scholarly journals Periplasmic nitrate reduction in Wolinella succinogenes: cytoplasmic NapF facilitates NapA maturation and requires the menaquinol dehydrogenase NapH for membrane attachment

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2784-2794 ◽  
Author(s):  
Melanie Kern ◽  
Jörg Simon
Keyword(s):  
Electron Transport ◽  
Nitrate Reductase ◽  
Deletion Mutant ◽  
Electron Flow ◽  
Gene Clusters ◽  
Nitrate Respiration ◽  
Wolinella Succinogenes ◽  
Periplasmic Nitrate Reductase ◽  
Iron Sulfur ◽  
Membrane Bound

Various nitrate-reducing bacteria produce proteins of the periplasmic nitrate reductase (Nap) system to catalyse electron transport from the membraneous quinol pool to the periplasmic nitrate reductase NapA. The composition of the corresponding nap gene clusters varies but, in addition to napA, genes encoding at least one membrane-bound quinol dehydrogenase module (NapC and/or NapGH) are regularly present. Moreover, some nap loci predict accessory proteins such as the iron–sulfur protein NapF, whose function is poorly understood. Here, the role of NapF in nitrate respiration of the Epsilonproteobacterium Wolinella succinogenes was examined. Immunoblot analysis showed that NapF is located in the membrane fraction in nitrate-grown wild-type cells whereas it was found to be a soluble cytoplasmic protein in a napH deletion mutant. This finding indicates the formation of a membrane-bound NapGHF complex that is likely to catalyse NapH-dependent menaquinol oxidation and electron transport to the iron–sulfur adaptor proteins NapG and NapF, which are located on the periplasmic and cytoplasmic side of the membrane, respectively. The cysteine residues of a CX3CP motif and of the C-terminal tetra-cysteine cluster of NapH were found to be required for interaction with NapF. A napF deletion mutant accumulated the catalytically inactive cytoplasmic NapA precursor, suggesting that electron flow or direct interaction between NapF and NapA facilitated NapA assembly and/or export. On the other hand, NapA maturation and activity was not impaired in the absence of NapH, demonstrating that soluble NapF is functional. Each of the four tetra-cysteine motifs of NapF was modified but only one motif was found to be essential for efficient NapA maturation. It is concluded that the NapGHF complex plays a multifunctional role in menaquinol oxidation, electron transfer to periplasmic NapA and maturation of the cytoplasmic NapA precursor.

scholarly journals NapF Is a Cytoplasmic Iron-Sulfur Protein Required for Fe-S Cluster Assembly in the Periplasmic Nitrate Reductase

2004 ◽  
Vol 279 (48) ◽  
pp. 49727-49735 ◽  
Author(s):  
M. Francisca Olmo-Mira ◽  
Mónica Gavira ◽  
David J. Richardson ◽  
Francisco Castillo ◽  
Conrado Moreno-Vivián ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Cluster Assembly ◽  
Periplasmic Nitrate Reductase ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Iron Sulfur Protein

scholarly journals Purification of Helicobacter pylori NCTC 11637 Cytochrome bc1 and Respiration with d-Proline as a Substrate

2009 ◽  
Vol 192 (5) ◽  
pp. 1410-1415 ◽  
Author(s):  
Minoru Tanigawa ◽  
Tomomitsu Shinohara ◽  
Katsushi Nishimura ◽  
Kumiko Nagata ◽  
Morio Ishizuka ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Amino Acid ◽  
Electron Transport ◽  
Electron Transport Chain ◽  
Electron Flow ◽  
Gene Clusters ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
Transport Chain ◽  
H Pylori

