| Bacteria sense and respond to a variety of environmental stimuli through two-component systems, which consist of an extensive network of histidine kinases and response regulators. Many bacteria possess a large number of two-component systems. Given the high level of sequence and structural similarity between different systems, the existence of cross-talk or interference between systems is possible. This work explores cross-talk between the CpxA-CpxR and EnvZ-OmpR two-component systems in E. coli and describes mechanisms that provide insulation against such interference. In particular, two mechanisms appear to suppress cross-talk between these systems, which depend on their cognate partners and on the bifunctional nature of the histidine kinases. This also gives rise to mutational robustness, i.e. it masks the effects of mutations that would otherwise lead to increased cross-talk. While the molecular specificity associated with interacting domains of histidine kinases and response regulators is likely to be the greatest contributor to the high level of signaling specificity, the mechanisms described here likely enhance this effect. In addition, this work explores the molecular determinants of specificity using results from a co-evolution analysis of histidine kinase and response regulator residues and also through an extensive saturation mutagenesis screen. Substitutions at key residues along the lower portion of the DHp domain alter the specificity of EnvZ and substantially increase cross-talk between non-cognate pairs. In contrast, random mutation at other sites, most notably those in close proximity to the conserved histidine involved in auto-phosphorylation, impact negatively on cross-talk. While this work is chiefly focused on characterizing cross-talk from kinase activity, it also lays the foundation for future work exploring the possibility of phosphatase crosstalk. |
