Carbon Nutrition of E. coli in the Mouse Intestine

Chang, et al., 2004, Proc. Nat. Acad. Sci. 101: 7427-7432 (.pdf)

This page provides access to DNA array and mouse colonization supplemental data.

Mucus Array experiment design

Mouse Colonization experiment design

Project summary. Whole-genome expression profiling revealed E. coli MG1655 genes induced by growth on mucus, conditions designed to mimic nutrient availability in the mammalian intestine. Most were nutritional genes corresponding to catabolic pathways for nutrients found in mucus. We knocked out several pathways and tested the mutants in competition with their wildtype parent for their relative fitness for colonization of the mouse intestine. We found that only mutations in sugar pathways affected colonization, not phospholipid and amino acid catabolism, not gluconeogenesis, not the tricarboxylic acid cycle, and not the pentose phosphate pathway. Gluconate appeared to be a major carbon source used by E. coli MG1655 to colonize, impacting both the initiation and maintenance stages. N-acetylglucosamine and N-acetylneuraminic acid appeared to be involved in initiation, but not maintenance. Glucuronate, mannose, fucose, and ribose appeared to be involved in maintenance, but not initiation. The in vitro order of preference for these seven sugars paralleled the relative impact of the corresponding metabolic lesions on colonization: gluconate > N-acetylglucosamine > N-acetylneuraminic acid = glucuronate > mannose > fucose > ribose. The results of this systematic analysis of nutrients used by E. coli MG1655 to colonize the mouse intestine are intriguing in light of the nutrient-niche hypothesis, which states that the ecological niches within the intestine are defined by nutrient availability. Since humans are presumably colonized with different commensal strains, differences in nutrient availability may provide an open niche for infecting E. coli pathogens in some individuals and a barrier to infection in others.

Mucus array experiments

MG1655_MG_MM_PH1 minimal glucose vs. 10 mg/ml minimal mucus: phase 1
MG1655_MG_MM_PH2 minimal glucose vs. 10 mg/ml minimal mucus: phase 2
MG1655_MG_V_MM minimal glucose vs. 5 mg/ml minimal mucus
MG1655_MG_V_CG minimal glucose vs. glucose complete (supplemented)
MG1655_MG_V_CM minimal glucose vs. mucus complete (supplemented)

E. coli Gene Expresssion Database (Oracle) Interface

Rationale and culture conditions. Since E. coli is known to grow rapidly in vivo within the mucus layer of the intestine, presumably using nutrients derived from the mucus, we grew E. coli in vitro in minimal salts medium containing lyophilized mucus as the sole source of carbon and energy. E. coli MG1655 was grown in triplicate at 37 degrees C, without shaking, in 18 mm test tubes containing 5 ml of the MOPS-based culture medium designed by Neidhardt for proteome studies. Mouse cecal mucus was prepared from streptomycin-treated CD-1 mice (not colonized with E. coli). Cultures were grown in triplicate to early (A600 = 0.1) or late (A600 = 0.3) logarithmic phase in 5 or 10 mg/ml of lyophilized mucus. Duplicate or quaduplicate arrays were obtained for each condition. For further information regarding culture conditions and preparation of mucus, please refer to Moller, et al.

MG1655-StrR grown on mucus (5 mg/ml)

Mouse Colonization Assays (Go to all public datasets)
Gluconate (edd)
N-acetylglucosamine(nagE manXYZ)
N-acetylneuraminic (sialic) acid (nanAT)
Glucuronate (uxaC)
Mannose (manA)
Fucose (fucK and fucAO)
Ribose (rbsK)
Galacturonate (uxaB)
Ethanolamine (eutBC)
Glycerol-3-phosphate from phospholipids (glpTQ)
Glycerol (glpK)
Fructuronate (gntP)
B-glucuronides (uidA)
Tryptophan (tnaA)
Glycolysis (pgi phosphoglucose isomerase)
Pentose phosphate pathway (gnd 6-phosphogluconate dehydrogenase)
TCA cycle (sdhB succinate dehydrogenase)
Gluconeogenesis (ppsA pckA phosphoenolpyruvate synthase and phosphoenolpyruvate carboxykinase)

