Nitrite and nitrate regulation at the promoters of two Escherichia coli operons encoding nitrite reductase: identification of common target heptamers for both NarP- and NarL-dependent regulation

1994 ◽  
Vol 13 (6) ◽  
pp. 1045-1055 ◽  
Author(s):  
K. L. Tyson ◽  
J. A. Cole ◽  
S. J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Common Target

scholarly journals The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors

1982 ◽  
Vol 203 (2) ◽  
pp. 505-510 ◽  
Author(s):  
R H Jackson ◽  
J A Cole ◽  
A Cornish-Bowden
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Nitrite Reductase ◽  
Maximum Velocity ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Cytochrome C Reduction ◽  
Pattern Of Variation ◽  
Rate Limiting

The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.

scholarly journals Electron-spin-resonance studies of the NADH-dependent nitrite reductase from Escherichia coli K12

1982 ◽  
Vol 207 (2) ◽  
pp. 333-339 ◽  
Author(s):  
R Cammack ◽  
R H Jackson ◽  
A Cornish-Bowden ◽  
J A Cole
Keyword(s):  
Escherichia Coli ◽  
Electron Spin Resonance ◽  
Low Temperature ◽  
Nitrite Reductase ◽  
Hyperfine Splitting ◽  
Midpoint Potential ◽  
Spin Resonance ◽  
Nitrite Reductases ◽  
Haem Iron ◽  
Nitrosyl Derivative

The NADH-dependent nitrite reductase of Escherichia coli, which contains sirohaem, flavin, non-haem iron and labile sulphide, was examined by low-temperature e.s.r. spectroscopy. The enzyme, stored in the presence of nitrite and ascorbate, gave the spectrum of a nitrosyl derivative, with hyperfine splitting due to the nitrosyl nitrogen. On removal of these reagents, a series of signals centred around g = 6 was observed, typical of high-spin ferric haem. Cyanide converted this into a low-spin form. On reduction of the enzyme with NADH, an axial spectrum at g = 1.92, 2.01 was observed. The temperature-dependence of this signal is indicative of a [2Fe-2S] iron-sulphur cluster. The midpoint potential of this cluster was estimated to be −230 +/- 15 mV by two independent methods. Reduction of the enzyme with dithionite yielded further signals, which are at present unidentified, at g = 2.1-2.28. No signals were observed that could be assigned to a [4Fe-4S] cluster, such as is found in other sulphite reductases and nitrite reductases that contain sirohaem.

scholarly journals The nrfA and nirB Nitrite Reductase Operons in Escherichia coli Are Expressed Differently in Response to Nitrate than to Nitrite

2000 ◽  
Vol 182 (20) ◽  
pp. 5813-5822 ◽  
Author(s):  
Henian Wang ◽  
Robert P. Gunsalus
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
Physiological Role ◽  
Growth Conditions ◽  
Potent Inducer ◽  
Culture Techniques ◽  
Nitrite Reductase Gene

ABSTRACT Escherichia coli possesses two distinct nitrite reductase enzymes encoded by the nrfA and nirBoperons. The expression of each operon is induced during anaerobic cell growth conditions and is further modulated by the presence of either nitrite or nitrate in the cells' environment. To examine how each operon is expressed at low, intermediate, and high levels of either nitrate or nitrite, anaerobic chemostat culture techniques were employed using nrfA-lacZ and nirB-lacZ reporter fusions. Steady-state gene expression studies revealed a differential pattern of nitrite reductase gene expression where optimalnrfA-lacZ expression occurred only at low to intermediate levels of nitrate and where nirB-lacZ expression was induced only by high nitrate conditions. Under these conditions, the presence of high levels of nitrate suppressed nrfA gene expression. While either NarL or NarP was able to inducenrfA-lacZ expression in response to low levels of nitrate, only NarL could repress at high nitrate levels. The different expression profile for the alternative nitrite reductase operon encoded by nirBDC under high-nitrate conditions was due to transcriptional activation by either NarL or NarP. Neither response regulator could repress nirB expression. Nitrite was also an inducer of nirB and nrfA gene expression, but nitrate was always the more potent inducer by >100-fold. Lastly, since nrfA operon expression is only induced under low-nitrate concentrations, the NrfA enzyme is predicted to have a physiological role only where nitrate (or nitrite) is limiting in the cell environment. In contrast, the nirB nitrite reductase is optimally synthesized only when nitrate or nitrite is in excess of the cell's capacity to consume it. Revised regulatory schemes are presented for NarL and NarP in control of the two operons.

scholarly journals Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12

1981 ◽  
Vol 193 (3) ◽  
pp. 861-867 ◽  
Author(s):  
R H Jackson ◽  
A Cornish-Bowden ◽  
J A Cole
Keyword(s):  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soret Band ◽  
Horse Heart Cytochrome ◽  
E Coli ◽  
Highly Active ◽  
Covalently Bound