ABSTRACT Helicobacter pylori is a microaerophilic bacterium associated with gastric inflammation and peptic ulcers. Knowledge of how pathogenic organisms produce energy is important from a therapeutic point of view. We found d-amino acid dehydrogenase-mediated electron transport from d-proline or d-alanine to oxygen via the respiratory chain in H. pylori. Coupling of the electron transport to ATP synthesis was confirmed by using uncoupler reagents. We reconstituted the electron transport chain to demonstrate the electron flow from the d-amino acids to oxygen using the recombinant cytochrome bc 1 complex, cytochrome c-553, and the terminal oxidase cytochrome cbb 3 complex. Upon addition of the recombinant d-amino acid dehydrogenase and d-proline or d-alanine to the reconstituted electron transport system, reduction of cytochrome cbb 3 and oxygen consumption was revealed spectrophotometrically and polarographically, respectively. Among the constituents of H. pylori's electron transport chain, only the cytochrome bc 1 complex had been remained unpurified. Therefore, we cloned and sequenced the H. pylori NCTC 11637 cytochrome bc 1 gene clusters encoding Rieske Fe-S protein, cytochrome b, and cytochrome c 1, with calculated molecular masses of 18 kDa, 47 kDa, and 32 kDa, respectively, and purified the recombinant monomeric protein complex with a molecular mass of 110 kDa by gel filtration. The absorption spectrum of the recombinant cytochrome bc 1 complex showed an α peak at 561 nm with a shoulder at 552 nm.

The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3395-3403 ◽  
Author(s):  
María J. Delgado ◽  
Nathalie Bonnard ◽  
Alvaro Tresierra-Ayala ◽  
Eulogio J. Bedmar ◽  
Peter Müller
Keyword(s):  
Nitrate Reductase ◽  
Bradyrhizobium Japonicum ◽  
Nitrate Reductase Activity ◽  
Transmembrane Protein ◽  
Reductase Activity ◽  
Protein Band ◽  
Anaerobic Growth ◽  
Nitrate Respiration ◽  
Western Blots ◽  
Periplasmic Nitrate Reductase

The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66·5 bp downstream of the centre of a putative FNR-like binding site.

scholarly journals The Periplasmic Nitrate Reductase inPseudomonas sp. Strain G-179 Catalyzes the First Step of Denitrification

1999 ◽  
Vol 181 (9) ◽  
pp. 2802-2806 ◽  
Author(s):  
Laura Bedzyk ◽  
Tao Wang ◽  
Rick W. Ye
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Primary Role ◽  
Anaerobic Conditions ◽  
It Use ◽  
Alternative Substrate ◽  
Periplasmic Nitrate Reductase ◽  
Reduction Activity ◽  
Membrane Bound

ABSTRACT Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase inPseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap− mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.

scholarly journals Catabolite Repression Control of napF (Periplasmic Nitrate Reductase) Operon Expression in Escherichia coli K-12

2008 ◽  
Vol 191 (3) ◽  
pp. 996-1005 ◽  
Author(s):  
Valley Stewart ◽  
Peggy J. Bledsoe ◽  
Li-Ling Chen ◽  
Amie Cai
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Nitrate Reductase ◽  
Catabolite Repression ◽  
Carbon Sources ◽  
Anaerobic Growth ◽  
Periplasmic Nitrate Reductase ◽  
Membrane Bound ◽  
Operon Expression ◽  
K 12

ABSTRACT Escherichia coli, a facultative aerobe, expresses two distinct respiratory nitrate reductases. The periplasmic NapABC enzyme likely functions during growth in nitrate-limited environments, whereas the membrane-bound NarGHI enzyme functions during growth in nitrate-rich environments. Maximal expression of the napFDAGHBC operon encoding periplasmic nitrate reductase results from synergistic transcription activation by the Fnr and phospho-NarP proteins, acting in response to anaerobiosis and nitrate or nitrite, respectively. Here, we report that, during anaerobic growth with no added nitrate, less-preferred carbon sources stimulated napF operon expression by as much as fourfold relative to glucose. Deletion analysis identified a cyclic AMP receptor protein (Crp) binding site upstream of the NarP and Fnr sites as being required for this stimulation. The napD and nrfA operon control regions from Shewanella spp. also have apparent Crp and Fnr sites, and expression from the Shewanella oneidensis nrfA control region cloned in E. coli was subject to catabolite repression. In contrast, the carbon source had relatively little effect on expression of the narGHJI operon encoding membrane-bound nitrate reductase under any growth condition tested. Carbon source oxidation state had no influence on synthesis of either nitrate reductase. The results suggest that the Fnr and Crp proteins may act synergistically to enhance NapABC synthesis during growth with poor carbon sources to help obtain energy from low levels of nitrate.

