O2 and Reactive Oxygen Species Detoxification Complex, Composed of O2-Responsive NADH:Rubredoxin Oxidoreductase-Flavoprotein A2-Desulfoferrodoxin Operon Enzymes, Rubperoxin, and Rubredoxin, in Clostridium acetobutylicum

ABSTRACT Clostridium acetobutylicum, an obligate anaerobe, grows normally under continuous-O2-flow culture conditions, where the cells consume O2 proficiently. An O2-responsive NADH:rubredoxin oxidoreductase operon composed of three genes (nror, fprA2, and dsr), encoding NROR, functionally uncharacterized flavoprotein A2 (FprA2), and the predicted superoxide reductase desulfoferrodoxin (Dsr), has been proposed to participate in defense against O2 stress. To functionally characterize these proteins, native NROR from C. acetobutylicum, recombinant NROR (rNROR), FprA2, Dsr, and rubredoxin (Rd) expressed in Escherichia coli were purified. Purified native NROR and rNROR both exhibited weak H2O2-forming NADH oxidase activity that was slightly activated by Rd. A mixture of NROR, Rd, and FprA2 functions as an efficient H2O-forming NADH oxidase with a high affinity for O2 (the Km for O2 is 2.9 ï¿½ 0.4 μM). A mixture of NROR, Rd, and Dsr functions as an NADH-dependent O2 − reductase. A mixture of NROR, Rd, and rubperoxin (Rpr, a rubrerythrin homologue) functions as an inefficient H2O-forming NADH oxidase but an efficient NADH peroxidase with a low affinity for O2 and a high affinity for H2O2 (the Km s for O2 and H2O2 are 303 ï¿½ 39 μM and ≤1 μM, respectively). A gene encoding Rd is dicistronically transcribed with a gene encoding a glutaredoxin (Gd) homologue, and the expression levels of the genes encoding Gd and Rd were highly upregulated upon exposure to O2. Therefore, nror operon enzymes, together with Rpr, efficiently function to scavenge O2, O2 −, and H2O2 by using an O2-responsive rubredoxin as a common electron carrier protein.

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Pathway for H2O2 and O2 detoxification in Clostridium acetobutylicum

Microbiology ◽

10.1099/mic.0.022756-0 ◽

2009 ◽

Vol 155 (1) ◽

pp. 16-24 ◽

Cited By ~ 66

Author(s):

Oliver Riebe ◽

Ralf-Jörg Fischer ◽

David A. Wampler ◽

Donald M. Kurtz ◽

Hubert Bahl

Keyword(s):

Oxidative Stress ◽

Electron Transfer ◽

Clostridium Acetobutylicum ◽

Consumption Rate ◽

Nadh Oxidase ◽

Strictly Anaerobic ◽

Nadh Peroxidase ◽

Overexpressing Strain

An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upregulated in response to oxidative stress in the strictly anaerobic bacterium Clostridium acetobutylicum. In the present study both in vivo and in vitro results demonstrate an H2O2 and O2 detoxification pathway in C. acetobutylicum involving revRbr, rubredoxin (Rd) and NADH : rubredoxin oxidoreductase (NROR). RevRbr exhibited both NADH peroxidase (NADH : H2O2 oxidoreductase) and NADH oxidase (NADH : O2 oxidoreductase) activities in in vitro assays using NROR as the electron-transfer intermediary from NADH to revRbr. Rd increased the NADH consumption rate by serving as an intermediary electron-transfer shuttle between NROR and revRbr. While H2O2 was found to be the preferred substrate for revRbr, its relative oxidase activity was found to be significantly higher than that reported for other Rbrs. A revRbr-overexpressing strain of C. acetobutylicum showed significantly increased tolerance to H2O2 and O2 exposure. RevRbr thus appears to protect C. acetobutylicum against oxidative stress by functioning as the terminal component of an NADH peroxidase and NADH oxidase.

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Biochemical evidence for the reversed polarity of the outer membrane of the bacterial forespore

Biochemical Journal ◽

10.1042/bj1520561 ◽

1975 ◽

Vol 152 (3) ◽

pp. 561-569 ◽

Cited By ~ 20

Author(s):

B J Wilkinson ◽

J A Deans ◽

D J Ellar

Keyword(s):

Outer Membrane ◽

Nadh Dehydrogenase ◽

Nadh Oxidase ◽

Permeability Barrier ◽

Cell Plasma Membrane ◽

High Affinity ◽

Cell Plasma ◽

Membrane Bound ◽

Reversed Polarity ◽

Nadh Oxidase Activity

Measurement of certain membrane-bound enzymic activities was used to study the orientation of the outer membrane of the double-membraned forespore of Bacillus megaterium KM. 2. Adenosine triphosphatase, NADH dehydrogenase and L-malate intact protoplasts, but were readily detected in intact stage II or IV forespores, consistent with reversed polarity of the outer forespore membrane relative to the mother-cell plasma membrane. 3. Measurement of NADH oxidase activity revealed that intact stage III forespores had the same high affinity for NADH as protoplast membrane preparations and protoplast lystates, consistent with ready access of NADH to oxidation sites on the outer forespores membrane. 4. Forespores and protoplasts showed osmometric behaviour in solutions of non-permanent solutes consistent with the presence of an intact permeability barrier in these structures.