The Streptomycin-treated Mouse Model of Intestinal Colonization

Overview: The streptomycin-treated mouse is the model of choice for determining the relative fitness of enteric bacteria for intestinal colonization, which can be subdivided in two distinct stages, initiation and maintenance (Miranda, et al., 2004; Moeller, et. al., 2003). During initiation (5 h to three days post-feeding), small numbers of E. coli grow rapidly to high numbers. A few days following initiation, the growth rate and slough rate become balanced to achieve a stable population in the maintenance stage (day 7 post-feeding and beyond). Streptomycin treatment preferentially eliminates the facultative flora and leaves the anaerobic flora largely intact. Like the conventional mouse, the streptomycin-treated mouse large intestine contains a myriad of species, each competing for available nutrients. It is therefore likely that the rapid growth of E. coli in the intestine during the initiation stage depends on utilization of non-limiting nutrients made available by removing the streptomycin-sensitive facultative microflora, i.e., nutrients which the anaerobic flora apparently do not utilize.

Rationale: The way to find out what an individual strain chooses to grow on in the intestine - in the absence of competition from other E. coli strains -- is to simultaneously feed the wildtype parent with an isogenic mutant which is blocked in a specific metabolic pathway. If that pathway and the corresponding nutrient are important, then the mutant will be less fit to compete with the wildtype. Mutant strains that do not compete effectively for nutrients involved in initiation will fail to reach the same high numbers as the wildtype. Since rapid growth during initiation occurs when nutrients are not limiting, these experiments reveal the in vivo nutrient preference. Mutants which do not compete effectively for nutrients involved in maintenance (whether or not they initiate efficiently) will decline in numbers relative to the wildtype during the maintenance stage. The streptomycin-treated mouse colonization assay provides a relative measure of fitness - the nutrients involved and whether the nutrient has a major, significant, or minor role. These experiments are attractive in that even a negative result - a mutation that has no effect on fitness for colonization - is useful.

Experimental approach: Streptomycin-treated mouse colonization assays can be designed in several different ways to determine the relative fitness of two or more strains for colonization. The method used to compare the large intestine colonizing abilities of E. coli strains in mice has been described previously (Sweeney, et al., 1996). Briefly, three male CD-1 mice (5-8 weeks old, from Jackson Laboratories) are given drinking water containing streptomycin sulfate (5 g/liter) for 24 h to eliminate resident facultative bacteria (Hentges, et al., 1984; Miller and Bonhoff, 1963). Following 18 h of starvation for food and water, the mice are fed 1 ml of 20% (w/v) sucrose containing Luria broth-grown E. coli strains, depending on the experiment. After ingesting the bacterial suspension, both the food (Charles River Valley Rat, Mouse, and Hamster Formula) and streptomycin-water were returned to the mice, and 1 gram of feces is collected after 5 h, 24 h, and on odd numbered days at the indicated times. Mice are housed individually in cages without bedding and placed in clean cages daily. Fecal samples (no older than 24 h) are homogenized in 1% Bacto Tryptone, diluted in the same medium, and plated on Luria agar plates with appropriate antibiotics. Plates contain streptomycin sulfate (100 µg/ml), streptomycin sulfate (100 µg/ml) and nalidixic acid (50 µg/ml), or streptomycin sulfate (100 µg/ml) and chloramphenicol (30 µg/ml). All plates are incubated for 18 to 24 h at 37*C prior to counting. The NalR and other antibiotic resistance markers have no effect on colonization in control experiments.

Nutrients used for initiation: (simultaneous low feeding experiments) Three mice are fed 100,000 cfu each of two E. coli strains. The strains are counted in feces at 5 hours, 1 day, and at 2 day intervals post-feeding. A mutant strain that is defective in its ability to initiate colonization will reach lower numbers, relative to the wildtype parent, within 3 days post-feeding, whereas the wildtype normally grows from 100 thousand to about 10 million cfu per gram of feces within 1 day post-feeding. A strain that has no trouble initiating colonization but is defective in the maintenance stage will grow to the same high level as the wildtype strain for 1-3 days and then will decline in numbers relative to the wildtype. This experiment is designed to reveal the relative contribution of nutrients that are involved in the initiation and/or maintenance stages of colonization in competition with the wildtype parent.

