Ren Ge, Wang Zhou, Li Ye, Hu Xiaoqing, Wang Xiaoyuan
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
School of Biotechnology, Jiangnan University, Wuxi, China.
J Bacteriol. 2016 May 13;198(11):1576-1584. doi: 10.1128/JB.00094-16. Print 2016 Jun 1.
When 10 Escherichia coli mutant strains with defects in lipopolysaccharide (LPS) core biosynthesis were grown on agar medium at 30°C, four of them, the ΔwaaF, ΔwaaG, ΔwaaP, and ΔwaaB strains, formed mucoid colonies, while the other six, the ΔwaaU, ΔwaaR, ΔwaaO, ΔwaaC, ΔwaaQ, and ΔwaaY strains, did not. Using light microscopy with tannin mordant staining, the presence of exopolysaccharide around the cells of the mutants that formed mucoid colonies could be discerned. The ΔwaaF mutant produced the largest amounts of exopolysaccharide, regardless of whether it was grown on agar or in liquid medium. The exopolysaccharide was isolated from the liquid growth medium of ΔwaaF cells, hydrolyzed, and analyzed by high-performance liquid chromatography with an ion-exchange column, and the results indicated that the exopolysaccharide was consistent with colanic acid. When the key genes related to the biosynthesis of colanic acid, i.e., wza, wzb, wzc, and wcaA, were deleted in the ΔwaaF background, the exopolysaccharide could not be produced any more, further confirming that it was colanic acid. Colanic acid could not be produced in strains in which rcsA, rcsB, rcsD, or rcsF was deleted in the ΔwaaF background, but a reduced level of colanic acid production was detected when the rcsC gene was deleted, suggesting that a change of lipopolysaccharide structure in ΔwaaF cells might be sensed by the RcsCDB phosphorelay system, leading to the production of colanic acid. The results demonstrate that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural deficiency of lipopolysaccharide.
Lipopolysaccharide and colanic acid are important forms of exopolysaccharide for Escherichia coli cells. Their metabolism and biological significance have been investigated, but their interrelation with the cell stress response process is not understood. This study demonstrates, for the first time, that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural change of lipopolysaccharide, suggesting that bacterial cells can monitor the outer membrane integrity, which is essential for cell survival and damage repair.
当10株脂多糖（LPS）核心生物合成存在缺陷的大肠杆菌突变株在30°C的琼脂培养基上生长时，其中4株，即ΔwaaF、ΔwaaG、ΔwaaP和ΔwaaB菌株，形成了黏液状菌落，而其他6株，即ΔwaaU、ΔwaaR、ΔwaaO、ΔwaaC、ΔwaaQ和ΔwaaY菌株则没有。使用单宁媒染染色的光学显微镜，可以辨别出形成黏液状菌落的突变株细胞周围存在胞外多糖。无论在琼脂上还是在液体培养基中生长，ΔwaaF突变株产生的胞外多糖量最大。从ΔwaaF细胞的液体生长培养基中分离出胞外多糖，进行水解，并通过离子交换柱高效液相色谱分析，结果表明该胞外多糖与结肠酸一致。当在ΔwaaF背景中缺失与结肠酸生物合成相关的关键基因，即wza、wzb、wzc和wcaA时，不再产生胞外多糖，进一步证实其为结肠酸。在ΔwaaF背景中缺失rcsA、rcsB、rcsD或rcsF的菌株中不能产生结肠酸，但当缺失rcsC基因时，检测到结肠酸产量降低，这表明ΔwaaF细胞中脂多糖结构的变化可能被RcsCDB磷酸化传递系统感知，从而导致结肠酸的产生。结果表明，大肠杆菌细胞可通过RcsCDB磷酸化传递系统响应脂多糖的结构缺陷而激活结肠酸的产生。
脂多糖和结肠酸是大肠杆菌细胞胞外多糖的重要形式。它们的代谢和生物学意义已得到研究，但它们与细胞应激反应过程的相互关系尚不清楚。本研究首次表明，大肠杆菌细胞可通过RcsCDB磷酸化传递系统响应脂多糖的结构变化而激活结肠酸的产生，这表明细菌细胞可以监测外膜完整性，这对细胞存活和损伤修复至关重要。
