Preferences help
enabled [disable] Abstract
Number of results
2008 | 55 | 4 | 761-766
Article title

Nitrate-related down-regulation of respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus)

Title variants
Languages of publication
Previously, we showed that anaerobic induction of respiratory nitrate reductase (NR) activity in Bradyrhizobium sp. (Lupinus) USDA 3045 is strongly enhanced by nitrate or nitrite through de novo synthesis. Here, multiple NR-active soluble forms, ranging from 75 kDa to 190 kDa, were observed under anaerobic conditions. Electrophoretic activity band patterns differed depending on the level and the type of the N oxyanion added. The intensity of the membrane-bound NR activity band of 230 kDa changed with time along with consumption of 2 mM nitrate. It was associated with a parallel 5-fold increase and then 2-fold reduction in the amount of membrane-bound NR protein. In contrast, on 4 mM nitrate, the level of NR protein was much more stable, apparently due to slower nitrate depletion. Moreover, in cells anaerobically grown without nitrate addition, a 42-kDa derivative of NR degradation was immunodetected, which was not observed if nitrate was present in the medium. These findings suggest that the amount of the respiratory NR protein could be negatively regulated by endogenous proteases in relation to the level of nitrate available. It seems, therefore, that multiple native forms might be not different isoenzymes but immature complexes or derivatives of the enzyme protein turnover. This report adds to a modest list of bacterial enzymes apparently regulated by proteolysis, such as GS, MurAA, EnvA, GdhA, and MetA.

Physical description
  • Institute of Experimental Biology, Adam Mickiewicz University, Poznań, Poland
  • Biran D, Gur E, Gollan L, Ron EZ (2000) Control of methionine biosynthesis in Escherichia coli by proteolysis. Mol Microbiol 37: 1436-1443.
  • Blasco F, Dos Santos JP, Magalon A, Frixon C, Guigliarelli B, Santini CL, Giordano G (1998) NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli. Mol Microbiol 28: 435-447.
  • Blümle S, Zumft WG (1991) Respiratory nitrate reductase from denitrifying Pseudomonas stutzeri, purification, properties and target of proteolysis. Biochim Biophys Acta 1057: 102-108.
  • Chamber-Pérez MA, Camacho M, Burgos AR, Mercedes-Lucas M, Fernandez-Pascual M, Manclús JJ, Rosario M (2002) Nitrate reductase isozymes in Bradyrhizobium sp. (Lupinus) bacteroids: localisation, biochemical and kinetic characteristics. J Plant Physiol 159: 525-533.
  • Delgado MJ, Fernandez-Lopez M, Bedmar EJ (1998) Soluble and membrane-bound nitrate reductase from Bradyrhizobium japonicum bacteroids. Plant Physiol Biochem 36: 279-283.
  • Delgado MJ, Bonnard N, Tresierra-Ayala A, Bedmar EJ, Muller P (2003) The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration. Microbiology 149: 3395-3403.
  • Fernández-López M, Olivares J, Bedmar EJ (1994) Two differentially regulated nitrate reductases required for nitrate-dependent, microaerobic growth of Bradyrhizobium japonicum. Arch Microbiol 162: 310-315.
  • Gottesman S (1996) Proteases and their targets in Escherichia coli. Annu Rev Genet 30: 465-506.
  • Hackett CS, MacGregor CH (1981) Synthesis and degradation of nitrate reductase in Escherichia coli. J Bacteriol 146: 352-359.
  • Hettmann T, Anemuller S, Borcherding H, Mathe L, Steinrucke P, Diekmann S (2003) Pseudomonas stutzeri soluble nitrate reductase αβ-subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane-bound αβγ holoenzyme. FEBS Lett 534: 143-150.
  • Jenal U, Hengge-Aronis R (2003) Regulation by proteolysis in bacterial cells. Curr Opin Microbiol 6: 163-172.
  • Kock H, Gerth U, Hecker M (2004) MurAA, catalysing the first committed step in peptidoglycan biosynthesis, is a target of Clp-dependent proteolysis in Bacillus subtilis. Mol Microbiol 51: 1087-1102.
  • Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680-685.
  • Lee YS, Park SC, Goldberg AL, Chung CH (1988) Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase. J Biol Chem 263: 6643-6646.
  • Maurizi MR, Rasulova F (2003) Degradation of L-glutamate dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability. Arch Biochem Biophys 397: 206-216.
  • Millipore (2000) Rapid immunodetection of blotted proteins without blocking -Immobilon-P transfer membrane. Application Note RP562.
  • Ogura T, Inoue K, Tatsuta T et al. (1999) Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli. Mol Microbiol 31: 833-844.
  • Polcyn W (2008) Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus). Acta Biochim Polon 55: 753-760.
  • Polcyn W, Luciński R (2003) Aerobic and anaerobic nitrate and nitrite reduction in free-living cells of Bradyrhizobium sp. (Lupinus). FEMS Microbiol Lett 226: 331-337.
  • Polcyn W, Luciński R (2006) Dissimilatory nitrate reductase from Bradyrhizobium sp. (Lupinus): subcellular location, catalytic properties, and characterization of the active enzyme forms. Curr Microbiol 52: 231-237.
  • Roseman JE, Levine RL (1987) Purification of a protease from Escherichia coli with specificity for oxidized glutamine synthetase. J Biol Chem 262: 2101-2110.
  • Somasegaran P, Hoben HJ (1994) Handbook for Rhizobia: Methods in Legume-Rhizobium Technology. pp 366-369. Springer, New York.
  • Vergnes A, Gouffi-Belhabich K, Blasco F, Giordano G, Magalon A (2004) Involvement of the molybdenum cofactor biosynthetic machinery in the maturation of the Escherichia coli nitrate reductase A. J Biol Chem 279: 41398-41403.
  • Wang H, Tseng CP, Gunsalus RP (1999) The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite. J Bacteriol 181: 5303-5308.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.