Nutrients used for maintenance: (pre-colonization experiments) The simultaneous low feeding experiment does not always provide an accurate measure of the impact of a particular mutation on maintenance; the following experiment does. Three mice are fed 100 thousand CFU of the E. coli wildtype and allowed to reach the maintenance stage (10 days after feeding); then the mice are held overnight without food and water and the next morning fed 10 billion cfu of the mutant strain. In a control experiment, mice are challenged with a differentially marked wildtype strain, which would be expected to colonize. Post-feeding, feces are collected from each mouse, and counted as described above, at 5 hours, 1 day, and 2 day intervals for 15 days. If the mutant strain has no trouble in maintenance, it will remain in the intestine and co-colonize with the wildtype parent in high numbers. If the mutant strain has difficulty in maintaining colonization, it will decline in numbers, relative to the wildtype parent, to the degree that the nutrient in question affects maintenance.

Feeding alone: Mutants with a severe defect in initiation, maintenance, or both are tested to see if they are able to colonize in the absence of competition with the wildtype parent, i.e., when fed alone. Three mice are fed 100 thousand CFU of the E. coli mutant and the strain counted in feces at 5 hours post-feeding, 1 day post-feeding, and at 2 day intervals thereafter. Strains that cannot colonize will be lost from feces. This experiment implicates a nutrient as being important, but indicates that an alternative strategy may be required (i.e., mutation of a different step in the pathway to be sure that toxic metabolites are not involved).


Chang, D.E., D. Smalley, D. L. Tucker, M. P. Leatham, W. E. Norris, S. J. Stevenson, A. B. Anderson, J. E. Grissom, D. C. Laux, P. S. Cohen, and T. Conway. Carbon nutrition of Escherichia coli in the mouse intestine. 2004. Proc. Nat. Acad. Sci. 101: 7427-7432 .pdf PubMed

Hentges, D. J., J. U. Que, et al. (1984). "The influence of streptomycin on colonization in mice." Microecol Theor 14: 53-62.

Miller, C. P., and M. Bohnhoff. 1963. Changes in the mouse's enteric microflora associated with enhanced susceptibility to Salmonella infection following streptomycin-treatment. J. Infect. Dis. 113:59-66.

Miranda, R. L., T. Conway, M. P. Leatham, D. E. Chang, D. L. Tucker, W. E. Norris, J. H. Allen, S. J. Stevenson, D. C. Laux, and P. S. Cohen. 2004. Glycolytic and gluconeogenic growth of Escherichia coli O157:H7 (EDL933) and E. coli K-12 (MG1655) in the mouse intestine. Infect. Immun. 72: 1666-76. .pdf PubMed

Møller, A., T. Conway, P. Nuijten, K. A. Krogfelt and P. S. Cohen. 2003. An Escherichia coli MG1655 lipopolysaccharide deep-rough core mutant grows and survives in mouse cecal mucus, but fails to colonize the mouse large intestine. Infect. Immun. 71: 2142-2152. .pdf PubMed

Sweeney, N. J., P. Klemm, B. A. McCormick, E. Moller-Nielsen, M. Utley, M. A. Schembri, D. C. Laux, and P. S. Cohen. 1996. The Escherichia coli K-12 gntP gene allows E. coli F-18 to occupy a distinct nutritional niche in the streptomycin-treated mouse large intestine. Infect. Immun. 64:3497-3503.

Sweeney, N. J., D. C. Laux, and P. S. Cohen. 1996. Escherichia coli F-18 and K-12 eda mutants do not colonize the streptomycin-treated mouse large intestine. Infect. Immun. 64:3504-3511.

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OU Bioinformatics Core Facility @ Advanced Center for Genome Technology | Credits | updated:21 Dec 2005