A substantially improved purification of Escherichia coli NADH-dependent nitrite reductase was obtained by purifying it in presence of 1 mM-NO2- and 10 microM-FAD. The enzyme was obtained in 20% yield with a maximum specific activity of 1.04 kat . kg-1: more than 95% of this sample subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis migrated as a single band of protein. This highly active enzyme contained one non-covalently bound FAD molecule, and, probably, 5 Fe atoms and 4 acid-labile S atoms per subunit. No FMN, covalently bound flavin or Mo was detected. The spectrum of the enzyme shows absorption maxima at 386, 455, 530 and about 575 nm with a shoulder at 480–490 nm. The Soret-band/alpha-band absorbance ratio is about 4:1. These spectral features are characteristic of sirohaem, apart from the maximum at 455nm, which is attributed to flavin. The enzyme also catalyses the NADH-dependent reduction of horse heart cytochrome c, 2,6-dichlorophenol-indophenol and K3Fe(CN)6. The presence of sirohaem in E. coli nitrite reductase explains the apparent identity of the cysG and nirB gene of E. coli and inability of hemA mutants to reduce nitrite.

scholarly journals A Triphosphopyridine Nucleotide-specific Nitrite Reductase from Escherichia coli

1961 ◽  
Vol 236 (12) ◽  
pp. 3330-3335
Author(s):  
Robert A. Lazzarini ◽  
Daniel E. Atkinson
Keyword(s):  
Escherichia Coli ◽  
Nitrite Reductase

scholarly journals Activation of nitrite reductase from Escherichia coli K12 by oxidized nicotinamide-adenine dinucleotide

1978 ◽  
Vol 175 (2) ◽  
pp. 495-499 ◽  
Author(s):  
K J Coleman ◽  
A Cornish-Bowden ◽  
J A Cole
Keyword(s):  
Escherichia Coli ◽  
Electron Acceptor ◽  
Nitrite Reductase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Maximum Rate ◽  
Initial Rate ◽  
Nadh Oxidation ◽  
Michaelis Constant ◽  
Escherichia Coli K12 ◽  
High Concentrations

Nitrite reductase from Escherichia coli K12 requires the presence of NAD+, one of the products of the reduction of NO2-by NADH, for full activity. The effect is observed with both crude extracts and purified enzyme. NAD+ also acts as a product inhibitor at high concentrations, and plots of initial rate against NAD+ concentration are bell-shaped. The maximum occurs at about 1 mM-NAD+, but increases with increasing NADH concentration. In the presence of 1 mM-NAD+ and saturating NO2-(2mM) the Michaelis constant for NADH is about 16 micron. The Michaelis constant for NO2-is about 5 micron and is largely independent of the NAD+ concentration. Similar but more pronounced effects of NAD+ are observed with hydroxylamine as electron acceptor instead of NO2-. The maximum rate of NADH oxidation by hydroxylamine is about 5.4 times greater than the maximum rate of NADH oxidation by NO2- when assayed with the same volume of the same preparation of purified enzyme. The Michaelis constant for hydroxylamine is 5.3 mM, however, about 1000 times higher than for NO2-. These results are consistent with a mechanism in which the same enzyme-hydroxylamine complex occurs as an intermediate in both reactions.

scholarly journals The NsrR Regulon of Escherichia coli K-12 Includes Genes Encoding the Hybrid Cluster Protein and the Periplasmic, Respiratory Nitrite Reductase

2007 ◽  
Vol 189 (12) ◽  
pp. 4410-4417 ◽  
Author(s):  
Nina Filenko ◽  
Stephen Spiro ◽  
Douglas F. Browning ◽  
Derrick Squire ◽  
Tim W. Overton ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Reactive Nitrogen Species ◽  
Defense Mechanism ◽  
Regulatory Protein ◽  
Reactive Nitrogen ◽  
Nitrogen Species ◽  
Hybrid Cluster ◽  
K 12

ABSTRACT Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defense mechanisms or as products of their own metabolism. The regulatory protein NsrR (a member of the Rrf2 family of transcription factors) plays key roles in this stress response. Microarray analysis revealed that NsrR represses nine operons encoding 20 genes in Escherichia coli MG1655, including the hmpA, ytfE, and ygbA genes that were previously shown to be regulated by NsrR. Novel NsrR targets revealed by this study include hcp-hcr (which were predicted in a recent bioinformatic study to be NsrR regulated) and the well-studied nrfA promoter that directs the expression of the periplasmic respiratory nitrite reductase. Conversely, transcription from the ydbC promoter is strongly activated by NsrR. Regulation of the nrf operon by NsrR is consistent with the ability of the periplasmic nitrite reductase to reduce nitric oxide and hence protect against reactive nitrogen species. Gel retardation assays were used to show that both FNR and NarL bind to the hcp promoter. The expression of hcp and the contiguous gene hcr is not induced by hydroxylamine. As hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair iron-sulfur centers damaged by nitric oxide, the demonstration that hcp-hcr, hmpA, and ytfE are the three transcripts most tightly regulated by NsrR highlights the possibility that the hybrid cluster protein, HCP, might also be part of a defense mechanism against reactive nitrogen stress.

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