Zhu, Z.; Zhao, N.; Song, Y.; Fan, D.; Ying, J.; Nong, C.; Liu, T.; Duan, C.; Zheng, Y.; Zou, S.; Mou, X.; Ma, H.; Liu, H.; Bao, R.

Two-component systems convert environmental signals into transcriptional responses, but how one response-regulator phosphorylation state produces different outputs at different promoters remains unclear. Here we show that promoter architecture determines how the ParR response regulator in Pseudomonas aeruginosa interprets phosphorylation. Phenotypic, transcriptomic, biochemical and promoter-engineering analyses showed that phosphorylation of the conserved ParR receiver residue Asp57 lowered DNA-occupancy thresholds across target promoters. Individual promoters, however, converted this shared increase in binding into activation, repression or phosphorylation-dependent sign switching. Chromosomally D57 ParR variants reproduced these behaviours at endogenous loci and altered antibiotic susceptibility, biofilm formation and virulence-associated phenotypes. Mapping and engineering of representative promoters identified a two-tier cis-regulatory code: half-site sequence compatibility determines productive ParR engagement, whereas spacer length determines regulatory sign and magnitude. Spacer swaps were sufficient to reprogram promoter logic between regulatory modes. These findings separate regulator state from promoter decoding and show how bacterial promoter architecture can diversify the outputs of a shared phosphorylation signal.