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. 1995 May;177(9):2416–2424. doi: 10.1128/jb.177.9.2416-2424.1995

The NarX and NarQ sensor-transmitter proteins of Escherichia coli each require two conserved histidines for nitrate-dependent signal transduction to NarL.

R Cavicchioli 1, I Schröder 1, M Constanti 1, R P Gunsalus 1
PMCID: PMC176900  PMID: 7730273

Abstract

The NarX, NarQ, and NarL proteins of Escherichia coli constitute a two-component regulatory system that controls the expression of a number of anaerobic respiratory pathway genes in response to nitrate. NarX and NarQ are sensor-transmitter proteins that can independently detect the presence of nitrate in the cell environment and transmit this signal to the response regulator, NarL. Upon activation, NarL binds DNA and modulates the expression of its target genes by the repression or activation of transcription. NarX and NarQ each contain a conserved histidine residue that corresponds to the site of autophosphorylation of other sensor-transmitter proteins. They also contain a second conserved histidine residue that is present in the NarX, NarQ, UhpB, DegS, and ComP subfamily of sensor-transmitter proteins. The second histidine is located near a universally conserved asparagine residue, the role of which in signal transduction is unknown. To investigate the role of these conserved amino acids in the NarX and NarQ proteins, we mutated the narX and narQ genes by site-directed mutagenesis. In vivo, each mutation severely impaired NarL-dependent activation or repression of reporter gene expression in response to nitrate. The in vivo data suggest that the environmental signal nitrate controls both the kinase and phosphatase activities of the two sensor-transmitter proteins. The altered NarX and NarQ proteins were purified and shown to be defective in their ability to autophosphorylate in the presence of [gamma-32P]ATP. The NarX and NarQ proteins with amino acid substitutions at the first conserved histidine position were also unable to dephosphorylate NarL-phosphate in vitro. In contrast, the proteins containing amino acid substitutions at the second conserved histidine or at the conserved asparagine residue retained NarL-phosphate dephosphorylation activity. The conserved histidine and asparagine residues are essential for NarX and NarQ function, and this suggests that other two-component sensor-transmitter proteins may function in a similar fashion.

