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Journal of Bacteriology, September 1999, p. 5309-5316, Vol. 181, No. 17
Department of Microbiology and Molecular
Genetics and Molecular Biology Institute, University of California,
Los Angeles, California 90095-1489
Received 19 January 1999/Accepted 17 June 1999
The Nar two-component regulatory system, consisting of the dual
sensor-transmitters NarX and NarQ and the dual response regulators NarL
and NarP, controls the expression of various anaerobic respiratory pathway genes and fermentation pathway genes. Although both NarX and
NarQ are known to detect the two environmental signals nitrate and
nitrite, little is known regarding the sensitivity and selectivity of
ligand for detection or activation of the sensor-transmitters. In this
study, we have developed a sensitive anion-specific in vitro assay for
NarX autophosphorylation by using Escherichia coli
membranes highly enriched in the full-length NarX protein. In this ATP-
and magnesium-dependent reaction, nitrate elicited a greater signal
output (i.e., NarX autophosphorylation) than did nitrite. Nitrate
stimulation occurred at concentrations as low as 5 µM, and the
half-maximal level of NarX autophosphorylation occurred at
approximately 35 µM nitrate. In contrast, nitrite-dependent stimulation was detected only at 500 µM, while 3.5 mM nitrite was
needed to achieve half-maximal NarX autophosphorylation. Maximal nitrate- and nitrite-stimulated levels of NarX phosphorylation were
five and two times, respectively, over the basal level of NarX
autophosphorylation. The presence of Triton X-100 eliminated the
nitrate-stimulated kinase activity and lowered the basal level of
activity, suggesting that the membrane environment plays a crucial role
in nitrate detection and/or regulation of kinase activity. These
results provide in vitro evidence for the differential detection of
dual signaling ligands by the NarX sensor-transmitter protein, which
modulates the cytoplasmic NarX autokinase activity and phosphotransfer
to NarL, the cognate response regulator.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Signal-Dependent Phosphorylation of the
Membrane-Bound NarX Two-Component Sensor-Transmitter Protein of
Escherichia coli: Nitrate Elicits a Superior Anion
Ligand Response Compared to Nitrite
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Genetics, University of California, Los
Angeles, CA 90095-1489. Phone: (310) 206-8201. Fax: (310) 206-5231. E-mail: robg{at}microbio.ucla.edu.
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