Systemic deconstruction of bacterial signaling networks unmasks hidden layers of translational complexity and ancestral sensing logic

Large-scale genome engineering requires rapid, scarless, and multi-locus editing. We developed the HoSeI method, a CRISPR-Cas9-supported one-step recombination system, and established a validated library of 1,360 sgRNA plasmids for Escherichia coli . Systematic analysis unmasked critical design principles, identifying that a T/A at the 3rd position of the guide sequence and ~40% GC content ensure optimal cleavage. Leveraging this resource , we simultaneously rewrote 34 loci to engineer strains entirely devoid of the two-component signaling repertoire. This systemic deconstruction unmasked hidden biological backup mechanisms: pervasive overlapping translation bypassing nonsense mutations and ancestral, sensor-independent activation of response regulators by cellular metabolites. Our findings redefine bacterial signaling logic and provide a robust platform for investigating the foundational principles of microbial physiology and robustness.