Kachlany S C, Levery S B, Kim J S, Reuhs B L, Lion L W, Ghiorse W C
Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Environ Microbiol. 2001 Dec;3(12):774-84. doi: 10.1046/j.1462-2920.2001.00248.x.
The aromatic hydrocarbon-degrading bacterium, Pseudomonas putida G7, produces exopolymers of potential interest in biotechnological applications. These exopolymers have been shown to have significant metal-binding ability. To initiate the study of the metal-polymer interactions, we explored the physical and chemical nature of the P. putida G7 exopolysaccharide, a major component of the exopolymer. A capsular structure was observed by light microscopy surrounding both planktonic and attached cells in biofilms after immunofluorescence staining with polyclonal antiserum raised against planktonic cells. Further work with planktonic cells showed that the immunostained capsule remained associated with young (log phase) cells, whereas older (stationary phase) cells lost their capsular material to the external milieu. Visualization of frozen, hydrated stationary phase cells by cryo-field emission scanning electron microscopy (cryoFESEM) revealed highly preserved extracellular material. In contrast, conventional scanning electron microscopy (SEM) of stationary phase cells showed rope-like material that most probably results from dehydrated and collapsed exopolymer. Both capsular and released exopolymers were separated from cells, and the released extracellular polysaccharide (EPS) was purified. Deoxycholate-polyacrylamide gel electrophoresis (PAGE) and silver/alcian blue staining of the partially purified material showed that it contained both EPS and lipopolysaccharide (LPS). Further purification of the EPS using a differential solubilization technique to remove LPS yielded highly purified EPS. Gas chromatography-mass spectrometry revealed that the purified EPS contained the monosaccharides, glucose, rhamnose, ribose, N-acetylgalactosamine and glucuronic acid. The structural and chemical properties of the P. putida EPS described here increase our understanding of the mechanisms of toxic metal binding by this well-known Proteobacterium.
芳香烃降解细菌恶臭假单胞菌G7能产生在生物技术应用中具有潜在价值的胞外聚合物。这些胞外聚合物已被证明具有显著的金属结合能力。为了启动对金属 - 聚合物相互作用的研究，我们探索了恶臭假单胞菌G7胞外多糖的物理和化学性质，胞外多糖是胞外聚合物的主要成分。在用针对浮游细胞产生的多克隆抗血清进行免疫荧光染色后，通过光学显微镜观察到生物膜中浮游细胞和附着细胞周围都有荚膜结构。对浮游细胞的进一步研究表明，免疫染色的荚膜与年轻（对数期）细胞相关联，而较老（稳定期）细胞则将其荚膜物质释放到外部环境中。通过低温场发射扫描电子显微镜（cryoFESEM）对冷冻、水合的稳定期细胞进行可视化观察，发现细胞外物质保存完好。相比之下，对稳定期细胞进行传统扫描电子显微镜（SEM）观察时，显示出绳状物质，这很可能是脱水和塌陷的胞外聚合物。荚膜和释放的胞外聚合物都从细胞中分离出来，并且对释放的细胞外多糖（EPS）进行了纯化。用脱氧胆酸盐 - 聚丙烯酰胺凝胶电泳（PAGE）和银/阿尔辛蓝染色对部分纯化的物质进行分析，结果表明其同时含有EPS和脂多糖（LPS）。使用差异溶解技术进一步纯化EPS以去除LPS，得到了高度纯化的EPS。气相色谱 - 质谱分析表明，纯化的EPS含有单糖葡萄糖、鼠李糖、核糖、N - 乙酰半乳糖胺和葡萄糖醛酸。本文所述的恶臭假单胞菌EPS的结构和化学性质增进了我们对这种著名变形杆菌结合有毒金属机制的理解。
