Catalase activity in Campylobacter jejuni: comparison of a wild-type strain with an aerotolerant variant

1990 ◽  
Vol 36 (6) ◽  
pp. 449-451 ◽  
Author(s):  
Pamela A. Vercellone ◽  
Robert M. Smibert ◽  
Noel R. Krieg
Keyword(s):  
Campylobacter Jejuni ◽  
Catalase Activity ◽  
Specific Activity ◽  
Wild Type ◽  
Oxygen Tolerance ◽  
Variant Strain ◽  
Cultural Conditions ◽  
Cell Extracts ◽  
Bactericidal Effects

A comparison of Campylobacter jejuni VPI strain H840 (ATCC 29428), which can grow at O2 levels up to 15%, with variant strain MC711-01 (which can grown at O2 levels up to 21–26%) indicated that the specific activity of catalase in crude cell extracts was higher in the variant by a factor of 1.6 to 2.5, depending on cultural conditions. Smaller differences occurred with superoxide dismutase activity, while peroxidase activities were invariably lower in the variant strain. The variant strain was much more resistant than the wild type to the bactericidal effects of H2O2. The results suggest that catalase activity might be one of the factors associated with the greater tolerance of O2 by the variant strain. However, both strains became more susceptible to H2O2 when cultures were initially grown at 6% O2 and then shifted to 21% O2; thus the role of catalase in the oxygen tolerance of C. jejuni is probably minor. Key words: Campylobacter jejuni, catalase, oxygen tolerance.

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

2001 ◽  
Vol 47 (3) ◽  
pp. 222-228 ◽  
Author(s):  
Anne J Anderson ◽  
Charles D Miller
Keyword(s):  
Hydrogen Peroxide ◽  
Pseudomonas Putida ◽  
Peracetic Acid ◽  
Catalase Activity ◽  
Specific Activity ◽  
Active Oxygen Species ◽  
Oxygen Species ◽  
Deficient Mutant ◽  
Wild Type ◽  
Cell Extracts

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.Key words: Pseudomonas putida (Pp), activated oxygen species (AOS), hydrogen peroxide, luciferase, colonization.

scholarly journals Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

2004 ◽  
Vol 384 (3) ◽  
pp. 507-513 ◽  
Author(s):  
Salvador HERRERO ◽  
Joel GONZÁLEZ-CABRERA ◽  
Juan FERRÉ ◽  
Petra L. BAKKER ◽  
Ruud A. de MAAGD
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Exigua ◽  
Brush Border Membrane ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Oligomeric Form ◽  
Specific Receptors ◽  
Domain Iii

Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541–544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins.

scholarly journals The role of the 5′-flanking sequence of a human tRNAGlu gene in modulation of its transcriptional activity in vitro

1990 ◽  
Vol 272 (3) ◽  
pp. 797-803 ◽  
Author(s):  
E S Gonos ◽  
J P Goddard
Keyword(s):  
Transcriptional Activity ◽  
Potential Model ◽  
Trna Gene ◽  
Deletion Mutants ◽  
Wild Type ◽  
Flanking Sequence ◽  
Cell Extracts ◽  
Transcription In Vitro

The role of a tRNA-like structure within the 5′-flanking sequence of a human tRNA(Glu) gene in the modulation of its transcription in vitro by HeLa cell extracts has been investigated using several deletion mutants of a recombinant of the gene which lacked part or all of the tRNA-like structure. The transcriptional efficiency of four mutants was the same as that of the wild-type recombinant, two mutants had decreased transcriptional efficiency, one was more efficient, and one, lacking part of the 5′ intragenic control region, was inactive. Correlation of the transcriptional efficiencies with the position and the size of the 5′-flanking sequence that was deleted indicated that the tRNA-like structure may be deleted without loss of transcriptional efficiency. Current models for the modulation of tRNA gene transcription by the 5′-flanking sequence are assessed in the light of the results obtained, and a potential model is presented.

