Fig. 1: Mutation of HK phosphatase catalytic residue activates TCS signalling.

A Conserved motif of the HisKA and HisKA_3 histidine kinases with the phospho-acceptor histidine (blue) and the predicted residue specifically involved in the phosphatase activity (red). The phosphatase residue is substituted by an alanine in the HK⁺ mutants. Genes ID, proteins ID, and alternative names of TCSs are provided in Supplementary Data 1. B Fitness of HK⁺ mutants. The violin plots represent the distribution of the relative doubling time (Fitness (F) = doubling time WT mean / doubling time mutant) in an exponential growth phase in THY with the median (bar) and the interquartile range (dashed lines). Individual dots are shown for biological replicate (n = 16 for the WT, n = 8 for mutants), and significant differences are highlighted (*, | F | > 0.1, two-tailed Mann Whitney test p < 10⁻⁴). The bimodal distribution due to the occurrence of faster-growing VicK_T221A suppressors is highlighted (#). Corresponding growth curves and doubling times are shown in Supplementary Fig. 1. C Activation of transcriptional feedback loops in HK⁺ mutants. Fold changes (FC) for all genes encoding TCS (n = 41) in each HK⁺ mutant after RNA-seq analysis are shown as dots. The HK-RR gene pair in the corresponding HK⁺ mutant is highlighted in red (e.g., saeRS in SaeS_T133A). Cross-regulations, defined as significant differential expression of a TCS gene pair not corresponding to the HK⁺ mutation, are highlighted in blue (hk11050-rr11055 in VicKT_221A and relRS in CiaH_T228A). D Activation of the VicR and the RelR response regulators by phosphorylation in the corresponding HK⁺ mutants. Upper: representative Phos-Tag western-blots with anti-FLAG antibodies allowing to separate phosphorylated and non-phosphorylated forms of the ectopically expressed epitope-tagged RR. Bottom: quantification of the proportion of phosphorylated regulators in the WT (black) and the cognate HK⁺ mutant (red). Bars represent the mean with SD of biological replicate (n = 3). Source data are provided in Supplementary Data 4E for panel (C) and as a Source Data file for panels (B) and (D).