that must ensue. Measurements indicated that phenylethylamine-fed cells generate H₂O₂ at 30 times the rate of glucose-fed cells. Two tolerance mechanisms were identified. First, in enclosed laboratory cultures, growth on phenylethylamine triggered induction of the OxyR H₂O₂ stress response. Null mutants (ΔoxyR) that could not induce that response were unable to grow. This is the first demonstration that OxyR plays a role in protecting cells against endogenous H₂O₂. The critical element of the OxyR response was the induction of H₂O₂ scavenging enzymes, since mutants that lacked NADH peroxidase (Ahp) grew poorly, and those that additionally lacked catalase did not grow at all. Other OxyR-controlled genes were expendable. Second, phenylethylamine oxidase is an unusual catabolic enzyme in that it is localized in the periplasm. Calculations showed that when cells grow in an open environment, virtually all of the oxidase-generated H₂O₂ will diffuse across the outer membrane and be lost to the external world, rather than enter the cytoplasm where H₂O₂-sensitive enzymes are located. In this respect, the periplasmic compartmentalization of phenylethylamine oxidase serves the same purpose as the peroxisomal compartmentalization of oxidases in eukaryotic cells.     

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H₂O₂ system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H₂O₂ was 86-times lower than that of HRP (K_m = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H₂O₂ as electron donor was 2838-times lower than that of HRP (k_cat/K_m = 15.8 and 44 833 mM⁻¹ min⁻¹, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H₂O₂ concentration, and that it poorly protects recombinant Escherichia coli from H₂O₂ stress. These observations indicate that rice Hb1 inefficiently scavenges H₂O₂ as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type="Italic">katA</Emphasis> from <Emphasis Type="Italic">Lactobacillus Sakei</Emphasis> Protects <Emphasis Type="Italic">Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.     

Expression of a glutathione reductase from <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> enhanced cellular redox homeostasis by modulating antioxidant proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H₂O₂, menadione, and heavy metal (CdCl₂, ZnCl₂ and AlCl₂)-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H₂O₂ stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.     

Responsibility of Hydrogen Peroxide for the Lethality of Resting Escherichia coli B Cells Exposed to Alternating Current in Phosphate Buffer Solution

Surviving fractions of Escherichia coli B exposed to an alternating current (AC) of 50 Hz in a phosphate buffer solution of pH 7.0 at 29°C were closely related to the amount of H₂O₂ formed in cell suspensions. At a definite current density, the amount of H₂O₂ in the suspensions or in buffer solution without cells increased with increasing AC-exposure time under aerobic conditions. On the other hand, the formation of H₂O₂ on AC-exposure was not detected under anaerobic conditions. It was considered that H₂O₂ was formed on the surface of carbon electrodes by AC-electrolytic reduction of dissolved oxygen. The amount of H₂O₂ formed decreased with increasing concentration of cells suspended or of catalase added to the suspension. When the formation of H₂O₂ was significantly suppressed, surviving fractions of cells exposed to AC remained almost unchanged. Growth conditions, modifying the intracellular level of catalase of E. coli, affected the sensitivity of cells to AC-exposure.     

Catalase activity is modulated by calcium and calmodulin in detached mature leaves of sweet potato

Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species and affects the H₂O₂ homeostasis in plants. In sweet potato, a major catalase isoform was detected, and total catalase activity showed the highest level in mature leaves (L3) compared to immature (L1) and completely yellow, senescent leaves (L5). The major catalase isoform as well as total enzymatic activity were strongly suppressed by ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). This inhibition could be specifically and significantly mitigated in mature L3 leaves by exogenous CaCl₂, but not MgCl₂ or CoCl₂. EGTA also inhibited the activity of the catalase isoform in vitro. Furthermore, chlorpromazine (CPZ), a calmodulin (CAM) inhibitor, drastically suppressed the major catalase isoform as well as total enzymatic activity, and this suppression was alleviated by exogenous sweet potato calmodulin (SPCAM) fusion protein in L3 leaves. CPZ also inhibited the activity of the catalase isoform in vitro. Protein blot hybridization showed that both anti-catalase SPCAT1 and anti-calmodulin SPCAM antibodies detect a band at the same position, which corresponds to the activity of the major catalase isoform from unboiled, but not boiled crude protein extract of L3 leaves. An inverse correlation between the major catalase isoform/total enzymatic activity and the H₂O₂ level was also observed. These data suggest that sweet potato CAT activity is modulated by CaCl₂ and SPCAM, and plays an important role in H₂O₂ homeostasis in mature leaves. Association of SPCAM with the major CAT isoform is required and regulates the in-gel CAT activity band.     

Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H₂O₂ elevation

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2-80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24 h, then decreased gradually until 72 h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H₂O₂ amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H₂O₂, NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H₂O₂ elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H₂O₂ homeostasis in leaves caused by developmental cues and environmental stimuli.     

Down‐regulation of catalase activity allows transient accumulation of a hydrogen peroxide signal in Chlamydomonas reinhardtii

In photosynthetic organisms, excess light is a stress that induces production of reactive oxygen species inside the chloroplasts. As a response, the capacity of antioxidative defence mechanisms increases. However, when cells of Chlamydomonas reinhardtii were shifted from dark to high light, a reversible partial inactivation of catalase activity was observed, which correlated with a transient increase in the level of H₂O₂ in the 10 μm range. This concentration range seems to be necessary to activate H₂O₂‐dependent signalling pathways stimulating the expression of H₂O₂ responsive genes, such as the heat shock protein HSP22C. Catalase knock‐down mutants had lost the transient accumulation of H₂O₂, suggesting that a decrease in catalase activity was the key element for establishing a transient H₂O₂ burst. Catalase was inactivated by a one‐electron event consistent with the reduction of a single cysteine. We propose that under high light intensity, the redox state of the photosynthetic electron transport chain is sensed and transmitted to the cytosol to regulate the catalase activity. This allows a transient accumulation of H₂O₂, inducing a signalling event that is transmitted to the nucleus to modulate the expression of chloroplast‐directed protection enzymes.     

Response of Plant-Colonizing Pseudomonads to Hydrogen Peroxide

Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H₂O₂) at concentrations up to 10 mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1 mM were killed. Extracts of logarithmic-phase cells displayed only band A catalase activity. Protection against 5 mM H₂O₂ was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentrations (30 to 300 μM) of H₂O₂. Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.