Genetic Analysis of the Plasmid-Based Temperature-Lethal Mutant in .

Many enzymes in the Raetz pathway for lipid A biosynthesis in are essential. A homologous protein Pa1792|LpxH in is known to complement the loss of LpxH in . Genome-wide transposon-insertion sequencing analysis indicates that is essential in . However, genetic analysis of in 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 or in PAO1. Spot-plating assay indicated that was lethal at a restrictive temperature, confirming its essentiality for growth. Microscopic analysis revealed that exhibited an oval-shaped morphology, suggesting that was required for rod-shape formation. SDS-PAGE and Western blotting analysis showed that failed to synthesize lipid A, consistent with its function in lipid A biosynthesis. Strong expression of but not the non-homologous isoenzyme or impeded growth and caused cell lysis, implying that -specific cofactors were required for this toxic effect in . Together, our results demonstrate that is essential for lipid A biosynthesis, rod-shaped growth, and viability in . We propose that this plasmid-based conditional allele is a useful tool for the genetic study of essential genes in .