Asymmetric cross‐regulation between the nitrate‐responsive NarX–NarL and NarQ–NarP two‐component regulatory systems from Escherichia coli K‐12

Abstract

SummaryThe NarX-NarL and NarQ-NarP sensor-response regulator pairs control Escherichia coli gene expression in response to nitrate and nitrite. Previous analysis suggests that the Nar two-component systems form a cross-regulation network in vivo. Here we report on the kinetics of phosphoryl transfer between different sensor-regulator combinations in vitro. NarX exhibited a noticeable kinetic preference for NarL over NarP, whereas NarQ exhibited a relatively slight kinetic preference for NarL. These findings were substantiated in reactions containing one sensor and both response regulators, or with two sensors and a single response regulator. We isolated 21 NarX mutants with missense substitutions in the cytoplasmic central and transmitter modules. These confer phenotypes that reflect defects in phosphoNarL dephosphorylation. Five of these mutants, all with substitutions in the transmitter DHp domain, also exhibited NarP-blind phenotypes. Phosphoryl transfer assays in vitro confirmed that these NarX mutants have defects in catalysing NarP phosphorylation. By contrast, the corresponding NarQ mutants conferred phenotypes indicating comparable interactions with both NarP and NarL. Our overall results reveal asymmetry in the Nar cross-regulation network, such that NarQ interacts similarly with both response regulators, whereas NarX interacts preferentially with NarL.

Conserved mechanism for sensor phosphatase control of two-component signaling revealed in the nitrate sensor NarX

“…Missense substitutions and amino acid deletions conferring the K + P -phenotype have been identified throughout the DHp and CA domains (11,22,32). Most of these substitutions change nonconserved residues and likely influence transmitter phosphatase function indirectly by affecting interactions between the DHp and CA domains, DHp and receiver domains, or transmitter and auxiliary proteins (such as NtrB-PII).…”

“…We denote negative-function phenotypes as P + for near-wild type and as P -for deficient (22). Reporter strains for both negative-and positive-function assays carry a null allele of narQ, encoding the paralogous nitrate sensor (11).…”

“…MBP-NarX retains an intact GAF domain and transmitter module and displays transmitter autokinase, phosphotransfer, and transmitter phosphatase activities (11,25). Fig.…”

Sensor–response regulator interactions in a cross-regulated signal transduction network

“…The dashed arrow represents relatively low phosphoryltransfer rates between NarX and NarP. Adapted from Noriega et al (2010). (Table 1), as described previously Williams & Stewart, 1997a).…”

Determinants of specificity in two-component signal transduction

“…Consistently, an examination of the specificity residues in two-component proteins from an individual organism typically reveals significant differences in almost all possible pairwise comparisons of kinases or regulators [37]. In rare cases, the specificity residues of some two-component proteins are similar, indicating potentially advantageous cross-regulation [41]. For instance, the sporulation kinases in B. subtilis, KinA/B/C/D/E, have similar specificity residues and each phosphorylates Spo0F, effectively integrating different signals into a common response -the initiation of sporulation [42].…”