Klaassens Eline S, Boesten Rolf J, Haarman Monique, Knol Jan, Schuren Frank H, Vaughan Elaine E, de Vos Willem M
Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.
Appl Environ Microbiol. 2009 May;75(9):2668-76. doi: 10.1128/AEM.02492-08. Epub 2009 Mar 13.
Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from young infants that were fed either breast milk or a formula containing a mixture of galacto- and fructo-oligosaccharides were studied. The presence and diversity of the bifidobacterial fecal communities were determined using PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR for specific species. Changes in the total number of bifidobacteria as well as in species diversity were observed, indicating the metabolic activities of the bifidobacteria within the infant gut. In addition, total RNAs isolated from infant feces were labeled and hybridized to a bifidobacterium-specific microarray comprising approximately 6,000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones that showed the most prominent hybridization with the samples were sequenced. Fewer than 10% of the hybridizing clones contained rRNA genes, whereas the vast majority of the inserts showed matches with protein-encoding genes predicted to originate from bifidobacteria. Although a wide range of functional groups was covered by the obtained sequences, the largest fraction (14%) of the transcribed genes assigned to a functional category were predicted to be involved in carbohydrate metabolism, while some were also implicated in exopolysaccharide production or folate production. A total of three of the above-described protein-encoding genes were selected for quantitative PCR and sequence analyses, which confirmed the expression of the corresponding genes and the expected nucleotide sequences. In conclusion, the results of this study show the feasibility of obtaining insight into the transcriptional responses of intestinal bifidobacteria by analyzing fecal RNA and highlight the in vivo expression of bifidobacterial genes implicated in host-related functions.
尽管双歧杆菌的确切功能仍不清楚，但它们是新生儿肠道最早的定植菌之一，并会进一步发展成为丰富的群落，尤其是对饮食做出反应时。因此，研究了双歧杆菌在快速处理的来自以母乳或含有低聚半乳糖和低聚果糖混合物的配方奶喂养的幼儿粪便样本中的转录反应。使用PCR-变性梯度凝胶电泳和针对特定菌种的定量实时PCR来确定双歧杆菌粪便群落的存在和多样性。观察到双歧杆菌总数以及物种多样性的变化，这表明了婴儿肠道内双歧杆菌的代谢活性。此外，从婴儿粪便中分离出的总RNA被标记并与包含约6000个人类肠道主要双歧杆菌菌种克隆的双歧杆菌特异性微阵列杂交。对与样本杂交最显著的约270个克隆进行了测序。杂交克隆中少于10%含有rRNA基因，而绝大多数插入片段与预测源自双歧杆菌的蛋白质编码基因匹配。尽管获得的序列涵盖了广泛的功能组，但分配到一个功能类别的转录基因中最大的一部分（14%）预计参与碳水化合物代谢，同时一些基因也与胞外多糖产生或叶酸产生有关。总共选择了上述三个蛋白质编码基因进行定量PCR和序列分析，证实了相应基因的表达以及预期的核苷酸序列。总之，本研究结果表明通过分析粪便RNA来深入了解肠道双歧杆菌转录反应的可行性，并突出了与宿主相关功能有关的双歧杆菌基因的体内表达。
