Escherichia coli Nissle 1917 (EcN) is an efficient probiotic strain extensively used worldwide because of its several health benefits. Adhesion to the intestinal cells is one of the prerequisites for a probiotic strain. To identify the genes essential for the adhesion of EcN on the intestinal cells, we utilized a quantitative genetic footprinting approach called transposon insertion sequencing (INSeq). A transposon insertion mutant library of EcN comprising of ~17,000 mutants was used to screen the adherence to the intestinal epithelial cells, Caco-2. The transposon insertion sites were identified from the input and output population by employing next-generation sequencing using the Ion torrent platform. Based on the relative abundance of reads in the input and output pools, we identified 113 candidate genes that are essential for the fitness of EcN during the adhesion and colonization on the Caco-2 cells. Functional categorization revealed that these fitness genes are associated with carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, post-translational modification, stress response, motility and adhesion, and signal transduction. To further validate the genes identified in our INSeq analysis, we constructed individual knock-out mutants in five genes (cyclic di-GMP phosphodiesterase (gmp), hda, uidC, leuO, and hypothetical protein-coding gene). We investigated their ability to adhere to Caco-2 cells. Evaluation of these mutants showed reduced adhesion on Caco-2 cells, confirming their role in adhesion. Understanding the functions of these genes may provide novel insights into molecular regulation during colonization of probiotic bacteria to the intestinal cells, and useful to develop designer probiotic strains.

大肠杆菌Nissle 1917 (EcN) 是一种高效的益生菌菌株，在世界范围内广泛使用，因为它具有多种健康益处。对肠道细胞的粘附是益生菌菌株的先决条件之一。为了鉴定 EcN 在肠细胞上粘附所必需的基因，我们使用了一种称为转座子插入测序 (INSeq) 的定量遗传足迹方法。包含约 17,000 个突变体的 EcN 转座子插入突变体文库用于筛选对肠上皮细胞 Caco-2 的粘附。通过使用 Ion Torrent 平台的下一代测序，从输入和输出群体中识别出转座子插入位点。根据输入和输出池中reads的相对丰度，我们确定了 113 个候选基因，这些基因对 Caco-2 细胞粘附和定植期间 EcN 的适应性至关重要。功能分类表明，这些适应性基因与碳水化合物转运和代谢、细胞壁/膜/包膜生物发生、翻译后修饰、应激反应、运动和粘附以及信号转导有关。为了进一步验证我们的 INSeq 分析中鉴定的基因，我们在五个基因（环状二 GMP 磷酸二酯酶（gmp )、hda、uidC、leuO和假设的蛋白质编码基因)。我们研究了它们粘附在 Caco-2 细胞上的能力。对这些突变体的评估显示对 Caco-2 细胞的粘附减少，证实了它们在粘附中的作用。了解这些基因的功能可能会为益生菌在肠道细胞定植过程中的分子调控提供新的见解，并有助于开发设计益生菌菌株。