State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Biotechnol Appl Biochem. 2022 Jun;69(3):1080-1093. doi: 10.1002/bab.2179. Epub 2021 May 9.
Many genes in the biosynthetic pathway of lipopolysaccharide in Cronobacter sakazakii have not been identified. In this study, we demonstrate that an operon containing four genes ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 is responsible for L-glycero-D-mannoheptose addition on the inner core of lipopolysaccharide in C. sakazakii. The proteins encoded by these four genes are homologous to E. coli WaaQ, WaaC, WaaF, and WaaD. Lipopolysaccharide from the deletion mutants of ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 (named as △RS18945, △RS18950, △RS18955 and △RS18960, respectively) were analyzed by SDS-PAGE. △RS18945 synthesized lipopolysaccharide with similar length to the wildtype BAA-894, whereas △RS18950, △RS18955, and △RS18960 synthesized much shorter lipopolysaccharide. This suggests that the enzyme encoded by ESA_RS18945 might function as E. coli WaaQ on the sidechain of lipopolysaccharide. When E. coli WaaC, WaaF, and WaaD were overexpressed in △RS18950, △RS18955, and △RS18960, respectively, the full length of lipopolysaccharide was recovered. Mass spectrometry analysis indicates that △RS18950 and △RS18960 only synthesized Kdo -lipid A, confirming that enzymes encoded by ESA_RS18950 and ESA_RS18960 have similar functions to E. coli WaaC and WaaD, respectively. Hep-Kdo -lipid A with a phosphoethanolamine was produced in △RS18955, suggesting that the enzyme encoded by ESA_RS18955 has similar function to E. coli WaaF.
在阪崎克罗诺杆菌的脂多糖生物合成途径中，有许多基因尚未被鉴定。在本研究中，我们证明了一个包含四个基因 ESA_RS18945、ESA_RS18950、ESA_RS18955 和 ESA_RS18960 的操纵子负责在阪崎克罗诺杆菌脂多糖的内核心上添加 L-甘油-D-甘露庚糖。这些基因编码的蛋白质与大肠杆菌的 WaaQ、WaaC、WaaF 和 WaaD 同源。缺失突变体 ESA_RS18945、ESA_RS18950、ESA_RS18955 和 ESA_RS18960 的脂多糖（分别命名为△RS18945、△RS18950、△RS18955 和△RS18960）通过 SDS-PAGE 进行分析。与野生型 BAA-894 相比，△RS18945 合成的脂多糖长度相似，而△RS18950、△RS18955 和△RS18960 合成的脂多糖短得多。这表明 ESA_RS18945 编码的酶可能在脂多糖侧链上充当大肠杆菌的 WaaQ。当大肠杆菌的 WaaC、WaaF 和 WaaD 分别在△RS18950、△RS18955 和△RS18960 中过表达时，脂多糖全长得到恢复。质谱分析表明，△RS18950 和△RS18960 仅合成 Kdo-脂酰基 A，证实 ESA_RS18950 和 ESA_RS18960 编码的酶分别具有与大肠杆菌的 WaaC 和 WaaD 相似的功能。在△RS18955 中产生了带有磷酸乙醇胺的 Hep-Kdo-脂酰基 A，表明 ESA_RS18955 编码的酶具有与大肠杆菌 WaaF 相似的功能。
