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.