GSE128611
Engineering orthogonal signaling pathways reveals the sparse distribtion of protein protein interactions in sequence space [RNA-seq]
To test the global insulation of selected PhoQ*-PhoP* variants, we used RNA-Seq to examine gene expression in strains carrying one of six different PhoQ-PhoP variant pairs. In each case, cells were grown in medium with either excess or limiting extracellular Mg2+ to repress or stimulate PhoQ, respectively, before harvesting RNA. Each system produced a similar induction of known PhoP-dependent genes. To assess whether a variant PhoQ cross-phosphorylated other response regulators, we took advantage of the fact that, when active, most response regulators autoregulate and promote expression of themselves and their cognate histidine kinase2. Notably, none of the six strains tested showed significant induction of other two-component systems relative to a wild-type control. Similar results are seen for the chimeric pathway AQ4-PhoP4, which was induced with its ligand: trans-zeatin. Overall design: We performed RNAseq on strains containing variants of PhoQP with re-engineered, functional interfaces in order to measure potential interference with other two-component signaling pathways. RNA was harvested from each strain after 30 min induction with low MgSO4 or 30 min repression with high MgSO4. An identical experiment was conducted on the chimeric AQ4-PhoP4 pathway, with induction by trans-zeatin in place of low MgS04.