scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

scholarly journals Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro

1998 ◽  
Vol 180 (16) ◽  
pp. 4192-4198 ◽  
Author(s):  
Andrew J. Darwin ◽  
Eva C. Ziegelhoffer ◽  
Patricia J. Kiley ◽  
Valley Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Binding Site ◽  
Anaerobic Growth ◽  
Periplasmic Nitrate Reductase ◽  
Operon Expression ◽  
Nitrate And Nitrite ◽  
K 12

ABSTRACT The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, thenapF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napFcontrol region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

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 Periplasmic Nitrate Reductase (NapABC Enzyme) Supports Anaerobic Respiration by Escherichia coli K-12

2002 ◽  
Vol 184 (5) ◽  
pp. 1314-1323 ◽  
Author(s):  
Valley Stewart ◽  
Yiran Lu ◽  
Andrew J. Darwin
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Null Alleles ◽  
E Coli ◽  
Periplasmic Nitrate Reductase ◽  
Nitrate Concentrations ◽  
K 12

ABSTRACT Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of Φ(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that Φ(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.

Cloning and expression of the fumarate reductase gene of Escherichia coli

1981 ◽  
Vol 59 (3) ◽  
pp. 158-164 ◽  
Author(s):  
Elke Lohmeier ◽  
D. Scott Hagen ◽  
Peter Dickie ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Reductase Activity ◽  
Fumarate Reductase ◽  
Cloning And Expression ◽  
Kinetic Properties ◽  
Structural Genes ◽  
E Coli ◽  
Reductase Gene ◽  
Mutant Host

Mutants of Escherichia coli deficient in fumarate reductase activity and therefore unable to grow anaerobically with fumarate as an electron acceptor have been isolated. By F+-mediated conjugation and complementation with the mutant host, two E. coli: Col E1 recombinant DNA plasmids have been identified from the Clarke and Carbon Colony Bank which carry the structural genes for fumarate reductase. Bacteria harboring either of these plasmids express about ten times the normal level of fumarate reductase. Enzyme purified from the two sources, plasmid-carrying and plasmidless E. coli, have identical physical and kinetic properties indicating that both the 69 000 and 25 000 dalton polypeptides are amplified. Regulation of plasmid-encoded enzyme, like the chromosomally encoded enzyme, is dependent upon the presence of fumarate and anaerobiosis.

scholarly journals Dual Overlapping Promoters Control napF (Periplasmic Nitrate Reductase) Operon Expression in Escherichia coli K-12

2003 ◽  
Vol 185 (19) ◽  
pp. 5862-5870 ◽  
Author(s):  
Valley Stewart ◽  
Peggy J. Bledsoe ◽  
Stanly B. Williams
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Control Region ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Periplasmic Nitrate Reductase ◽  
Fnr Protein ◽  
Operon Expression ◽  
K 12 ◽  
A Site

ABSTRACT Escherichia coli elaborates a flexible respiratory metabolism, involving differential synthesis of isoenzymes for many oxidation and reduction reactions. Periplasmic nitrate reductase, encoded by the napFDAGHBC operon, functions with concentrations of nitrate that are too low to support respiration by membrane-bound nitrate reductase. The napF operon control region exhibits unusual organization of DNA binding sites for the transcription regulators Fnr and NarP, which activate transcription in response to anaerobiosis and nitrate, respectively. Previous studies have shown that the napF operon control region directs synthesis of two transcripts whose 5′ ends differ by about 3 nucleotides. We constructed mutant control regions in which either of the two promoter −10 regions is inactivated. Results indicate that the downstream promoter (P1) was responsible for Fnr- and NarP-regulated napF operon expression, whereas transcription from the upstream promoter (P2) was activated only weakly by the Fnr protein and was inhibited by phospho-NarP and -NarL proteins. The physiological function of promoter P2 is unknown. These results establish the unconventional napF operon control region architecture, in which the major promoter P1 is activated by the Fnr protein bound to a site centered at −64.5 with respect to the transcription initiation site, working in conjunction with the phospho-NarP protein bound to a site centered at −44.5.

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