Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves

Planta ◽  
1979 ◽  
Vol 146 (1) ◽  
pp. 83-89 ◽  
Author(s):  
A. F. Mann ◽  
D. P. Hucklesby ◽  
E. J. Hewitt
Keyword(s):  
Nitrate Reductase ◽  
Anaerobic Conditions ◽  
Nitrate Reductase Assay ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions ◽  
Spinach Leaves

Further evidence for the existence of cyclic and non-cyclic photophosphorylation in vivo by means of desaspidin and DCMU

1967 ◽  
Vol 22 (5) ◽  
pp. 537-540 ◽  
Author(s):  
W. Urbach ◽  
W. Simonis
Keyword(s):  
Antimycin A ◽  
Anaerobic Conditions ◽  
Intact Cells ◽  
Cyclic Photophosphorylation ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

The effect of desaspidin and DCMU on photophosphorylation in intact cells under aerobic and anaerobic conditions has been studied. Desaspidin is mainly effective in N2 and inhibits under these conditions the DCMU-insensitive cyclic photophosphorylation in vivo like antimycin A. The inhibition of the phosphorylation in light by DCMU is stronger in N2 than in air which suggests a partial existence of oxydative phosphorylation during illumination.

scholarly journals In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 4063-4070 ◽  
Author(s):  
David P. Dibden ◽  
Jeffrey Green
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Oxygen Availability ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Fnr Protein ◽  
Transcription Regulators ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

FNR proteins are transcription regulators that sense changes in oxygen availability via assembly–disassembly of [4Fe–4S] clusters. The Escherichia coli FNR protein is present in bacteria grown under aerobic and anaerobic conditions. Under aerobic conditions, FNR is isolated as an inactive monomeric apoprotein, whereas under anaerobic conditions, FNR is present as an active dimeric holoprotein containing one [4Fe–4S] cluster per subunit. It has been suggested that the active and inactive forms of FNR are interconverted in vivo, or that iron–sulphur clusters are mostly incorporated into newly synthesized FNR. Here, experiments using a thermo-inducible fnr expression plasmid showed that a model FNR-dependent promoter is activated under anaerobic conditions by FNR that was synthesized under aerobic conditions. Immunoblots suggested that FNR was more prone to degradation under aerobic compared with anaerobic conditions, and that the ClpXP protease contributes to this degradation. Nevertheless, FNR was sufficiently long lived (half-life under aerobic conditions, ∼45 min) to allow cycling between active and inactive forms. Measuring the abundance of the FNR-activated dms transcript when chloramphenicol-treated cultures were switched between aerobic and anaerobic conditions showed that it increased when cultures were switched to anaerobic conditions, and decreased when aerobic conditions were restored. In contrast, measurement of the abundance of the FNR-repressed ndh transcript under the same conditions showed that it decreased upon switching to anaerobic conditions, and then increased when aerobic conditions were restored. The abundance of the FNR- and oxygen-independent tatE transcript was unaffected by changes in oxygen availability. Thus, the simplest explanation for the observations reported here is that the FNR protein can be switched between inactive and active forms in vivo in the absence of de novo protein synthesis.

scholarly journals Kinetic Analysis of the Oxidative Conversion of the [4Fe-4S]2+ Cluster of FNR to a [2Fe-2S]2+ Cluster

2004 ◽  
Vol 186 (23) ◽  
pp. 8018-8025 ◽  
Author(s):  
Victoria R. Sutton ◽  
Erin L. Mettert ◽  
Helmut Beinert ◽  
Patricia J. Kiley
Keyword(s):  
Oxidative Conversion ◽  
Anaerobic Conditions ◽  
Protein Levels ◽  
Detectable Difference ◽  
Fnr Protein ◽  
Sense And Respond ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

ABSTRACT The ability of FNR to sense and respond to cellular O2 levels depends on its [4Fe-4S]2+ cluster. In the presence of O2, the [4Fe-4S]2+ cluster is converted to a [2Fe-2S]2+ cluster, which inactivates FNR as a transcriptional regulator. In this study, we demonstrate that ∼2 Fe2+ ions are released from the reaction of O2 with the [4Fe-4S]2+ cluster. Fe2+ release was then used as an assay of reaction progress to investigate the rate of [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion in vitro. We also found that there was no detectable difference in the rate of O2-induced cluster conversion for FNR free in solution compared to its DNA-bound form. In addition, the rate of FNR inactivation was monitored in vivo by measuring the rate at which transcriptional regulation by FNR is lost upon the exposure of cells to O2; a comparison of the in vitro and in vivo rates of conversion suggests that O2-induced cluster conversion is sufficient to explain FNR inactivation in cells. FNR protein levels were also compared for cells grown under aerobic and anaerobic conditions.

Properties of the in vivo Nitrate Reductase Assay in Maize, Soybean, and Spinach Leaves

1986 ◽  
Vol 124 (1-2) ◽  
pp. 123-130 ◽  
Author(s):  
S.G. Mauriño ◽  
C. Echevarria ◽  
J.A. Mejias ◽  
M.A. Vargas ◽  
J.M. Maldonado
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Assay ◽  
Spinach Leaves

scholarly journals Mycobacterium smegmatis does not display functional redundancy in nitrate reductase enzymes

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245745
Author(s):  
Nicole C. Cardoso ◽  
Andrea O. Papadopoulos ◽  
Bavesh D. Kana
Keyword(s):  
Nitrate Reductase ◽  
Mycobacterium Smegmatis ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
Anaerobic Conditions ◽  
Relative Contribution ◽  
Allelic Replacement ◽  
Encoding Genes ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller, Mycobacterium smegmatis is able to assimilate nitrate and herein we set out to confirm the genetic basis for this by probing NR activity in mutants defective for putative nitrate reductase (NR) encoding genes. In addition to the annotated narB and narGHJI, bioinformatics identified three other putative NR-encoding genes: MSMEG_4206, MSMEG_2237 and MSMEG_6816. To assess the relative contribution of each, the corresponding gene loci were deleted using two-step allelic replacement, individually and in combination. The resulting strains were tested for their ability to assimilate nitrate and reduce nitrate under aerobic and anaerobic conditions, using nitrate assimilation and modified Griess assays. We demonstrated that narB, narGHJI, MSMEG_2237 and MSMEG_6816 were individually dispensable for nitrate assimilation and for nitrate reductase activity under aerobic and anaerobic conditions. Only deletion of MSMEG_4206 resulted in significant reduction in nitrate assimilation under aerobic conditions. These data confirm that in M. smegmatis, narB, narGHJI, MSMEG_2237 and MSMEG_6816 are not required for nitrate reduction as MSMEG_4206 serves as the sole assimilatory NR.

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