A protein phosphatase positively regulates the trafficking of phosphate transporters in response to phosphate levels.

Background/Objectives: Rhamnolipids (RLs) are biosurfactants with significant industrial and environmental potential, which physicochemical properties depend greatly on their fatty acyl chain composition. This study investigated the impact of genetically modulating the fatty acid synthesis genes fabA and fabZ on RL composition and functionality in Pseudomonas aeruginosa PAO1. Methods and Results: Using temperature-sensitive mutants and suppressor strains for these essential genes, we successfully engineered RLs with altered fatty acyl chain lengths and saturation levels. LC–MS/MS analyses showed that deletion and overexpression of fabA and fabZ significantly shifted RL fatty acid profiles. Functional analyses indicated that these structural changes markedly influenced RL emulsification activity and critical micelle concentration (CMC). Conclusions: These findings demonstrate the feasibility of optimizing RL properties through targeted genetic manipulation, offering valuable insights for designing customized biosurfactants for diverse industrial and environmental applications.

Microbial essential genes encode nondispensable function for cell growth and therefore are ideal targets for the development of new drugs. Essential genes are readily identified using transposon-sequencing technology at the genome scale.

Pseudomonas aeruginosa is an opportunistic pathogen whose multidrug resistance demands new drug development. Fatty acids are essential for viability, and essential genes are ideal drug targets.

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.

Pa0665 in Pseudomonas aeruginosa shares homologous sequences with that of the essential A-type iron–sulfur (Fe-S) cluster insertion protein ErpA in Escherichia coli. However, its essentiality in P. aeruginosa and its complementation with E. coli erpA has not been experimentally examined. To fulfill this task, we constructed plasmid-based ts-mutant Δpa0665/pTS-pa0665 using a three-step protocol. The mutant displayed growth defects at 42 °C, which were complemented by expressing ec.erpA. Microscopic observations indicated a petite cell phenotype for Δpa0665/pTS-pa0665 at 42 °C, correlated with the downregulation of the oprG gene. RNA sequencing revealed significant transcriptional changes in genes associated with the oxidative phosphorylation (OXPHOS) system, aligning with reduced ATP levels in Δpa0665/pTS-pa0665 under 42 °C. Additionally, the ts-mutant showed heightened sensitivity to H₂O₂ at 42 °C. Overall, our study demonstrates the essential role of pa0665 for OXPHOS function and is complemented by ec.erpA. We propose that the plasmid-based ts-allele is useful for genetic analysis of essential genes of interest in P. aeruginosa.