Whole-genome expression profiling revealed Escherichia 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 relative fitness of the mutants for colonization of the mouse intestine in competition with their wild-type parent. 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, having an impact on 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 nutrientniche hypothesis, which states that the ecological niches within the intestine are defined by nutrient availability. Because 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.

The carbon sources that support the growth of pathogenic Escherichia coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, the pathogenic E. coli EDL933 grows primarily as single cells dispersed within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogenic strain and the commensal E. coli strain MG1655 modes of metabolism in vitro, using a mixture of the sugars known to be present in cecal mucus, and found that the two strains used the 13 sugars in a similar order and cometabolized as many as 9 sugars at a time. We conducted systematic mutation analyses of E. coli EDL933 and E. coli MG1655 by using lesions in the pathways used for catabolism of 13 mucus-derived sugars and five other compounds for which the corresponding bacterial gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both bacterial genetic backgrounds was fed to streptomycin-treated mice, together with the respective wild-type parent strain, and their colonization was monitored by fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations that caused colonization defects, we determined that both E. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coli EDL933 used galactose, hexuronates, mannose, and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive with E. coli EDL933 but not with E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota.

Infect. Immun. 58: [2438][2439][2440][2441][2442][2443][2444][2445] 1990), but what nutrients and metabolic pathways are employed during colonization has not been determined. In this study, when the wild-type EDL933 strain was fed to mice along with an EDL933 ⌬ppsA ⌬pckA mutant, which is unable to utilize tricarboxylic acid cycle intermediates and gluconeogenic substrates for growth, both strains colonized the mouse intestine equally well. Therefore, EDL933 utilizes a glycolytic substrate(s) for both initial growth and maintenance when it is the only E. coli strain fed to the mice. However, in the presence of large numbers of MG1655, a K-12 strain, it is shown that EDL933 utilizes a glycolytic substrate(s) for initial growth in the mouse intestine but appears to utilize both glycolytic and gluconeogenic substrates in an attempt to maintain colonization. It is further shown that MG1655 predominantly utilizes glycolytic substrates for growth in the mouse intestine whether growing in the presence or absence of large numbers of EDL933. Data are presented showing that although small numbers of EDL933 grow to large numbers in the intestine in the presence of large numbers of MG1655 when both strains are fed to mice simultaneously, precolonization with MG1655 affords protection against subsequent colonization by EDL933. Moreover, in mice that are precolonized with EDL933, small numbers of MG1655 are able to grow rapidly in the intestine and EDL933 is eliminated. In situ hybridization experiments using E. coli-specific rRNA probes showed that while MG1655 is found only in mucus, EDL933 is found both in mucus and closely associated with intestinal epithelial cells. The data are discussed with respect to competition for nutrients and to the protection that some intestinal commensal E. coli strains might afford against infection by O157:H7 strains.Escherichia coli strains of serotype O157:H7 cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans (reviewed in reference 14). E. coli O157:H7 initiates infection by binding to intestinal epithelial cells and producing Shiga toxins Stx1 and/or Stx2, depending on the strain (reviewed in reference 14). Stx1 and Stx2 depurinate a critical residue in the eucaryotic 28S rRNA of 60S ribosomes, resulting in the inhibition of protein synthesis and consequent cell death (33). E. coli EDL933, an O157:H7 strain, does not normally kill streptomycin-treated mice and appears to colonize the mouse intestine by growing in intestinal mucus (38, 40), but little is known about the nutrients that are utilized for growth or the metabolic pathways involved. If these pathways were defined, it is likely that preventative measures or more effective treatments for patients infected with O157:H7 strains could be developed. With this goal in mind, we isolated an EDL933 ⌬ppsA ⌬pckA mutant, which grows normally on glycolytic substrates but is unable to utilize tricarboxylic acid (TCA) cycle intermediates and gluconeogenic substrates for growth, and tested its ability to colonize the m...

In Salmonella enterica serovar Typhimurium, the Cra protein (catabolite repressor/activator) regulates utilization of gluconeogenic carbon sources by activating transcription of genes in the gluconeogenic pathway, the glyoxylate bypass, the tricarboxylic acid (TCA) cycle, and electron transport and repressing genes encoding glycolytic enzymes. A serovar Typhimurium SR-11 ⌬cra mutant was recently reported to be avirulent in BALB/c mice via the peroral route, suggesting that gluconeogenesis may be required for virulence. In the present study, specific SR-11 genes in the gluconeogenic pathway were deleted (fbp, glpX, ppsA, and pckA), and the mutants were tested for virulence in BALB/c mice. The data show that SR-11 does not require gluconeogenesis to retain full virulence and suggest that as yet unidentified sugars are utilized by SR-11 for growth during infection of BALB/c mice. The data also suggest that the TCA cycle operates as a full cycle, i.e., a sucCD mutant, which prevents the conversion of succinyl coenzyme A to succinate, and an ⌬sdhCDA mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 ⌬aspA mutant and an SR-11 ⌬frdABC mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. Moreover, although it appears that SR-11 replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since an SR-11 ⌬fadD mutant and an SR-11 ⌬aceA mutant were both fully virulent.In sensitive mice, Salmonella enterica serovar Typhimurium causes a systemic, often fatal disease, similar to human typhoid fever (43). After ingestion, serovar Typhimurium survives passage through the acidic environment of the stomach and reaches the terminal ileum where, within 30 min, it invades M cells in the Peyer's patches (22). Within 60 min, the M cells are destroyed and serovar Typhimurium gains access to both adjacent enterocytes and to underlying lymphoid cells in the mesenteric lymph follicles of the Peyer's patches (22,24). Serovar Typhimurium grows logarithmically in Peyer's patches for 2 days (20) and simultaneously disseminates systemically to the liver and spleen, where it grows in macrophages (40, 43). Mice usually become ruffled and lethargic 3 to 5 days post-oral infection and usually die within 7 to 12 days. Despite much work relating to the relevant virulence mechanisms, little is known about the nutrition of serovar Typhimurium during infection.The Cra protein (catabolite repressor/activator) is a regulator of central carbon metabolism in salmonellae and Escherichia coli. Cra is a transcription factor that activates genes encoding key enzymes in the glyoxylate bypass, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and electron transport and represses genes encoding key enzymes in the EmbdenMeyerhof and Entner-Doudoroff pathways (42). It has recently been reported that a cra mutant of serovar Typhimurium SR-11 is totally avirulent and immunogenic in...