Larsen Poul, Nielsen Jeppe Lund, Dueholm Morten Simonsen, Wetzel Ronald, Otzen Daniel, Nielsen Per Halkjaer
Department of Biotechnology, Aalborg University, Sohngaardsholmvej 57, DK-9000, Aalborg, Denmark.
Environ Microbiol. 2007 Dec;9(12):3077-90. doi: 10.1111/j.1462-2920.2007.01418.x.
Surface-associated amyloid fibrils have been described by bacteria in the family Enterbacteriaceae, but it is unknown to what extent amyloid adhesins are present in natural biofilms. In this study, amyloid adhesins were specifically stained with Thioflavin T and two conformationally specific antibodies targeting amyloid fibrils. These three independent detection methods were each combined with fluorescence in situ hybridization using fluorescently labelled oligonucleotide probes in order to link phenotype with identity. Escherichia coli mutants with and without amyloid adhesins (curli) served as controls. In biofilms from four different natural habitats, bacteria producing extracellular amyloid adhesins were identified within several phyla: Proteobacteria (Alpha-, Beta-, Gamma- and Deltaproteobacteria), Bacteriodetes, Chloroflexi and Actinobacteria, and most likely also in other phyla. Quantification of the microorganisms producing amyloid adhesins showed that they constituted at least 5-40% of all prokaryotes present in the biofilms, depending on the habitat. Particularly in drinking water biofilms, a high number of amyloid-positive bacteria were identified. Production of amyloids was confirmed by environmental isolates belonging to the Gammaproteobacteria, Bacteriodetes, Firmicutes and Actinobacteria. The new approach is a very useful tool for further culture-independent studies in mixed microbial communities, where the abundance and diversity of bacteria expressing amyloid adhesins seems much greater than hitherto anticipated.
肠杆菌科细菌已被描述能产生表面相关淀粉样纤维，但尚不清楚天然生物膜中淀粉样黏附素的存在程度。在本研究中，淀粉样黏附素用硫黄素T以及两种靶向淀粉样纤维的构象特异性抗体进行特异性染色。这三种独立的检测方法分别与使用荧光标记寡核苷酸探针的荧光原位杂交相结合，以便将表型与身份联系起来。有无淀粉样黏附素（卷曲菌毛）的大肠杆菌突变体用作对照。在来自四个不同自然栖息地的生物膜中，在几个门中鉴定出了产生细胞外淀粉样黏附素的细菌：变形菌门（α-、β-、γ-和δ-变形菌纲）、拟杆菌门、绿弯菌门和放线菌门，很可能在其他门中也有。对产生淀粉样黏附素的微生物进行定量分析表明，根据栖息地不同，它们占生物膜中所有原核生物的比例至少为5%-40%。特别是在饮用水生物膜中，鉴定出了大量淀粉样蛋白阳性细菌。属于γ-变形菌纲、拟杆菌门、厚壁菌门和放线菌门的环境分离株证实了淀粉样蛋白的产生。这种新方法是在混合微生物群落中进行进一步非培养研究的非常有用的工具，在这些群落中，表达淀粉样黏附素的细菌的丰度和多样性似乎比迄今预期的要大得多。