scholarly journals The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha

1995 ◽  
Vol 309 (3) ◽  
pp. 983-992 ◽  
Author(s):  
B C Berks ◽  
D J Richardson ◽  
A Reilly ◽  
A C Willis ◽  
S J Ferguson
Keyword(s):  
Nitrate Reductase ◽  
Sequence Similarity ◽  
Alcaligenes Eutrophus ◽  
Integral Membrane Protein ◽  
Periplasmic Nitrate Reductase ◽  
Thiosphaera Pantotropha ◽  
Cysteine Motif ◽  
Membrane Bound ◽  
K 12 ◽  
Nitrate Reductases

The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced. The large and small subunits of the enzyme are coded by napA and napB. The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor. Cysteine-181 is proposed to ligate the molybdenum atom. It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation. napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family. NapC may be the direct electron donor to the NapAB complex. napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.

scholarly journals Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans, Hyphomicrobium denitrificans, and Hyphomicrobium zavarzinii

2015 ◽  
Vol 81 (15) ◽  
pp. 5003-5014 ◽  
Author(s):  
Christine Martineau ◽  
Florian Mauffrey ◽  
Richard Villemur
Keyword(s):  
Gene Expression ◽  
Nitrate Reductase ◽  
Gene Clusters ◽  
Whole Genome Sequence ◽  
Nitrite Accumulation ◽  
Content Type ◽  
Denitrification Gene ◽  
Pure Cultures ◽  
Genetic Level ◽  
Membrane Bound

ABSTRACTHyphomicrobiumspp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifyingHyphomicrobiumspecies are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifyingHyphomicrobiumspecies,H. denitrificansATCC 51888,H. zavarziniiZV622, and a newly described species,H. nitrativoransNL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) inH. nitrativorans, a membrane-bound nitrate reductase (Nar) inH. denitrificans, and one Nap and two Nar enzymes inH. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of theseHyphomicrobiumspecies were determined.H. nitrativoransgrew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted forH. nitrativoransat higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in bothH. nitrativoransandH. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential ofH. nitrativoransas an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.

scholarly journals Restoration of respiratory electron-transport reactions in quinone-depleted particle preparations from Anacystis nidulans

1980 ◽  
Vol 186 (2) ◽  
pp. 515-523 ◽  
Author(s):  
G A Peschek
Keyword(s):  
Steady State ◽  
Electron Transport ◽  
Electron Flow ◽  
Anacystis Nidulans ◽  
Isolation And Identification ◽  
Respiratory Quinone ◽  
State Reduction ◽  
Membrane Bound ◽  
Ubiquinone 10 ◽  
Respiratory Electron Transport

Electron transport from H2, NADPH, NADH and succinate to O2 or ferricytochrome c in respiratory particles isolated from Anacystis nidulans in which hydrogenase had been induced was abolished after extraction of the membranes with n-pentane; oxidation of ascorbate plus NNN‘N’-tetramethyl-p-phenylenediamine remained unaffected. Incorporation of authentic ubiquinone-10, plastoquinone-9, menaquinone-7 and phylloquinone (in order of increasing efficiency) restored the electron-transport reactions. ATP-dependent reversed electron flow from NNN‘N’-tetramethyl-p-phenylenediamine to NADP+ or, via the membrane-bound hydrogenase, to H+ was likewise abolished by pentane extraction and restored by incorporation of phylloquinone. Participation of the incorporated quinones in the respiratory electron-transport reactions of reconstituted particles was confirmed by measuring the degree of steady-state reduction of the quinones. Isolation and identification of the quinones present in native Anacystis membranes yielded mainly plastoquinone-9 and phylloquinone; neither menaquinone nor alpha-tocopherolquinone could be detected. Together with the results from reconstitution experiments this suggests that phylloquinone might function as the main respiratory quinone in Anacystis nidulans.

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