Analysis of the plasmid-based ts-mutant Δpa0947 (pTS- pa0947 ) reveals pa0947 essentiality and dnaA -centered suppressor bypass in Pseudomonas aeruginosa

ABSTRACT PA0947 is annotated as an essential hypothetical protein in Pseudomonas aeruginosa and shares substantial sequence identity with the Escherichia coli replication regulator Hda, yet its cellular function and genetic bypass logic in P. aeruginosa have not been experimentally characterized. Here, we constructed a plasmid-based temperature-sensitive complementation strain, Δpa0947 (pTS- pa0947 ), to enable conditional depletion of pa0947 . The mutant grew like wild type at 30°C but failed to proliferate at 42°C, demonstrating pa0947 as essential for growth. pa0947 depletion triggered rapid filamentation, increased PicoGreen-associated fluorescence, and an elevated oriC/ter ratio, consistent with dysregulated replication initiation. In addition, E. coli hda did not complement pa0947 depletion, and overexpression of either pa0947 or E. coli hda impaired growth, indicating species-dependent incompatibility and dosage sensitivity. To identify suppressors capable of bypassing pa0947 loss, we performed a spontaneous suppressor screen and isolated four independent suppressor mutants that grew without pa0947 . Genome resequencing of these suppressors revealed convergent mutations in the dnaA locus, including upstream regulatory indels and C-terminal coding changes. Upstream mutations reduced dnaA transcript levels by qRT-PCR, whereas coding changes compromised DnaA–oriC binding in EMSA. Together, these results identify dnaA as the convergent bypass locus for pa0947 essentiality and link pa0947 function to tight control of DnaA-driven initiation in P. aeruginosa . IMPORTANCE Many bacterial essential genes remain poorly understood, limiting efforts to exploit them as antibiotic targets. PA0947 is predicted to be essential in the opportunistic pathogen P. aeruginosa , yet its essentiality and physiological role have not been experimentally defined. Using a conditional depletion strategy based on plasmid-borne, temperature-sensitive complementation, we show that PA0947 loss causes severe growth failure, filamentation, and abnormal DNA accumulation, consistent with disrupted control of chromosome duplication. Suppressor analysis identified four independent suppressors, including promoter lesions that reduced dnaA expression and coding changes that compromised DnaA–oriC binding. Together, our results establish in P. aeruginosa that PA0947 is required to keep replication initiation within a narrow activity window compatible with robust growth, and they highlight conditional depletion plus suppressor mapping as a broadly useful strategy to uncover the function and bypass logic of essential bacterial genes.