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.

In cyanobacterial Nostoc species, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm that Nostoc punctiforme hormogonia are positively phototactic to white light over a wide range of intensities. N. punctiforme contains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome-methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis in N. punctiforme hormogonia; here these genes are designated ptx (for phototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of the ptx genes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpA and gvpC), which also are expressed only in the hormogonium filaments of N. punctiforme.