Shank Elizabeth Anne
Department of Biology, University of North Carolina at Chapel Hill.
J Vis Exp. 2013 Oct 31(80):e50863. doi: 10.3791/50863.
In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.
在自然界中，细菌很少单独存在；相反，它们被各种各样的其他微生物所包围，这些微生物通过分泌代谢产物来改变局部环境。这些代谢产物有可能调节其微生物邻居的生理和分化，并且很可能是复杂微生物群落建立和维持的重要因素。我们开发了一种基于荧光的共培养筛选方法来识别这种化学介导的微生物相互作用。该筛选方法包括将荧光转录报告菌株与环境微生物在固体培养基上混合，并让菌落进行共培养生长。荧光转录报告菌株的设计使得当所选细菌菌株表达特定的感兴趣表型（即生物膜形成、孢子形成、毒力因子产生等）时会发出荧光。筛选是在该表型不表达的生长条件下进行的（因此报告菌株通常不发荧光）。当环境微生物分泌激活该表型的代谢产物时，它会扩散穿过琼脂并激活荧光报告构建体。这使得产生诱导代谢产物的微生物能够被检测到：它们是最靠近荧光菌落的非荧光菌落。因此，该筛选方法能够识别产生可扩散代谢产物从而激活报告菌株中特定生理反应的环境微生物。本出版物讨论了如何：a）选择合适的共培养筛选条件，b）准备用于筛选的报告菌株和环境微生物，c）进行共培养筛选，d）分离假定的诱导生物体，以及e）在二次筛选中确认它们的活性。我们开发这种方法是为了筛选能够激活枯草芽孢杆菌生物膜基质产生的土壤生物体；然而，我们也讨论了将这种方法应用于其他遗传上易于处理的细菌时的注意事项。