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Selected References

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  1. Atkinson M. R., Ninfa A. J. Mutational analysis of the bacterial signal-transducing protein kinase/phosphatase nitrogen regulator II (NRII or NtrB). J Bacteriol. 1993 Nov;175(21):7016–7023. doi: 10.1128/jb.175.21.7016-7023.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chiang R. C., Cavicchioli R., Gunsalus R. P. Identification and characterization of narQ, a second nitrate sensor for nitrate-dependent gene regulation in Escherichia coli. Mol Microbiol. 1992 Jul;6(14):1913–1923. doi: 10.1111/j.1365-2958.1992.tb01364.x. [DOI] [PubMed] [Google Scholar]
  4. Choe M., Reznikoff W. S. Anaerobically expressed Escherichia coli genes identified by operon fusion techniques. J Bacteriol. 1991 Oct;173(19):6139–6146. doi: 10.1128/jb.173.19.6139-6146.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cotter P. A., Chepuri V., Gennis R. B., Gunsalus R. P. Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product. J Bacteriol. 1990 Nov;172(11):6333–6338. doi: 10.1128/jb.172.11.6333-6338.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cotter P. A., Darie S., Gunsalus R. P. The effect of iron limitation on expression of the aerobic and anaerobic electron transport pathway genes in Escherichia coli. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):227–232. doi: 10.1111/j.1574-6968.1992.tb14045.x. [DOI] [PubMed] [Google Scholar]
  7. Egan S. M., Stewart V. Nitrate regulation of anaerobic respiratory gene expression in narX deletion mutants of Escherichia coli K-12. J Bacteriol. 1990 Sep;172(9):5020–5029. doi: 10.1128/jb.172.9.5020-5029.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gunsalus R. P. Control of electron flow in Escherichia coli: coordinated transcription of respiratory pathway genes. J Bacteriol. 1992 Nov;174(22):7069–7074. doi: 10.1128/jb.174.22.7069-7074.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Iuchi S., Lin E. C. The narL gene product activates the nitrate reductase operon and represses the fumarate reductase and trimethylamine N-oxide reductase operons in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3901–3905. doi: 10.1073/pnas.84.11.3901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jones H. M., Gunsalus R. P. Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product. J Bacteriol. 1987 Jul;169(7):3340–3349. doi: 10.1128/jb.169.7.3340-3349.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kalman L. V., Gunsalus R. P. Identification of a second gene involved in global regulation of fumarate reductase and other nitrate-controlled genes for anaerobic respiration in Escherichia coli. J Bacteriol. 1989 Jul;171(7):3810–3816. doi: 10.1128/jb.171.7.3810-3816.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kalman L. V., Gunsalus R. P. The frdR gene of Escherichia coli globally regulates several operons involved in anaerobic growth in response to nitrate. J Bacteriol. 1988 Feb;170(2):623–629. doi: 10.1128/jb.170.2.623-629.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kanamaru K., Aiba H., Mizushima S., Mizuno T. Signal transduction and osmoregulation in Escherichia coli. A single amino acid change in the protein kinase, EnvZ, results in loss of its phosphorylation and dephosphorylation abilities with respect to the activator protein, OmpR. J Biol Chem. 1989 Dec 25;264(36):21633–21637. [PubMed] [Google Scholar]
  15. Kolesnikow T., Schröder I., Gunsalus R. P. Regulation of narK gene expression in Escherichia coli in response to anaerobiosis, nitrate, iron, and molybdenum. J Bacteriol. 1992 Nov;174(22):7104–7111. doi: 10.1128/jb.174.22.7104-7111.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ninfa A. J., Bennett R. L. Identification of the site of autophosphorylation of the bacterial protein kinase/phosphatase NRII. J Biol Chem. 1991 Apr 15;266(11):6888–6893. [PubMed] [Google Scholar]
  19. Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
  20. Rabin R. S., Stewart V. Either of two functionally redundant sensor proteins, NarX and NarQ, is sufficient for nitrate regulation in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8419–8423. doi: 10.1073/pnas.89.18.8419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schröder I., Darie S., Gunsalus R. P. Activation of the Escherichia coli nitrate reductase (narGHJI) operon by NarL and Fnr requires integration host factor. J Biol Chem. 1993 Jan 15;268(2):771–774. [PubMed] [Google Scholar]
  23. Schröder I., Wolin C. D., Cavicchioli R., Gunsalus R. P. Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli. J Bacteriol. 1994 Aug;176(16):4985–4992. doi: 10.1128/jb.176.16.4985-4992.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  25. Stewart V., Berg B. L. Influence of nar (nitrate reductase) genes on nitrate inhibition of formate-hydrogen lyase and fumarate reductase gene expression in Escherichia coli K-12. J Bacteriol. 1988 Oct;170(10):4437–4444. doi: 10.1128/jb.170.10.4437-4444.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Walker M. S., DeMoss J. A. Phosphorylation and dephosphorylation catalyzed in vitro by purified components of the nitrate sensing system, NarX and NarL. J Biol Chem. 1993 Apr 25;268(12):8391–8393. [PubMed] [Google Scholar]
  28. Westenberg D. J., Gunsalus R. P., Ackrell B. A., Cecchini G. Electron transfer from menaquinol to fumarate. Fumarate reductase anchor polypeptide mutants of Escherichia coli. J Biol Chem. 1990 Nov 15;265(32):19560–19567. [PubMed] [Google Scholar]
  29. Yang Y., Inouye M. Requirement of both kinase and phosphatase activities of an Escherichia coli receptor (Taz1) for ligand-dependent signal transduction. J Mol Biol. 1993 May 20;231(2):335–342. doi: 10.1006/jmbi.1993.1286. [DOI] [PubMed] [Google Scholar]

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