ABSTRACTIt is uncertain whetherPA1610|fabAis essential or dispensable for growth on LB-agar plates under aerobic conditions inPseudomonas aeruginosaPAO1. To examine its essentiality, we disruptedfabAin the presence of a native promoter-controlled complementary copy onts-plasmid. In this analysis, we showed that the plasmid-basedts-mutant ΔfabA/pTS-fabAfailed to grow at a restrictive temperature, consistent with the observation by Hoang and Schweizer (T. T. Hoang, H. P. Schweizer, J Bacteriol 179:5326–5332, 1997,https://doi.org/10.1128/jb.179.17.5326-5332.1997), and expanded on this by showing that ΔfabAexhibited curved cell morphology. On the other hand, strong induction offabA-OEorPA3645|fabZ-OEimpeded the growth of cells displaying oval morphology. Suppressor analysis revealed a mutantsupgene that suppressed a growth defect but not cell morphology of ΔfabA. Genome resequencing and transcriptomic profiling ofsupidentifiedPA0286|desA, whose promoter carried a single-nucleotide polymorphism (SNP), and transcription was significantly upregulated (level increase of >2-fold,P< 0.05). By integration of the SNP-bearing promoter-controlleddesAgene into the chromosome of ΔfabA/pTS-fabA, we showed that the SNP is sufficient for ΔfabAto phenocopy thesupmutant. Furthermore, mild induction of thearaC-PBAD-controlleddesAgene but notdesBrescued ΔfabA. These results validated that mild overexpression ofdesAfully suppressed the lethality but not the curved cell morphology of ΔfabA. Similarly, Zhu et al. (Zhu K, Choi K-H, Schweizer HP, Rock CO, Zhang Y-M, Mol Microbiol 60:260–273, 2006,https://doi.org/10.1111/j.1365-2958.2006.05088.x) showed that multicopydesApartially alleviated the slow growth phenotype of ΔfabA, the difference in which was that ΔfabAwas viable. Taken together, our results demonstrate thatfabAis essential for aerobic growth. We propose that the plasmid-basedts-allele is useful for exploring the genetic suppression interaction of essential genes of interest inP. aeruginosa.IMPORTANCEPseudomonas aeruginosais an opportunistic pathogen whose multidrug resistance demands new drug development. Fatty acids are essential for viability, and essential genes are ideal drug targets. However, the growth defect of essential gene mutants can be suppressed. Suppressors tend to be accumulated during the construction of essential gene deletion mutants, hampering the genetic analysis. To circumvent this issue, we constructed a deletion allele offabAin the presence of a native promoter-controlled complementary copy in thets-plasmid. In this analysis, we showed that ΔfabA/pTS-fabAfailed to grow at a restrictive temperature, supporting its essentiality. Suppressor analysis revealeddesA, whose promoter carried a SNP and whose transcription was upregulated. We validated that both the SNP-bearing promoter-controlled and regulable PBADpromoter-controlleddesAsuppressed the lethality of ΔfabA. Together, our results demonstrate thatfabAis essential for aerobic growth. We propose that plasmid-basedts-alleles are suitable for genetic analysis of essential genes of interest.