Unit of Bacterial Genetics, Gdansk University of Technology, 80-233 Gdansk, Poland.
Int J Mol Sci. 2020 Nov 29;21(23):9088. doi: 10.3390/ijms21239088.
We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of Δ revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of Δ by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated , suggesting LapB and LapC act antagonistically. The same gene was identified during selection for mutations that induce transcription from LPS defects-responsive P3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of mutants that restored growth at elevated temperatures mapped to /, and genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in mutants. Interestingly, a deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.
我们之前曾表明，脂多糖（LPS）的组装需要必需的 LapB 蛋白来调节 FtsH 介导的 LpxC 蛋白的蛋白水解，LpxC 蛋白催化 LPS 合成的第一步。为了进一步了解 LapB 的必需功能及其在 LpxC 周转中的作用，Δ 的多拷贝抑制因子揭示了 HslV 蛋白酶亚基的过表达通过 LpxC 的蛋白水解降解来防止其致死性，提供了 LpxC 降解的替代途径。分离和鉴定外显子抑制突变，通过恢复正常 LPS 合成来防止 Δ 的致死性，鉴定出必需基因中 377 aa 后的移码突变，表明 LapB 和 LapC 拮抗作用。在选择诱导 LPS 缺陷反应 P3 启动子转录的突变时，也鉴定出相同的 基因，赋予对 LpxC 抑制剂 CHIR090 的敏感性和温度敏感表型。在升高的温度下恢复生长的 突变体的抑制剂突变体被定位到 / 、和 基因。这种抑制剂突变恢复了 LPS 的正常水平，并防止了 LpxC 在 突变体中的蛋白水解。有趣的是，在过表达 LpxC 或缺乏 LapB 的菌株中可以构建 缺失，这表明 FtsH、LapB 和 LapC 通过控制 LpxC 量共同调节 LPS 合成。
