氮是所有生物体的基本要素,包括大肠杆菌。肠道中潜在的氮源丰富,但是大肠杆菌专门用于定殖的知识仍然有限。这里,我们试图确定大肠杆菌用于定植链霉素处理的小鼠肠道的特定氮源。我们开始研究氮是否限制在肠道中。NtrBC双组分系统响应氮限制而上调约100个基因。我们发现NtrBC对大肠杆菌定植至关重要,虽然NtrBC调节子的大多数基因没有被诱导,这表明氮在肠道中不受限制。RNA-seq鉴定了定植大肠杆菌中7个氨基酸转运和分解代谢的上调基因,二肽和三肽,嘌呤,嘧啶,尿素,乙醇胺。竞争性定植实验表明,L-丝氨酸,N-乙酰神经氨酸,N-乙酰氨基葡萄糖,和二肽和三肽用作肠道中大肠杆菌的氮源。此外,L-丝氨酸脱氨酶突变体的定植缺陷是通过饮用水中过量的氮而不是通过过量的碳和能量来拯救的,证明L-丝氨酸主要作为氮源。类似的拯救实验表明,N-乙酰神经氨酸既可作为碳源,也可作为氮源。在很小的程度上,天冬氨酸和氨也作为氮源。总的来说,这些发现表明,大肠杆菌利用多种氮源成功定植小鼠肠道,其中最重要的是L-丝氨酸。重要的是,虽然人们对大肠杆菌用于定植哺乳动物肠道的碳和能源有很多了解,人们对氮源知之甚少。通过RNA-seq对定殖的大肠杆菌的询问表明,氮不是限制性的,表明肠道中氮源丰富。从几种氨基酸中同化氮的途径,二肽和三肽,嘌呤,嘧啶,尿素,和乙醇胺在小鼠中诱导。竞争性定植试验证实,缺乏L-丝氨酸分解代谢途径的突变体,N-乙酰神经氨酸,N-乙酰氨基葡萄糖,二肽和三肽有定植缺陷。小鼠的拯救实验表明,L-丝氨酸主要作为氮源,而N-乙酰神经氨酸提供碳和氮。在测试的许多氮同化突变体中,最大的定植缺陷是L-丝氨酸脱氨酶突变体,这表明L-丝氨酸是定殖大肠杆菌最重要的氮源。
Nitrogen is an essential element for all living organisms, including Escherichia coli. Potential nitrogen sources are abundant in the intestine, but knowledge of those used specifically by E. coli to colonize remains limited. Here, we sought to determine the specific nitrogen sources used by E. coli to colonize the streptomycin-treated mouse intestine. We began by investigating whether nitrogen is limiting in the intestine. The NtrBC two-component system upregulates approximately 100 genes in response to nitrogen limitation. We showed that NtrBC is crucial for E. coli colonization, although most genes of the NtrBC regulon are not induced, which indicates that nitrogen is not limiting in the intestine. RNA-seq identified upregulated genes in colonized E. coli involved in transport and catabolism of seven amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine. Competitive colonization experiments revealed that L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides serve as nitrogen sources for E. coli in the intestine. Furthermore, the colonization defect of a L-serine deaminase mutant was rescued by excess nitrogen in the drinking water but not by an excess of carbon and energy, demonstrating that L-serine serves primarily as a nitrogen source. Similar rescue experiments showed that N-acetylneuraminic acid serves as both a carbon and nitrogen source. To a minor extent, aspartate and ammonia also serve as nitrogen sources. Overall, these findings demonstrate that E. coli utilizes multiple nitrogen sources for successful colonization of the mouse intestine, the most important of which is L-serine.
OBJECTIVE: While much is known about the carbon and energy sources that are used by E. coli to colonize the mammalian intestine, very little is known about the sources of nitrogen. Interrogation of colonized E. coli by RNA-seq revealed that nitrogen is not limiting, indicating an abundance of nitrogen sources in the intestine. Pathways for assimilation of nitrogen from several amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine were induced in mice. Competitive colonization assays confirmed that mutants lacking catabolic pathways for L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides had colonization defects. Rescue experiments in mice showed that L-serine serves primarily as a nitrogen source, whereas N-acetylneuraminic acid provides both carbon and nitrogen. Of the many nitrogen assimilation mutants tested, the largest colonization defect was for an L-serine deaminase mutant, which demonstrates L-serine is the most important nitrogen source for colonized E. coli.