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The pathogenic Escherichia coli EDL933 is known to cause infection in the intestine. E. coli Nissle 1917 is used as a probiotic strain to prevent infection (63). When the mouse intestine is pre-colonized with E. coli Nissle 1917 it prevents E. coli EDL933 from growing up in the intestine. E. coli EDL933 is not eliminated from the intestine; it stays at the level in which it was fed to the mice. The P41L envZ missense mutation was previously shown to causes an increase in the level of phosporylated OmpR (OmpR~P) produced (67). E.coli MG1655 with the P41L missense envZ mutation is referred to as E. coli MG1655 mot-1. These changes result in an improvement of E. coli MG1655 colonization ability in the mouse intestine. The EnvZ/OmpR system positively regulates the small RNAs omrA and omrB (29). omrA and omrB were shown to negatively regulate a number of outer membrane genes including ompT, cirA, fepA, and fecA, (29). omrA and omrB work by hybridizing to mRNA thereby preventing translation from occurring (28). The mutant envZ gene was transferred to E. coli Nissle 1917 to create E. coli Nissle 1917 envZ(mot-1). The mutant envZ gene results in E. coli Nissle 1917 having improved colonization ability. E. coli Nissle 1917 envZ(mot-1) grows slower than E. coli Nissle 1917 wildtype on many sugars and in cecal mucus. The E. coli Nissle 1917 envZ(mot-1) mutant grows slower on galactose than E. coli Nissle 1917 wildtype. When the ability to utilize galactose is eliminated from E. coli Nissle 1917 envZ(mot1) it can no longer grow up in the mouse intestine in the presence of E. coli Nissle 1917 wildtype. This displays the importance of galactose utilization in the intestine for E. coli Nissle 1917 envZ(mot-1). E. coli Nissle 1917 envZ(mot-1) may have access to a niche where galactose is being utilized that E. coli Nissle 1917 is unable to inhabit. If E. coli Nissle 1917 was able to inhabit that niche then it would seem likely that E. coli Nissle 1917 would out compete E. coli Nissle 1917 envZ(mot-1) due to its superior ability to utilize galactose. E. coli Nissle 1917 envZ(mot-1) was then colonized against E. coli EDL933 to see if its improved colonization ability also results in improved probiotic potential. After performing multiple colonization experiments it is evident that E. coli Nissle 1917 envZ(mot-1) is not a better probiotic than E. coli Nissle 1917 wildtype.

Escherichia coli MG1655, a K-12 strain, uses glycolytic nutrients exclusively to colonize the intestines of streptomycin-treated mice when it is the only E. coli strain present or when it is confronted with E. coli EDL933, an O157:H7 strain. In contrast, E. coli EDL933 uses glycolytic nutrients exclusively when it is the only E. coli strain in the intestine but switches in part to gluconeogenic nutrients when it colonizes mice precolonized with E. coli MG1655 (R. L. Miranda et al., Infect Immun 72:1666–1676, 2004, http://dx.doi.org/10.1128/IAI.72.3.1666-1676.2004 ). Recently, J. W. Njoroge et al. (mBio 3:e00280-12, 2012, http://dx.doi.org/10.1128/mBio.00280-12 ) reported that E. coli 86-24, an O157:H7 strain, activates the expression of virulence genes under gluconeogenic conditions, suggesting that colonization of the intestine with a probiotic E. coli strain that outcompetes O157:H7 strains for gluconeogenic nutrients could render them nonpathogenic. Here we report that E. coli Nissle 1917, a probiotic strain, uses both glycolytic and gluconeogenic nutrients to colonize the mouse intestine between 1 and 5 days postfeeding, appears to stop using gluconeogenic nutrients thereafter in a large, long-term colonization niche, but continues to use them in a smaller niche to compete with invading E. coli EDL933. Evidence is also presented suggesting that invading E. coli EDL933 uses both glycolytic and gluconeogenic nutrients and needs the ability to perform gluconeogenesis in order to colonize mice precolonized with E. coli Nissle 1917. The data presented here therefore rule out the possibility that E. coli Nissle 1917 can starve the O157:H7 E. coli strain EDL933 of gluconeogenic nutrients, even though E. coli Nissle 1917 uses such nutrients to compete with E. coli EDL933 in the mouse intestine.

Previously we reported that the streptomycin-treated mouse intestine selected for two different Escherichia coli MG1655 mutants with improved colonizing ability: nonmotile E. coli MG1655 flhDC deletion mutants that grew 15% faster in vitro in mouse cecal mucus and motile E. coli MG1655 envZ missense mutants that grew slower in vitro in mouse cecal mucus yet were able to cocolonize with the faster-growing flhDC mutants. The E. coli MG1655 envZ gene encodes a histidine kinase that is a member of the envZ-ompR two-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, the envZ P41L gene was transferred from the intestinally selected E. coli MG1655 mutant to E. coli Nissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both the E. coli MG1655 and E. coli Nissle 1917 strains containing envZ P41L produced more phosphorylated OmpR than their parents. The E. coli Nissle 1917 strain containing envZ P41L also became more resistant to bile salts and colicin V and grew 50% slower in vitro in mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer than E. coli Nissle 1917. However, E. coli Nissle 1917 envZ P41L was not better at preventing colonization by enterohemorrhagic E. coli EDL933. The data can be explained according to our “restaurant” hypothesis for commensal E. coli strains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invading E. coli pathogens planktonically.

Previously, we reported that the streptomycin-treated mouse intestine selected nonmotile Escherichia coli MG1655 flhDC deletion mutants of E. coli MG1655 with improved colonizing ability that grow 15% faster in vitro in mouse cecal mucus and 15 to 30% faster on sugars present in mucus (M. P. Leatham et al., Infect. Immun. 73:8039–8049, 2005). Here, we report that the 10 to 20% remaining motile E. coli MG1655 are envZ missense mutants that are also better colonizers of the mouse intestine than E. coli MG1655. One of the flhDC mutants, E. coli MG1655 Δ flhD , and one of the envZ missense mutants, E. coli MG1655 mot-1, were studied further. E. coli MG1655 mot-1 is more resistant to bile salts and colicin V than E. coli MG1655 Δ flhD and grows ca. 15% slower in vitro in mouse cecal mucus and on several sugars present in mucus compared to E. coli MG1655 Δ flhD but grows 30% faster on galactose. Moreover, E. coli MG1655 mot-1 and E. coli MG1655 Δ flhD appear to colonize equally well in one intestinal niche, but E. coli MG1655 mot-1 appears to use galactose to colonize a second, smaller intestinal niche either not colonized or colonized poorly by E. coli MG1655 Δ flhD . Evidence is also presented that E. coli MG1655 is a minority member of mixed bacterial biofilms in the mucus layer of the streptomycin-treated mouse intestine. We offer a hypothesis, which we call the “Restaurant” hypothesis, that explains how nutrient acquisition in different biofilms comprised of different anaerobes can account for our results.