Ojima Yoshihiro, Nguyen Minh Hong, Yajima Reiki, Taya Masahito
Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka, Japan
Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka, Japan.
Appl Environ Microbiol. 2015 Sep 1;81(17):5900-6. doi: 10.1128/AEM.01011-15. Epub 2015 Jun 19.
Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.
微生物絮凝是分散的细菌细胞以絮状物或薄片形式聚集的现象。在本研究中，对通过过表达bcsB基因使大肠杆菌细胞自发絮凝的机制进行了研究。在所检测的各种大肠杆菌菌株（包括K-12、B和O菌株）中，bcsB过表达诱导的絮凝是一致的，形成的絮状物结构类似纸屑，大小约为1至2毫米。通过三维共聚焦激光扫描显微镜研究了绿色荧光蛋白标记的大肠杆菌细胞在絮状物结构中的分布。絮状物对蛋白酶K敏感，表明絮状物的主要成分是蛋白质。对絮状物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和纳升液相色谱串联质谱分析，有力地表明外膜囊泡（OMV）参与了大肠杆菌的絮凝过程。通过对絮状物的透射电子显微镜观察，证实了OMV参与絮凝。此外，在具有低囊泡化表型的菌株（ΔdsbA和ΔdsbB菌株）中，bcsB诱导的大肠杆菌絮凝受到极大抑制。因此，我们的结果表明自发絮凝与大肠杆菌细胞OMV产生增加之间存在密切关联。
