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.     

Gene transfection of H25A mutant heme oxygenase-1 protects cells against hydroperoxide-induced cytotoxicity.

Heme oxygenase (HO)-1 is a stress-inducible enzyme protecting cells against oxidative stress, and mechanisms have been considered to depend exclusively on its enzyme activity. This study aimed to examine if the protein lacking catalytic activities could also display such resistance against oxidative stress. Stable transfectants of rat wild type HO-1 cDNA (HO-1-U937) and those of its H25A mutant gene (mHO-1-U937) were established using human monoblastic lymphoma cell U937. HO-1-U937 and mHO-1-U937 used in the study exhibited similar levels of the protein expression, while only the former increased enzyme activities. HO-1- and mHO-1 U937 cells became more and less sensitive to H(2)O(2) than mock transfectants, respectively; such distinct susceptibility between the cells was ascribable to differences in the capacity to scavenge H(2)O(2) through catalase and to execute iron-mediated oxidant propagation. On the other hand, both cell lines exhibited greater resistance to tert-butyl hydroperoxide than mock transfectants. The resistance of HO-1-U937 to hydroperoxides appeared to result from antioxidant properties of bilirubin, an HO-derived product, while that of mHO-1-U937 was ascribable to increased contents of catalase and glutathione. These results provided evidence that gene transfection of the activity-lacking mutant HO-1 protects cells against oxidative stress through multiple mechanisms involving up-regulation of catalase and glutathione contents.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O(2) and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O(2) exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Mitochondria provide the main source of cytosolic ATP for activation of outward-rectifying K+ channels in mesophyll protoplast of chlorophyll-deficient mutant rice (OsCHLH) seedlings

The role of mitochondria in providing intracellular ATP that controls the activity of plasma membrane outward-rectifying K+ channels was evaluated. The OsCHLH rice mutant, which lacks chlorophyll in the thylakoids, was isolated by T-DNA gene trapping (Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., Hirochika, H., and An, G. (2003) Plant Cell Physiol. 44, 463-472). The OsCHLH mutant is unable to fix CO2 and exhibits reduced growth. Wild type and mutant plants exhibit similar rates of respiratory O2 uptake in the dark, whereas the rate of photosynthetic O2 evolution by the mutant was negligible during illumination. During dark respiration the wild type and mutant exhibited similar levels of cytoplasmic ATP. In the mutant oligomycin treatment (an inhibitor of mitochondrial F1F0-ATPase) drastically reduced ATP production. The fact that this was reversed by the addition of glucose suggested that the mutant produced ATP exclusively from mitochondria but not from chloroplasts. In whole cell patch clamp experiments, the activity of outward-rectifying K+ channels of rice mesophyll cells showed ATP-dependent currents, which were 1.5-fold greater in wild type than in mutant cells. Channels in both wild type and mutant cells were deactivated by the removal of cytosolic ATP, whereas in the presence of ATP the channels remained active. We conclude that mesophyll cells in the OsCHLH rice mutant derive ATP from mitochondrial respiration, and that this is critical for the normal function of plasma membrane outward-rectifying K+ channels.     

耐辐射奇球菌dps突变株的构建和蛋白功能初步研究

Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H₂O₂)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H₂O₂(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。     

Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis

Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O₂-resistant photosynthesis. The apparent K_m for H₂O₂ was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level.     

The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene

In order to investigate the effects of a permanent increase in cellular H(2)O(2) on cation homeostasis we have studied a T-DNA insertion mutant of the Arabidopsis CATALASE 2 gene. This mutant (cat2-1) exhibits 20% of wild-type leaf catalase activity and accumulates more H(2)O(2) than the wild type under normal growth conditions. In addition to reduced size, a pale green color and great reduction in secondary roots, the cat2-1 mutant exhibited increased sensitivity to H(2)O(2), NaCl, norspermidine, high light and cold stress. On the other hand, the germination of the cat2-1 mutant is more tolerant to lithium than the wild type. This novel phenotype cannot be explained by changes in lithium transport. Actually, the uptake of lithium (and of other toxic cations such as sodium and norspermidine) is increased in the cat2-1 mutant while K(+) levels were decreased. The lithium tolerance of this mutant seems to result both from insensitivity to the inhibitory ethylene induced by this cation and a reduced capability for ethylene production. Accordingly, induction by ethylene of responsive genes such as PR4 and EBP/ERF72 is decreased in cat2-1. Mutants insensitive to ethylene such as etr1-1 and ein3-3 are lithium tolerant, and inhibition of ethylene biosynthesis with 2-aminoisobutyrate protects against lithium toxicity. Microarray analysis of gene expression indicates that the expression of genes related to cation transport and ethylene synthesis and perception was not altered in the cat2-1 mutant, suggesting that H(2)O(2) modulates these processes at the protein level. These results uncover a cross-talk between oxidative stress, cation homeostasis and ethylene.     

Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant.

