Promoter architecture decodes response regulator phosphorylation into distinct transcriptional logics in Pseudomonas aeruginosa

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. ImportancePseudomonas aeruginosa is an opportunistic pathogen that survives antimicrobial treatment and adapts to diverse host environments by rapidly changing gene expression. How a single sensory pathway generates different, and sometimes opposing, responses across many genes has remained unclear. We show that the regulatory protein ParR does not impose one fixed transcriptional program. Instead, the DNA architecture of each target promoter determines whether the same phosphorylation signal activates, represses, or reverses gene expression. The sequence and spacing of ParR recognition sites form a compact regulatory code, and changing spacer length alone can reprogram promoter behavior. Different ParR states were also associated with changes in antibiotic susceptibility, biofilm formation, and virulence-associated phenotypes. Our findings explain how one bacterial signaling pathway can generate diverse physiological outcomes without additional regulators and provide a framework for understanding regulatory flexibility in Pseudomonas aeruginosa and for engineering genetic circuits with multiple outputs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/740031v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1b6ec28org.highwire.dtl.DTLVardef@4a3f2eorg.highwire.dtl.DTLVardef@79e528org.highwire.dtl.DTLVardef@b3aa77_HPS_FORMAT_FIGEXP M_FIG C_FIG