Genetic Analysis of the Plasmid-Based Temperature-Lethal Mutant pa1792|lpxH(Ts) in Pseudomonas aeruginosa.

Abstract

Many enzymes in the Raetz pathway for lipid A biosynthesis in Escherichia coli are essential. A homologous protein Pa1792|LpxH in Pseudomonas aeruginosa is known to complement the loss of LpxH in E. coli. Genome-wide transposon-insertion sequencing analysis indicates that lpxH is essential in P. aeruginosa. However, genetic analysis of lpxH in P. aeruginosa has not been carried out, partly because the conditional alleles of essential genes are not readily constructed. In this study, we first constructed a plasmid-based temperature-sensitive mutant ΔlpxH/pTS-lpxH or lpxH(Ts) in P. aeruginosa PAO1. Spot-plating assay indicated that lpxH(Ts) was lethal at a restrictive temperature, confirming its essentiality for growth. Microscopic analysis revealed that lpxH(Ts) exhibited an oval-shaped morphology, suggesting that lpxH was required for rod-shape formation. SDS-PAGE and Western blotting analysis showed that lpxH(Ts) failed to synthesize lipid A, consistent with its function in lipid A biosynthesis. Strong expression of lpxH but not the non-homologous isoenzyme lpxI or lpxG impeded growth and caused cell lysis, implying that lpxH-specific cofactors were required for this toxic effect in P. aeruginosa. Together, our results demonstrate that lpxH is essential for lipid A biosynthesis, rod-shaped growth, and viability in P. aeruginosa. We propose that this plasmid-based conditional allele is a useful tool for the genetic study of essential genes in P. aeruginosa.