scholarly journals Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin

1993 ◽  
Vol 293 (1) ◽  
pp. 181-185 ◽  
Author(s):  
N J Watkins ◽  
A K Campbell
Keyword(s):  
Light Emission ◽  
Specific Activity ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Proline Residue ◽  
Long Term Stability ◽  
Recombinant Aequorin

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

The role of distal tryptophan in the bifunctional activity of catalase-peroxidases

2001 ◽  
Vol 29 (2) ◽  
pp. 99-105 ◽  
Author(s):  
G. Regelsberger ◽  
C. Jakopitsch ◽  
P. G. Furtmüller ◽  
F. Rueker ◽  
J. Switala ◽  
...  
Keyword(s):  
Catalase Activity ◽  
Intermediate Compound ◽  
Stopped Flow ◽  
Wild Type ◽  
Compound I ◽  
Wild Type Enzyme ◽  
Catalase Peroxidase ◽  
Electron Reduction ◽  
The One

Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate compound I by probing the role of the conserved tryptophan at the distal haem cavity site. Two wild-type proteins and three mutants of Synechocystis catalase-peroxidase (W122A and W122F) and Escherichia coli catalase-peroxidase (W105F) have been investigated by steady-state and stopped-flow spectroscopy. W122F and W122A completely lost their catalase activity whereas in W105F the catalase activity was reduced by a factor of about 5000. However, the mutations did not influence both formation of compound I and its reduction by peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceeded that of the wild-type enzyme. This study demonstrates that the indole ring of distal Trp in catalase-peroxidases is essential for the two-electron reduction of compound I by hydrogen peroxide but not for compound I formation or for peroxidase reactivity (i.e. the one-electron reduction of compound I).

scholarly journals A Helicobacter hepaticus catalase mutant is hypersensitive to oxidative stress and suffers increased DNA damage

2007 ◽  
Vol 56 (4) ◽  
pp. 557-562 ◽  
Author(s):  
Yang Hong ◽  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Catalase Activity ◽  
Insertional Mutagenesis ◽  
Specific Activity ◽  
Bacterial Species ◽  
Helicobacter Hepaticus ◽  
Wild Type ◽  
Crude Cell Extract ◽  
H Pylori

Catalase (KatA) is known to play an important role in oxidative stress resistance in many bacterial species and a homologue exists in Helicobacter hepaticus, a member of the enterohepatic Helicobacter species. Here, a katA mutant was constructed by insertional mutagenesis and its oxidative stress phenotype was investigated. Catalase activity was readily detected [196 units (mg protein crude cell extract)−1] in the wild-type, whereas the mutant strain was deficient in, but not devoid of, activity. In contrast, Helicobacter pylori katA strains lack detectable catalase activity and wild-type H. pylori generally contains higher specific activity than H. hepaticus. Wild-type H. hepaticus cells tolerated 6 % O2 for growth, whilst the katA mutant could not survive at this oxygen level. Even at the optimal O2 level, the growth of the H. hepaticus katA strain was severely inhibited, which is also in contrast to H. pylori katA strains. Wild-type H. hepaticus cells withstood exposure to 100 mM H2O2 but the katA mutant cells were killed by the same treatment. Wild-type cells suffered no significant DNA damage by H2O2 treatment (100 mM for 6 min), whilst the same treatment resulted in severe DNA fragmentation in the katA mutant. Thus H. hepaticus KatA plays an important role as an antioxidant protein.

scholarly journals Long-Term Survival of Campylobacter jejuni at Low Temperatures Is Dependent on Polynucleotide Phosphorylase Activity

2009 ◽  
Vol 75 (23) ◽  
pp. 7310-7318 ◽  
Author(s):  
Nabila Haddad ◽  
Christopher M. Burns ◽  
Jean Michel Bolla ◽  
Hervé Prévost ◽  
Michel Fédérighi ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Low Temperatures ◽  
Survival Rates ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
Term Survival ◽  
Poultry Products ◽  
Bacterial Gastroenteritis

