Cross Talk Inhibition Nullified by a Receiver Domain Missense Substitution.

UNLABELLED: In two-component signal transduction, a sensor protein transmitter module controls cognate receiver domain phosphorylation. Most receiver domain sequences contain a small residue (Gly or Ala) at position T + 1 just distal to the essential Thr or Ser residue that forms part of the active site. However, some members of the NarL receiver subfamily have a large hydrophobic residue at position T + 1. Our laboratory previously isolated a NarL mutant in which the T + 1 residue Val-88 was replaced with an orthodox small Ala. This NarL V88A mutant confers a striking phenotype in which high-level target operon expression is both signal (nitrate) and sensor (NarX and NarQ) independent. This suggests that the NarL V88A protein is phosphorylated by cross talk from noncognate sources. Although cross talk was enhanced in ackA null strains that accumulate acetyl phosphate, it persisted in pta ackA double null strains that cannot synthesize this compound and was observed also in narL(+) strains. This indicates that acetate metabolism has complex roles in mediating NarL cross talk. Contrariwise, cross talk was sharply diminished in an arcB barA double null strain, suggesting that the encoded sensors contribute substantially to NarL V88A cross talk. Separately, the V88A substitution altered the in vitro rates of NarL autodephosphorylation and transmitter-stimulated dephosphorylation and decreased affinity for the cognate sensor, NarX. Together, these experiments show that the residue at position T + 1 can strongly influence two distinct aspects of receiver domain function, the autodephosphorylation rate and cross talk inhibition. IMPORTANCE: Many bacterial species contain a dozen or more discrete sensor-response regulator two-component systems that convert a specific input into a distinct output pattern. Cross talk, the unwanted transfer of signals between circuits, occurs when a response regulator is phosphorylated inappropriately from a noncognate source. Cross talk is inhibited in part by the high interaction specificity between cognate sensor-response regulator pairs. This study shows that a relatively subtle missense change from Val to Ala nullifies cross talk inhibition, enabling at least two noncognate sensors to enforce an inappropriate output independently of the relevant input.

The defective proteasome but not substrate recognition function is responsible for the null phenotypes of the Arabidopsis proteasome subunit RPN10.

Ubiquitylated substrate recognition during ubiquitin/proteasome-mediated proteolysis (UPP) is mediated directly by the proteasome subunits RPN10 and RPN13 and indirectly by ubiquitin-like (UBL) and ubiquitin-associated (UBA) domain-containing factors. To dissect the complexity and functional roles of UPP substrate recognition in Arabidopsis thaliana, potential UPP substrate receptors were characterized. RPN10 and members of the UBL-UBA-containing RAD23 and DSK2 families displayed strong affinities for Lys-48-linked ubiquitin chains (the major UPP signals), indicating that they are involved in ubiquitylated substrate recognition. Additionally, RPN10 uses distinct interfaces as primary proteasomal docking sites for RAD23s and DSK2s. Analyses of T-DNA insertion knockout or RNA interference knockdown mutants of potential UPP ubiquitin receptors, including RPN10, RPN13, RAD23a-d, DSK2a-b, DDI1, and NUB1, demonstrated that only the RPN10 mutant gave clear phenotypes. The null rpn10-2 showed decreased double-capped proteasomes, increased 20S core complexes, and pleiotropic vegetative and reproductive growth phenotypes. Surprisingly, the observed rpn10-2 phenotypes were rescued by a RPN10 variant defective in substrate recognition, indicating that the defectiveness of RPN10 in proteasome but not substrate recognition function is responsible for the null phenotypes. Our results suggest that redundant recognition pathways likely are used in Arabidopsis to target ubiquitylated substrates for proteasomal degradation and that their specific roles in vivo require further examination.

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

The 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 phospho-NarL 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.

Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in Escherichia coli K-12.

The facultative aerobe Escherichia coli K-12 can use respiratory nitrate ammonification to generate energy during anaerobic growth. The toxic compound nitric oxide is a by-product of this metabolism. Previous transcript microarray studies identified the yeaR-yoaG operon, encoding proteins of unknown function, among genes whose transcription is induced in response to nitrate, nitrite, or nitric oxide. Nitrate and nitrite regulate anaerobic respiratory gene expression through the NarX-NarL and NarQ-NarP two-component systems. All known Nar-activated genes also require the oxygen-responsive Fnr transcription activator. However, previous studies indicated that yeaR-yoaG operon transcription does not require Fnr activation. Here, we report results from mutational analyses demonstrating that yeaR-yoaG operon transcription is activated by phospho-NarL protein independent of the Fnr protein. The phospho-NarL protein binding site is centered at position -43.5 with respect to the transcription initiation site. Expression from the Shewanella oneidensis MR-1 nnrS gene promoter, cloned into E. coli, similarly was activated by phospho-NarL protein independent of the Fnr protein. Recently, yeaR-yoaG operon transcription was shown to be regulated by the nitric oxide-responsive NsrR repressor (N. Filenko et al., J. Bacteriol. 189:4410-4417, 2007). Our mutational analyses reveal the individual contributions of the Nar and NsrR regulators to overall yeaR-yoaG operon expression and document the NsrR operator centered at position -32. Thus, control of yeaR-yoaG operon transcription provides an example of overlapping regulation by nitrate and nitrite, acting through the Nar regulatory system, and nitric oxide, acting through the NsrR repressor.