Virulence and pathogenicity of an Escherichia coli O157:H7 sorbitol-positive mutant were investigated with an infant rabbit animal model as well as a battery of in vitro assays. Total cell lysate protein profiles, outer membrane protein profiles, plasmid profiles, and levels of cytotoxic activity against Vero cells were similar in the wild-type and mutant strains. Both adhered to intestinal epithelial cells in culture and reacted with fluorescein isothiocyanate-labeled antiserum against E. coli O157:H7. The mutant appeared to be similar to the wild type in all respects except in its ability to ferment sorbitol. [14C]sorbitol uptake and sorbitol-6-phosphate dehydrogenase activities were notably increased in the mutant strain. Diarrhea developed in rabbits administered the wild-type strain and in those fed the sorbitol-positive mutant. There was greater bacterial attachment and mucosal damage in the cecum and large intestine than in the small intestine. Scanning electron microscopy revealed bacteria adhering as single cells and as aggregates closely associated with mucus. Mucosal lesions consisted of areas of tissue necrosis with sloughing of epithelial cells. By transmission electron microscopy, electron-dense necrotic epithelial cells were visible in areas where bacteria were present, and epithelial cell debris containing bacteria was observed between the villar luminal surfaces. Light microscopy of epithelial cells of intestinal sections of infected rabbits revealed noticeable vacuolation and spherical, pyknotic nuclei. These data indicate that the sorbitol-negative phenotype is not associated with the pathogenicity of E. coli O157:H7.     

Scavenging of extracellular H2O2 by catalase inhibits the proliferation of HER-2/Neu-transformed rat-1 fibroblasts through the induction of a stress response

High levels of reactive oxygen species (ROS) are associated with cytotoxicity. Alternatively, nontoxic levels of ROS like hydrogen peroxide (H(2)O(2)) can mediate the transmission of many intracellular signals, including those involved in growth and transformation. To identify pathways downstream of endogenous cellular H(2)O(2) production, the response of Rat-1 fibroblasts exhibiting differential HER-2/Neu receptor tyrosine kinase activity to removal of physiological H(2)O(2) concentrations was investigated. The proliferation of all cells was abolished by addition of the H(2)O(2) scavenger catalase to the culture medium. HER-2/Neu activity was not significantly affected by catalase treatment, suggesting that the target(s) of the H(2)O(2) signal lie downstream of the receptor in our model. ERK1/2 phosphorylation was blocked by catalase in fibroblasts expressing wild type Neu, however such a response did not occur in cells possessing activated mutant Neu. This indicates that the ERK1/2 response contributes little to the growth inhibition observed. By contrast, JNK1 activity increased following the addition of catalase or H(2)O(2), regardless of Neu activity or level of cell transformation. Phosphorylation of p38 MAPK was induced by H(2)O(2) but not by catalase. These observations suggest that scavenging of H(2)O(2) from the cellular environment blocks Rat-1 proliferation primarily through the activation of stress pathways.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.     

Factors affecting expression of enhanced catalase activity in a tobacco mutant with o(2)-resistant photosynthesis

Tobacco (Nicotiana tabacum) mutants with 40 to 50% more catalase activity than wild type show O₂-resistant photosynthesis under conditions of high photorespiration. More than 90% of the population of mutant plants of an M₇ and M₈ generation had enhanced catalase activity, and nearly 40% had activities >3 standard deviations above the mean of wild type. Superoxide dismutase activity was the same in mutant and wild-type leaves. The greater photosynthetic rate of mutant leaves previously observed in the laboratory was confirmed with field-grown plants that showed significantly higher rates (8%) than wild type during 8 days of measurements during a 19-day period of active growth. The tip region of expanding mutant leaves had higher catalase activity than the base of the lamina, and photosynthesis was O₂ resistant in 42% O₂ in the tip compared with the base, thus further supporting the hypothesis that there is a biochemical linkage between these traits. Plants grown in high light (270 micromole photons per square meter per second) had greater catalase activity and an activity ratio of mutant to wild type of 1.45 compared with 1.22 for those grown in low light (130 micromole photons per square meter per second). After acclimation for 3 weeks, plants transferred from low to high light showed increasing activities, and after 5 days the activity ratio of mutant to wild type was the same as in plants acclimated in higher light. The role of enhanced catalase activity in reducing photorespiratory CO₂ is discussed.     

In vivo role of catalase-peroxidase in synechocystis sp. strain PCC 6803

The katG gene coding for the only catalase-peroxidase in the cyanobacterium Synechocystis sp. strain PCC 6803 was deleted in this organism. Although the rate of H2O2 decomposition was about 30 times lower in the DeltakatG mutant than in the wild type, the strain had a normal phenotype and its doubling time as well as its resistance to H2O2 and methyl viologen were indistinguishable from those of the wild type. The residual H2O2-scavenging capacity was more than sufficient to deal with the rate of H2O2 production by the cell, estimated to be less than 1% of the maximum rate of photosynthetic electron transport in vivo. We propose that catalase-peroxidase has a protective role against environmental H2O2 generated by algae or bacteria in the ecosystem (for example, in mats). This protective role is most apparent at a high cell density of the cyanobacterium. The residual H2O2-scavenging activity in the DeltakatG mutant was a light-dependent peroxidase activity. However, neither glutathione peroxidase nor ascorbate peroxidase accounted for a significant part of this H2O2-scavenging activity. When a small thiol such as dithiothreitol was added to the medium, the rate of H2O2 decomposition in the DeltakatG mutant increased more than 10-fold, indicating that a thiol-specific peroxidase, for which thioredoxin may be the physiological electron donor, is present. Oxidized thioredoxin is likely to be reduced again by photosynthetic electron transport. Therefore, under laboratory conditions, there are only two enzymatic mechanisms for H2O2 decomposition present in Synechocystis sp. strain PCC 6803. One is catalyzed by a catalase-peroxidase, and the other is catalyzed by thiol-specific peroxidase.     

