Tannock Gerald W, Lawley Blair, Munro Karen, Sims Ian M, Lee Julian, Butts Christine A, Roy Nicole
Department of Microbiology, University of Otago, Dunedin, New Zealand.
Appl Environ Microbiol. 2014 Apr;80(7):2240-7. doi: 10.1128/AEM.03799-13. Epub 2014 Jan 31.
Knowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope ((13)C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [(13)C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect (13)C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed that Bacteroides uniformis, Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis were the main users of the (13)C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified. B. uniformis utilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA-stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.
为了理解肠道微生物群的复杂系统发育和功能，需要了解支撑其组成的营养趋向。为了检测盲肠微生物群中特定成员对菊粉衍生底物的利用情况，一次性将稳定同位素（(13)C）标记的菊粉添加到大鼠饮食中。在提供[(13)C]菊粉饮食之前（0小时）以及之后的6、12、18和24小时，收集食用含纤维菊粉饲料大鼠的盲肠消化物。从这些盲肠标本中提取RNA，并在等密度浮力密度梯度中进行分级分离，以检测源自代谢了标记饮食成分的细菌细胞的(13)C标记核酸。从提供标记饮食后收集的标本中提取的RNA比0小时的RNA密度更大。对从这些级分获得的cDNA扩增的16S rRNA基因进行测序表明，均匀拟杆菌、解葡糖布劳特氏菌、吲哚梭菌和动物双歧杆菌是(13)C标记底物的主要使用者。对这些细菌菌株进行基于培养的研究，能够确定与菊粉及其水解产物相关的营养趋向。均匀拟杆菌利用含纤维菊粉生长，而其他物种利用低聚果糖和单糖。因此，RNA稳定同位素探测（RNA-SIP）提供了关于微生物群代谢中菊粉碳利用的新信息。
