GROWTH AND COLONIZATION OF THE INTESTINE BY E COLI

要在动物宿主上定居，细菌必须获得生长所需的营养。然而，尽管进行了广泛的研究，但对于生活在哺乳动物大肠中的400-500种物种在原位使用的营养物质和代谢途径几乎一无所知。这项拟议的研究利用功能基因组学技术来研究肠道细菌在定植过程中对营养物质的获取。具体地说，我们将测试这一假设，即诱导用于粘液衍生糖的分解代谢的途径对大肠杆菌的定植至关重要。全球转录分析将被用来确定在肠道粘液中存在的复杂营养混合物上诱导生长的调节基因。从含有盲肠粘液的最小培养基上生长的细胞制备的探针将与所有大肠杆菌基因的DNA阵列杂交。通过分析实验性无菌小鼠盲肠中大肠杆菌细胞的整体转录模式，将确定在粘液上原位诱导生长的途径。将研究有助于大肠杆菌成功地与大肠中的400-500个其他物种竞争的途径。在特定分解代谢途径中被选择性阻断的突变菌株将被测试它们在小鼠身上的定植能力，绿色荧光蛋白报告融合将被用来确定这些调节子在定植过程中在单个细菌细胞中的时空表达。这些实验将确定单个粘膜糖在大肠杆菌大肠定植中的不同生理作用，为解决更一般的问题提供重要的知识，即代谢多样性是否允许致病大肠杆菌菌株在预定植菌株存在的情况下定植，从而导致它们致病的能力。准确地了解哪些营养物质对定居是必不可少的，这将导致改进抗击感染的战略。

To colonize an animal host, bacteria must acquire nutrients for growth. However, despite extensive research, next to nothing is known about the nutrients and metabolic pathways used in situ by the 400-500 species which inhabit the mammalian large intestine. The proposed research uses functional genomics technology to investigate the acquisition of nutrients by enteric bacteria during the colonization process. Specifically, we will test the hypothesis that induction of the pathways used for catabolism of mucus-derived sugars is essential for colonization of E. coli. Global transcription assays will be used to identify regulons induced for growth on the complex mixture of nutrients present in intestinal mucus. Probes prepared from cells grown on minimal media containing cecal mucus will be hybridized to DNA arrays of all E. coli genes. Pathways induced in situ for growth on mucus will be identified by analyzing global transcription patterns of E. coli cells in the ceca of experimentally colonized germ-free mice. Pathways which contribute to the ability of E. coli to successfully compete with the 400-500 other species in the large intestine will be examined. Mutant strains selectively blocked in specific catabolic pathways will be tested for their ability to colonize mice and green fluorescent protein reporter fusions will be used to determine the temporal and spatial expression of these regulons in individual bacterial cells during colonization. These experiments will identify the distinct physiological roles of individual mucosal sugars in colonization of the large intestine by E. coli, providing knowledge important for addressing the more general question of whether or not metabolic diversity allows pathogenic E. coli strains to colonize in the presence of precolonized strains, thus leading to their ability to cause disease. Knowing precisely which nutrients are essential for colonization should lead to improved strategies for combating infection.