ABSTRACT Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Infection generally occurs after ingestion of contaminated poultry products, usually conserved at low temperatures. The mechanisms promoting survival of C. jejuni in the cold remain poorly understood despite several investigations. The present study provides insight into the survival mechanism by establishing the involvement of polynucleotide phosphorylase (PNPase), a 3′-5′ exoribonuclease with multiple biological functions in cold survival. The role of PNPase was demonstrated genetically using strains with altered pnp genes (which encode PNPase) created in C. jejuni F38011 and C. jejuni 81-76 backgrounds. Survival assays carried out at low temperatures (4 and 10°C) revealed a difference of 3 log CFU/ml between the wild-type and the pnp deletion (Δpnp) strains. This did not result from a general requirement for PNPase because survival rates of the strains were similar at higher growth temperatures (37 or 42°C). trans-Complementation with plasmid pNH04 carrying the pnp gene under the control of its natural promoter restored the cold survival phenotype to the pnp deletion strains (at 4 and 10°C) but not to the same level as the wild type. In this study we demonstrate the role of PNPase in low-temperature survival of C. jejuni and therefore attribute a novel biological function to PNPase directly related to human health.

scholarly journals KatG Is the Primary Detoxifier of Hydrogen Peroxide Produced by Aerobic Metabolism in Bradyrhizobium japonicum

2004 ◽  
Vol 186 (23) ◽  
pp. 7874-7880 ◽  
Author(s):  
Heather R. Panek ◽  
Mark R. O'Brian
Keyword(s):  
Catalase Activity ◽  
Bradyrhizobium Japonicum ◽  
Aerobic Metabolism ◽  
Short Exposure ◽  
Wild Type ◽  
Gene Encoding ◽  
Whole Cells ◽  
Cell Extracts ◽  
Genes Encoding ◽  
High Concentrations

ABSTRACT Bacteria are exposed to reactive oxygen species from the environment and from those generated by aerobic metabolism. Catalases are heme proteins that detoxify H2O2, and many bacteria contain more than one catalase enzyme. Also, the nonheme peroxidase alkyl hydroperoxide reductase (Ahp) is the major scavenger of endogenous H2O2 in Escherichia coli. Here, we show that aerobically grown Bradyrhizobium japonicum cells express a single catalase activity. Four genes encoding putative catalases in the B. japonicum genome were identified, including a katG homolog encoding a catalase-peroxidase. Deletion of the katG gene resulted in loss of catalase activity in cell extracts and of exogenous H2O2 consumption by whole cells. The katG strain had a severe aerobic growth phenotype but showed improved growth in the absence of O2. By contrast, a B. japonicum ahpCD mutant grew well aerobically and consumed H2O2 at wild-type rates. A heme-deficient hemA mutant expressed about one-third of the KatG activity as the wild type but grew well aerobically and scavenged low concentrations of exogenous H2O2. However, cells of the hemA strain were deficient in consumption of high concentrations of H2O2 and were very sensitive to killing by short exposure to H2O2. In addition, KatG activity did not decrease as a result of mutation of the gene encoding the transcriptional activator OxyR. We conclude that aerobic metabolism produces toxic levels of H2O2 in B. japonicum, which is detoxified primarily by KatG. Furthermore, the katG level sufficient for detoxification does not require OxyR.

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

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

1999 ◽  
Vol 45 (2) ◽  
pp. 125-129 ◽  
Author(s):  
Norihiro Mutoh ◽  
Chiaki W Nakagawa ◽  
Kenichiro Yamada
Keyword(s):  
Hydrogen Peroxide ◽  
Schizosaccharomyces Pombe ◽  
Catalase Activity ◽  
Parent Strain ◽  
Normal Growth ◽  
Wild Type ◽  
Catalase Gene ◽  
In The Wild ◽  
High Concentrations

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 wild-type 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.Key words: catalase, gene disruption, induced hydrogen peroxide resistance, overexpression, Schizosaccharomyces pombe.

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