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Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.01026-10 ◽

2010 ◽

Vol 76 (19) ◽

pp. 6351-6359 ◽

Cited By ~ 7

Author(s):

Toshihiko Kitajima ◽

Yasunori Chiba ◽

Yoshifumi Jigami

Keyword(s):

Saccharomyces Cerevisiae ◽

Methylotrophic Yeast ◽

Culture Conditions ◽

Crystallographic Analysis ◽

Gene Encoding ◽

X Ray ◽

X Ray Crystallography ◽

High Affinity ◽

Standard Culture ◽

Resistant Mutants

ABSTRACT The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

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Skeletal muscle sarcoplasmic reticulum contains a NADH-dependent oxidase that generates superoxide

AJP Cell Physiology ◽

10.1152/ajpcell.00034.2002 ◽

2003 ◽

Vol 285 (1) ◽

pp. C215-C221 ◽

Cited By ~ 90

Author(s):

Ruohong Xia ◽

Jason A. Webb ◽

Lisa L. M. Gnall ◽

Kerry Cutler ◽

Jonathan J. Abramson

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Nadh Oxidase ◽

Release Mechanism ◽

Antimycin A ◽

High Affinity ◽

Diphenylene Iodonium ◽

Nadh Oxidase Activity ◽

Terminal Cisternae ◽

Stimulation Of

Skeletal muscle sarcoplasmic reticulum (SR) is shown to contain an NADH-dependent oxidase (NOX) that reduces molecular oxygen to generate superoxide. Its activity is coupled to an activation of the Ca2+ release mechanism, as evident by stimulation in the rate of high-affinity ryanodine binding. NOX activity, coupled to the production of superoxide, is not derived from the mitochondria but is SR in origin. The SR preparation also contains a significant NADH oxidase activity, which is not coupled to the production of superoxide and appears to be mitochondrial in origin. This mitochondrial component is preferentially associated with the terminal cisternae region of the SR. Its activity is inhibited by diphenylene iodonium (10 μM), antimycin A (200 nM), and rotenone (40 nM) but is not coupled to the generation of superoxide or the stimulation of the ryanodine receptor. The rate of superoxide production per milligram of protein is larger in SR than in mitochondria. This NOX may be a major source of oxidative stress in muscle.

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The Role of PerR in O2-Affected Gene Expression of Clostridium acetobutylicum

Journal of Bacteriology ◽

10.1128/jb.00351-09 ◽

2009 ◽

Vol 191 (19) ◽

pp. 6082-6093 ◽

Cited By ~ 42

Author(s):

Falk Hillmann ◽

Christina Döring ◽

Oliver Riebe ◽

Armin Ehrenreich ◽

Ralf-Jörg Fischer ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Clostridium Acetobutylicum ◽

Wild Type ◽

Gene Encoding ◽

Homologous Protein ◽

Strict Anaerobe ◽

Genes Encoding ◽

Highly Expressed Genes

ABSTRACT In the strict anaerobe Clostridium acetobutylicum, a PerR-homologous protein has recently been identified as being a key repressor of a reductive machinery for the scavenging of reactive oxygen species and molecular O2. In the absence of PerR, the full derepression of its regulon resulted in increased resistance to oxidative stress and nearly full tolerance of an aerobic environment. In the present study, the complementation of a Bacillus subtilis PerR mutant confirmed that the homologous protein from C. acetobutylicum acts as a functional peroxide sensor in vivo. Furthermore, we used a transcriptomic approach to analyze gene expression in the aerotolerant PerR mutant strain and compared it to the O2 stimulon of wild-type C. acetobutylicum. The genes encoding the components of the alternative detoxification system were PerR regulated. Only few other targets of direct PerR regulation were identified, including two highly expressed genes encoding enzymes that are putatively involved in the central energy metabolism. All of them were highly induced when wild-type cells were exposed to sublethal levels of O2. Under these conditions, C. acetobutylicum also activated the repair and biogenesis of DNA and Fe-S clusters as well as the transcription of a gene encoding an unknown CO dehydrogenase-like enzyme. Surprisingly few genes were downregulated when exposed to O2, including those involved in butyrate formation. In summary, these results show that the defense of this strict anaerobe against oxidative stress is robust and by far not limited to the removal of O2 and its reactive derivatives.

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Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

Scientific Reports ◽

10.1038/s41598-020-80125-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

José Francisco Cruz-Pérez ◽

Roxana Lara-Oueilhe ◽

Cynthia Marcos-Jiménez ◽

Ricardo Cuatlayotl-Olarte ◽

María Luisa Xiqui-Vázquez ◽

...

Keyword(s):

Azospirillum Brasilense ◽

Type Strain ◽

Wild Type Strain ◽

Soil Conditions ◽

Wild Type ◽

Gene Encoding ◽

Wheat Roots ◽

Genes Encoding ◽

In The Wild ◽

Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

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Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

Genetics ◽

10.1093/genetics/133.4.999 ◽

1993 ◽

Vol 133 (4) ◽

pp. 999-1007

Author(s):

R G Gregerson ◽

L Cameron ◽

M McLean ◽

P Dennis ◽

J Strommer

Keyword(s):

Chromosomal Location ◽

Higher Plants ◽

Gene Encoding ◽

Genomic Libraries ◽

Regulatory Strategy ◽

Adh1 Gene ◽

Adh Genes ◽

Genes Encoding ◽

Adh Gene ◽

Polymerase Chain

Abstract In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

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