Either of two functionally redundant sensor proteins, NarX and NarQ, is sufficient for nitrate regulation in Escherichia coli K-12.

Rabin, Ross S.; Stewart, Valley

doi:10.1073/pnas.89.18.8419

Cited by 78 publications

(83 citation statements)

References 26 publications

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“…The NarX and NarQ proteins have 32% amino acid identity (3,20) and exhibit the highest degree of amino acid conservation in four regions of their cytoplasmic domains and one region of their periplasmic domains (Fig. 1).…”

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“…Either NarX or NarQ can independently sense nitrate and activate NarL (3,20). However, disruption of both the narX ϩ and narQ ϩ nitrate sensor genes or disruption of the narL ϩ regulator gene completely abolishes nitrate control (3,10,12,20,26).…”

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confidence: 99%

“…Nitrate-dependent gene expression in Escherichia coli is mediated by a two-component regulatory system composed of the narX, narQ, and narL gene products (3,8,19,20,27,28). The NarX and NarQ proteins are cytoplasmic membrane proteins that detect the presence of nitrate in the cell's environment.…”

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

et al. 1995

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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 [␥-32 P]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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The NarX and NarQ sensor-transmitter proteins of Escherichia coli each require two conserved histidines for nitrate-dependent signal transduction to NarL

et al. 1995

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“…A complex system of proteins that is involved in sensing nitrate in the environment and transmitting its presence to the level of gene expression has been discovered (33). The proteins include two sensor-regulator pairs termed NarX-NarL and NarQ-NarP of which NarX and NarQ are membrane-associated histidine kinases and NarL and NarP are transcription regulators (3,7,14,20,21,28,35,39). The NarQ and NarX proteins are not only capable of sensing nitrate but they are also responsive to nitrite.…”

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Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP

Kaiser¹,

Sawers²

1995

J Bacteriol

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The pfl operon is expressed at high levels anaerobically. Growth of Escherichia coli in the presence of nitrate or nitrite led to a 45% decrease in expression when cells were cultivated in rich medium. Nitrate repression, however, was significantly enhanced (sevenfold) when the cells were cultured in minimal medium. Regulation of pfl expression by nitrate was dependent on the NarL, NarP, NarQ, and NarX proteins but independent of FNR, ArcA, and integration host factor, which are additional regulators of pfl expression. Strains unable to synthesize any one of the NarL, NarP, NarQ, or NarX proteins, but retaining the capacity to synthesize the remaining three, exhibited essentially normal nitrate regulation. In contrast, narL narP and narX narQ double null mutants were devoid of nitrate regulation when cultured in rich medium but they retained some nitrate repression (1.3-fold) when grown in minimal medium. By using lacZ fusions, it was possible to localize the DNA sequences required to mediate nitrate repression to the pfl promoter-regulatory region. DNase I footprinting studies identified five potential binding sites for the wild-type NarL protein in the pfl promoter-regulatory region. Specific footprints were obtained only when NarL was phosphorylated with acetyl phosphate before the binding reaction was performed. Each of the protected regions contained at least one heptamer sequence which has been deduced from mutagenesis studies to be essential for NarL binding (K. Tyson, A. Bell, J. Cole, and S. Busby, Mol. Microbiol. 7:151-157, 1993).Nitrate respiration is an efficient means of energy generation for Escherichia coli. The use of this electron acceptor naturally requires that a particular complement of enzymes is synthesized. Included in this set of enzymes is a respiratory pathway comprising a formate dehydrogenase termed FDH-N (encoded by the fdnGHI operon) and a nitrate reductase (encoded by narGHJI). The coordinate synthesis of these enzymes occurs only anaerobically and when nitrate is available (33). The presence of nitrate also reduces or prevents the expression of a number of genes, e.g., the frdABCD operon encoding fumarate reductase and the dmsABC operon encoding dimethylsulfoxide reductase, whose products either are not required during nitrate respiration or are required but in much reduced amounts. A complex system of proteins that is involved in sensing nitrate in the environment and transmitting its presence to the level of gene expression has been discovered (33). The proteins include two sensor-regulator pairs termed NarX-NarL and NarQ-NarP of which NarX and NarQ are membrane-associated histidine kinases and NarL and NarP are transcription regulators (3,7,14,20,21,28,35,39). The NarQ and NarX proteins are not only capable of sensing nitrate but they are also responsive to nitrite. Mutational studies have demonstrated that either NarX or NarQ is capable of modulating the DNA-binding activity of the NarL and NarP proteins in response to nitrate and nitrite (for a detailed review, see reference ...

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“…For example, HK NarQ activates NarL almost as efficiently as NarX (47). We constructed a pathway in which NarL is controlled by both NarX and NarQ and measured almost-perfect OR behavior as the function of NarX and NarQ presence (Fig.…”

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confidence: 99%

Transplantation of prokaryotic two-component signaling pathways into mammalian cells

Hansen

Mailand

Swaminathan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance Synthetic biology and genetic engineering would greatly benefit from engineered genetic elements that are orthogonal to the host in which they operate. Two-component signaling pathways are the prevalent signal processing modality in prokaryotes that is also found in low eukaryotes and plants but absent from vertebrate cells. Here we investigate whether the elements of prokaryotic two-component pathways are operational in mammalian cells. We find that the core biochemical processes are maintained, whereas the capacity to sense chemical ligands is diminished or obscured. We use the pathways for multiinput gene regulation and show that they can serve as a rich source of orthogonal building blocks for gene expression control in mammalian cells. Our findings open new avenues in synthetic circuit design.

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Either of two functionally redundant sensor proteins, NarX and NarQ, is sufficient for nitrate regulation in Escherichia coli K-12.

Abstract: ABSTRACT

Cited by 78 publications

References 26 publications

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

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

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP

Transplantation of prokaryotic two-component signaling pathways into mammalian cells

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