Conditional lethality and suppressor analysis of plasmid-based temperature-sensitive fabZ expression in Pseudomonas...

FabZ, a β-hydroxyacyl-acyl carrier protein dehydratase in the type II fatty acid synthesis pathway, is essential for the viability of Pseudomonas aeruginosa by ensuring proper fatty acid elongation and membrane stability. However, the precise genetic interactions between fabZ and lipid A biosynthesis genes, such as lpxA and lpxC, as well as the potential existence of other suppressor genes of fabZ in P. aeruginosa, remain unclear. To explore these genetic interactions and identify potential suppressor genes, we constructed a conditional fabZ mutant, ΔfabZ(p_ts-fabZ), by deleting the chromosomal fabZ gene and complementing it with a temperature-sensitive plasmid-borne copy. The ΔfabZ(p_ts-fabZ) mutant exhibited lethality and cell morphology defects at a restrictive temperature, confirming its essentiality. Genetic interaction analyses revealed that deletion of lpxA or lpxC failed to rescue ΔfabZ(p_ts-fabZ) lethality at restrictive temperature. Through suppressor screening, we isolated a mutant strain capable of rescuing ΔfabZ lethality and identified lpxH as the suppressor gene using genome resequencing. Further analysis revealed that the fabZ and lpxH double mutant (ΔfabZΔlpxH) produced odd-chain fatty acids, identified as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) through fatty acid methyl ester analysis coupled with GC-MS, and supplementation with these fatty acids restored the growth and morphology of ΔfabZ(p_ts-fabZ) and ΔlpxH(p_ts-lpxH) mutants at restrictive temperature, suggesting their critical role in membrane stability. These results indicate that deletion of lpxH serves as a genetic suppressor of ΔfabZ lethality, highlighting a previously unrecognized compensatory mechanism involving odd-chain fatty acid synthesis essential for membrane stability in P. aeruginosa.