Poly(ADP ribose) polymerase-defective mutant cell clone of mouse L1210 cells.

By a sequential mutation and selection utilizing N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen, we succeeded in separating a poly(ADP ribose) polymerase-defective mutant clone (Cl-3527) from a mouse L1210 cell clone (Cl-3). The enzyme activity per cell in Cl-3527 cells was only 8% of that in wild type L1210 (CCL 219) cells. Immunoblot analysis of the enzyme protein in crude extracts of the mutant and wild type cells revealed that the enzyme defect was manifested as the loss of a 113-kDa wild type enzyme band in Cl-3527. Further analysis of partially purified enzyme from Cl-3527 by immunoblotting revealed that the molecular size of the enzyme in Cl-3527 was 108 kDa and that the amount of the mutant enzyme protein was markedly decreased in Cl-3527. The mutant enzyme was much more heat-labile than the wild type enzyme but the Km for NAD+, requirements for Mg2+ and nicked DNA, and the inhibition by 3-aminobenzamide, a potent inhibitor of the enzyme, however, were not so different from those of wild type enzyme. The mutant cells showed prolonged doubling time, increased temperature-sensitivity, increased percentage of active enzyme on a treatment of cells at high temperature, and increased expression of plasma membrane NADase, compared to wild type cells. Introduction of wild type ADPR pol gene into Cl-3527 cells partially restored the ADPR pol activity and the heat-resistance.     

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 Xanthomonas alkyl hydroperoxide reductase subunit C (ahpC) mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for alkyl peroxide metabolism. A Xanthomonas ahpC mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H(2)O(2) killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional ahpC gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an oxyR mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in ahpC appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in Xanthomonas differed from that in other bacteria.     

Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a [4Fe-4S] cluster which is essential for activity. The enzyme also undergoes removal of 11 NH2-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff, C. A., Paluh, J. L., and Zalkin, H. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. Inactivation and degradation in vivo of wild type and mutant amidotransferases were characterized in these integrants. Mutants FeS1 (C448S) and FeS2 (C451S) failed to form active enzyme, assemble FeS clusters, or undergo NH2-terminal processing. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t1/2 = 16 min) in exponentially growing cells. In contrast the wild type enzyme was stable in growing cells, and activity and cross-reactive protein were lost from glucose-starved cells with a t1/2 of 57 min. Mutant FeS3 (F394V) contained an FeS cluster and was processed normally, but had only about 40% of normal specific activity. The FeS3 enzyme was also inactivated by reaction with O2 in vitro about twice as fast as the wild type. The amidotransferase produced by the FeS3 integrant was stable in growing cells but was inactivated and degraded in glucose-starved cells more rapidly (t1/2 = 35 min) than the wild type enzyme. Mutant FeS4 (C451S, D442C) also contained an FeS cluster and was processed; the enzyme had about 50% of wild type-specific activity and reacted with O2 in vitro at the same rate as the wild type. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded at a rate (t1/2 = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O2 and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O2-dependent inactivation is the rate-determining step in degradation of the enzyme. The fact that mutant FeS3 was processed normally but degraded rapidly argues against a role for NH2-terminal processing in controlling degradation rates.     

A mutation in the rnh-locus of Escherichia coli affects the structural gene for RNase H. Examination of the mutant and wild type protein

Ribonuclease H (RNase H, EC 3.1.26.4) was purified to homogeneity from Escherichia coli wild type strain KS 351 and the RNase H mutant strain FB 2. The specific activity of the wild type enzyme was 43,200 units/mg, while that of the mutant enzyme was 3,430 units/mg, less than 8% of the wild type activity. Isoelectric focusing also revealed differences in the protein from mutant and wild type. The activity of the wild type enzyme was separated into two peaks with isoelectric points of 9.6 and 9.0. In contrast, the activity of the mutant enzyme focused in a single peak with a pI of 9.4. These results indicate that the mutation in the FB2 strain affects the structural gene for RNase H. The molecular weight of both enzymes was determined by gel filtration as well as NaDodSO4-polyacrylamide gel electrophoresis and found to be identical. Both enzymes are very sensitive to increased temperatures and show indistinguishable rates of inactivation. The basis for the heterogeneity of the isoelectric point and the altered activity of the mutant enzyme is still unknown.     

Susceptibility to hydrogen peroxide and catalase activity of root nodule bacteria

The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H2O2) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H2O2 resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H2O2. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H2O2. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested.