Nitrogen assimilation by E. coli in the mammalian intestine.

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. IMPORTANCE: 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.

Nutrition of Escherichia coli within the intestinal microbiome.

In this chapter, we update our 2004 review of "The Life of Commensal Escherichia coli in the Mammalian Intestine" (https://doi.org/10.1128/ecosalplus.8.3.1.2), with a change of title that reflects the current focus on "Nutrition of E. coli within the Intestinal Microbiome." The earlier part of the previous two decades saw incremental improvements in understanding the carbon and energy sources that E. coli and Salmonella use to support intestinal colonization. Along with these investigations of electron donors came a better understanding of the electron acceptors that support the respiration of these facultative anaerobes in the gastrointestinal tract. Hundreds of recent papers add to what was known about the nutrition of commensal and pathogenic enteric bacteria. The fact that each biotype or pathotype grows on a different subset of the available nutrients suggested a mechanism for succession of commensal colonizers and invasion by enteric pathogens. Competition for nutrients in the intestine has also come to be recognized as one basis for colonization resistance, in which colonized strain(s) prevent colonization by a challenger. In the past decade, detailed investigations of fiber- and mucin-degrading anaerobes added greatly to our understanding of how complex polysaccharides support the hundreds of intestinal microbiome species. It is now clear that facultative anaerobes, which usually cannot degrade complex polysaccharides, live in symbiosis with the anaerobic degraders. This concept led to the "restaurant hypothesis," which emphasizes that facultative bacteria, such as E. coli, colonize the intestine as members of mixed biofilms and obtain the sugars they need for growth locally through cross-feeding from polysaccharide-degrading anaerobes. Each restaurant represents an intestinal niche. Competition for those niches determines whether or not invaders are able to overcome colonization resistance and become established. Topics centered on the nutritional basis of intestinal colonization and gastrointestinal health are explored here in detail.

OmpC-Dependent Bile Tolerance Contributes to E. coli Colonization of the Mammalian Intestine.

Escherichia coli persistently colonizes the mammalian intestine by mechanisms that are not fully understood. Previously, we found when streptomycin-treated mice were fed E. coli MG1655, the intestine selected for envZ missense mutants that outcompeted the wild type. The better-colonizing envZ mutants had a higher level of OmpC and reduced OmpF. This suggested the EnvZ/OmpR two-component system and outer membrane proteins play a role in colonization. In this study, we show that wild-type E. coli MG1655 outcompetes an envZ-ompR knockout mutant. Moreover, ompA and ompC knockout mutants are outcompeted by the wild type, while an ompF knockout mutant colonizes better than the wild type. Outer membrane protein gels show the ompF mutant overproduces OmpC. An ompC mutant is more sensitive to bile salts than the wild type and ompF mutant. The ompC mutant initiates colonization slowly because it is sensitive to physiological concentrations of bile salts in the intestine. Overexpression of ompC under the control of a constitutive promoter confers a colonization advantage only when ompF is deleted. These results indicate that fine-tuning of OmpC and OmpF levels is needed to maximize competitive fitness in the intestine. RNA sequencing reveals the EnvZ/OmpR two-component system is active in the intestine: ompC is upregulated and ompF is downregulated. While other factors could also contribute to the advantage provided by OmpC, we provide evidence that OmpC is important for E. coli to colonize the intestine because its smaller pore size excludes bile salts or other unknown toxic substances, while OmpF is deleterious because its larger pore size allows bile salts or other unknown toxic substances to enter the periplasm. IMPORTANCE Every mammalian intestine is colonized with Escherichia coli. Although E. coli is one of the most studied model organisms, how it colonizes the intestine is not fully understood. Here, we investigated the role of the EnvZ/OmpR two-component system and outer membrane proteins in colonization of the mouse intestine by E. coli. We report that an ompC mutant is a poor colonizer, while an ompF mutant, which overproduces OmpC, outcompetes the wild type. OmpF has a larger pore size that allows toxic bile salts or other toxic compounds into the cell and is deleterious for colonization of the intestine. OmpC has a smaller pore size and excludes bile salts. Our findings provide insights into why E. coli fine-tunes the levels of OmpC and OmpF during colonization via the EnvZ/OmpR two-component system.