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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.
摘要：
在大肠杆菌中脂质A生物合成的Raetz途径中的许多酶是必需的。已知铜绿假单胞菌中的同源蛋白Pa1792|LpxH补充了大肠杆菌中LpxH的损失。全基因组转座子插入测序分析表明lpxH在铜绿假单胞菌中是必需的。然而,尚未进行铜绿假单胞菌中lpxH的遗传分析，部分原因是必需基因的条件等位基因不容易构建。在这项研究中,我们首先在铜绿假单胞菌PAO1中构建了基于质粒的温度敏感突变体ΔlpxH/pTS-lpxH或lpxH（Ts）。点电镀分析表明lpxH(Ts)在限制性温度下是致命的，确认其增长的重要性。显微分析表明，lpxH(Ts)表现出椭圆形的形态，这表明棒状形成需要lpxH。SDS-PAGE和Western印迹分析显示lpxH(Ts)未能合成脂质A,与其在脂质A生物合成中的功能一致。lpxH而非同源同工酶lpxI或lpxG的强表达阻碍了生长并导致细胞裂解，这意味着铜绿假单胞菌的这种毒性作用需要lpxH特异性辅因子。一起,我们的结果表明，lpxH对脂质A的生物合成至关重要，杆状生长，和在铜绿假单胞菌中的生存力。我们认为，这种基于质粒的条件等位基因是铜绿假单胞菌必需基因遗传研究的有用工具。
