Characterization of the norB Gene, Encoding Nitric Oxide Reductase, in the Nondenitrifying Cyanobacterium Synechocystis sp. Strain PCC6803

ABSTRACT A norB gene encoding a putative nitric oxide reductase is present in the genome of the nondenitrifying cyanobacterium Synechocystis sp. strain PCC6803. The gene product belongs to the quinol-oxidizing single-subunit class of nitric oxide reductases, discovered recently in the denitrifier Ralstonia eutropha. Heterologous complementation of a nitric oxide reductase-negative mutant of R. eutropha with norB from Synechocystis restored nitric oxide reductase activity. With reduced menadione as the electron donor, an enzymatic activity of 101 nmol of NO per min per mg of protein was obtained with membrane fractions of Synechocystis wild-type cells. Virtually no nitric oxide reductase activity was present in a norB-negative mutant of Synechocystis. Growing cells of this mutant are more sensitive toward NO than wild-type cells, indicating that the presence of a nitric oxide reductase is beneficial for Synechocystis when the cells are exposed to NO. Transcriptional fusions with the chloramphenicol acetyltransferase reporter gene were constructed to monitor norB expression in Synechocystis. Transcription of norB was not enhanced by the addition of the NO-generating agent sodium nitroprusside.

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Gonococcal Nitric Oxide Reductase Is Encoded by a Single Gene, norB, Which Is Required for Anaerobic Growth and Is Induced by Nitric Oxide

Infection and Immunity ◽

10.1128/iai.68.9.5241-5246.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 5241-5246 ◽

Cited By ~ 62

Author(s):

Tracey C. Householder ◽

Elizabeth M. Fozo ◽

Jean A. Cardinale ◽

Virginia L. Clark

Keyword(s):

Nitric Oxide ◽

Ralstonia Eutropha ◽

Single Gene ◽

Nitrite Reduction ◽

Anaerobic Growth ◽

Nitric Oxide Donor ◽

Nitric Oxide Reductase ◽

Wild Type ◽

Gene Encoding ◽

Anaerobic Environment

ABSTRACT The gene encoding a nitric oxide reductase has been identified inNeisseria gonorrhoeae. The norB gene product shares significant identity with the nitric oxide reductases inRalstonia eutropha and Synechocystis sp. and, like those organisms, the gonococcus lacks a norC homolog. The gonococcal norB gene was found to be required for anaerobic growth, but the absence of norB did not dramatically decrease anaerobic survival. In a wild-type background, induction of norB expression was seen anaerobically in the presence of nitrite but not anaerobically without nitrite or aerobically. norB expression is not regulated by FNR or NarP, but a functional aniA gene (which encodes an anaerobically induced outer membrane nitrite reductase) is necessary for expression. When aniA is constitutively expressed,norB expression can be induced both anaerobically and aerobically, but only in the presence of nitrite, suggesting that nitric oxide, which is likely to be produced by AniA as a product of nitrite reduction, is the inducing agent. This was confirmed with the use of the nitric oxide donor, spermine-nitric oxide complex, in ananiA null background both anaerobically and aerobically. NorB is important for gonococcal adaptation to an anaerobic environment, a physiologically relevant state during gonococcal infection. The presence of this enzyme, which is induced by nitric oxide, may also have implications in immune evasion and immunomodulation in the human host.

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Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

Biochemistry and Cell Biology ◽

10.1139/o90-153 ◽

1990 ◽

Vol 68 (7-8) ◽

pp. 1037-1044 ◽

Cited By ~ 14

Author(s):

Peter C. Loewen ◽

Jacek Switala ◽

Mark Smolenski ◽

Barbara L. Triggs-Raine

Keyword(s):

Escherichia Coli ◽

Specific Activity ◽

Polymerase Chain Reaction Amplification ◽

Protein A ◽

Wild Type ◽

Wild Type Enzyme ◽

Gene Encoding ◽

Reaction Amplification ◽

Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

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Detergent inhibition of nitric-oxide reductase activity

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(87)90074-4 ◽

1987 ◽

Vol 911 (3) ◽

pp. 334-340 ◽

Cited By ~ 33

Author(s):

J.P. Shapleigh ◽

K.J.P. Davies ◽

W.J. Payne

Keyword(s):

Nitric Oxide ◽

Reductase Activity ◽

Nitric Oxide Reductase

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A DNA Region Recognized by the Nitric Oxide-Responsive Transcriptional Activator NorR Is Conserved in β- and γ-Proteobacteria

Journal of Bacteriology ◽

10.1128/jb.186.23.7980-7987.2004 ◽

2004 ◽

Vol 186 (23) ◽

pp. 7980-7987 ◽

Cited By ~ 20

Author(s):

Andrea Büsch ◽

Anne Pohlmann ◽

Bärbel Friedrich ◽

Rainer Cramm

Keyword(s):

Nitric Oxide ◽

Transcriptional Activation ◽

Target Genes ◽

Ralstonia Eutropha ◽

Upstream Region ◽

Nitric Oxide Reductase ◽

Wild Type ◽

Genome Database ◽

Nitric Oxide Metabolism ◽

Signaling Domain

ABSTRACT The σ54-dependent regulator NorR activates transcription of target genes in response to nitric oxide (NO) or NO-generating agents. In Ralstonia eutropha H16, NorR activates transcription of the dicistronic norAB operon that encodes NorA, a protein of unknown function, and NorB, a nitric oxide reductase. A constitutively activating NorR derivative (NorR′), in which the N-terminal signaling domain was replaced by MalE, specifically bound to the norAB upstream region as revealed by gel retardation analysis. Within a 73-bp DNA segment protected by MalE-NorR′ in a DNase I footprint assay, three conserved inverted repeats, GGT-(N7)-ACC (where N is any base), that we consider to be NorR-binding boxes were identified. Mutations altering the spacing or the base sequence of these repeats resulted in an 80 to 90% decrease of transcriptional activation by wild-type NorR. Genome database analyses demonstrate that the GT-(N7)-AC core of the inverted repeat is found in several proteobacteria upstream of gene loci encoding proteins of nitric oxide metabolism, including nitric oxide reductase (NorB), flavorubredoxin (NorV), NO dioxygenase (Hmp), and hybrid cluster protein (Hcp).

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Kinetic Characterization of the Escherichia coli Nitric Oxide Reductase Flavorubredoxin

Globins and Other Nitric Oxide-Reactive Proteins, Part B - Methods in Enzymology ◽

10.1016/s0076-6879(07)37003-1 ◽

2008 ◽

pp. 47-62 ◽

Cited By ~ 10

Author(s):

João B. Vicente ◽

Francesca M. Scandurra ◽

Elena Forte ◽

Maurizio Brunori ◽

Paolo Sarti ◽

...

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Kinetic Characterization ◽

Nitric Oxide Reductase

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Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis

Journal of Experimental Biology ◽

10.1242/jeb.205.9.1209 ◽

2002 ◽

Vol 205 (9) ◽

pp. 1209-1219 ◽

Cited By ~ 4

Author(s):

Natalie Perzov ◽

Vered Padler-Karavani ◽

Hannah Nelson ◽

Nathan Nelson

Keyword(s):

Sucrose Gradient ◽

Ph Sensor ◽

Wild Type ◽

Gene Encoding ◽

Yeast Saccharomyces Cerevisiae ◽

Vacuolar Acidification ◽

Atpase Subunits ◽

Immunological Assays ◽

Gradient Fractionation

SUMMARYSubunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 ▵ and stv1 ▵mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 ▵ strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 ▵ strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1▵ as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 ▵ strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 ▵ one, markedly slow down endocytosis of the dye.

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