Role of the periplasmic domain of the Escherichia coli NarX sensor‐transmitter protein in nitrate‐dependent signal transduction and gene regulation

Abstract

The narX, narQ and narL genes of Escherichia coli encode a nitrate-responsive two-component regulatory system that controls the expression of many anaerobic electron-transport- and fermentation-related genes. When nitrate is present, the NarX and NarQ sensor-transmitter proteins function to activate the response-regulator protein, NarL, which in turn binds to its DNA-recognition sites to modulate gene expression. The sensor-transmitter proteins are anchored in the cytoplasmic membrane by two transmembrane domains that are separated by a periplasmic region of approximately 115 amino acids. In this study we report the isolation and characterization of narX* (star) mutants that constitutively activate nitrate reductase (narGHJI) gene expression and repress fumarate reductase (frdABCD) gene expression when no nitrate is provided for the cell. An additional narX mutant was identified that has lost its ability to respond to environmental signals. Each narX defect was caused by a single amino acid substitution within a conserved 17 amino acid sequence, called the 'P-box', in the periplasmic exposed region of the NarX protein. As a result, DNA binding is then 'locked-on' or 'locked-off' to give the observed pattern of gene expression. Diploid analysis of these narX mutants showed that a NarX P-box mutant which conferred a 'locked-on' phenotype was trans dominant over wild-type NarX. Both were also trans dominant over the NarX P-box mutant which conferred a 'locked-off' phenotype. Certain narX P-box mutations, when combined with a narX 'linker' region mutation, were recessive to the NarX linker mutation. Finally, a truncated form of the NarX protein that lacked the periplasmic and membrane regions also showed a 'locked-on' phenotype in vivo. Thus, the periplasmic and membrane domains are essential for signal transduction to NarL. From these findings, we propose that nitrate is detected in the periplasmic space of the cell, and that a signal-transduction event through the cytoplasmic membrane into the interior of the cell modulates the NarX-dependent phosphorylation/dephosphorylation of NarL.

Mutational Analysis of the Residue at Position 48 in the Salmonella enterica Serovar Typhimurium PhoQ Sensor Kinase

“…The highly conserved I box that includes residues 41 to 53 of various EnvZ homologues has been shown to be involved in the signal transduction mechanism (30). The P box of the NarQ and NarX sensors that corresponds to a stretch of 17 conserved amino acids has been shown to be required for nitrate sensing and/or transmembrane signaling (4,5,31). Although the corresponding region in PhoQ homologues is not as highly conserved as the I box or the P box (Fig.…”

Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases

“…3) (242). Alanine substitutions of highly conserved residues in the P box (but not in PЈ) strongly affected signal detection and were able to render NarX and NarQ in a constitutively active ("locked-on") or inactive ("locked-off") form (35,242,275). In addition to the conserved extracellular boxes, both types of Nar sensors have an extended cytoplasmic linker region.…”

“…In addition to the conserved extracellular boxes, both types of Nar sensors have an extended cytoplasmic linker region. In the linker region, "locked-on" mutations that are dominant over "locked-off" mutations in the P box were identified (35,131). Therefore, it was proposed that signal processing requires nitrate (or nitrite) detection in the periplasm by the P box, followed by signal transfer across the membrane and to the kinase, with the latter depending on transmission by the linker region.…”

Autophosphorylation and Dephosphorylation by Soluble Forms of the Nitrate-Responsive Sensors NarX and NarQ from Escherichia coli K-12

“…The paralagous sensors NarX and NarQ are histidine protein kinases (HPKs) that autophosphorylate in response to nitrate and other signals (7,19,27,33,40). These sensors control the phosphorylation state of the paralogous NarL and NarP response regulators (8,27,30,37).…”

“…The sensory module includes the periplasmic domain, which is delimited by two transmembrane helices. The periplasmic domain contains the conserved P box element (18 residues), which is involved in sensing nitrate (7,40). The sensory module also contains a HAMP linker and a signaling helix, both of which are involved in signal transduction (1)(2)(3).…”