Escherichia coli is a single species consisting of many biotypes, some of which are commensal colonizers of mammals and others that cause disease. Humans are colonized on average with five commensal biotypes, and it is widely thought that the commensals serve as a barrier to infection by pathogens. Previous studies showed that a combination of three pre-colonized commensal E. coli strains prevents colonization of E. coli O157:H7 in a mouse model (Leatham, et al., 2010, Infect Immun 77: 2876–7886). The commensal biotypes included E. coli HS, which is known to successfully colonize humans at high doses with no adverse effects, and E. coli Nissle 1917, a human commensal strain that is used in Europe as a preventative of traveler's diarrhea. We hypothesized that commensal biotypes could exert colonization resistance by consuming nutrients needed by E. coli O157:H7 to colonize, thus preventing this first step in infection. Here we report that to colonize streptomycin-treated mice E. coli HS consumes six of the twelve sugars tested and E. coli Nissle 1917 uses a complementary yet divergent set of seven sugars to colonize, thus establishing a nutritional basis for the ability of E. coli HS and Nissle 1917 to occupy distinct niches in the mouse intestine. Together these two commensals use the five sugars previously determined to be most important for colonization of E. coli EDL933, an O157:H7 strain. As predicted, the two commensals prevented E. coli EDL933 colonization. The results support a model in which invading pathogenic E. coli must compete with the gut microbiota to obtain the nutrients needed to colonize and establish infection; accordingly, the outcome of the challenge is determined by the aggregate capacity of the native microbiota to consume the nutrients required by the pathogen.

The intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochrome bd oxidase and the anaerobic terminal reductases. We found that E. coli uses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bd oxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence of E. coli, we conclude that E. coli is the only bacterium in the streptomycintreated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases that E. coli uses to maximize its competitiveness and achieve the highest possible population in the intestine.The mouse colon is home to at least several hundred bacterial species and more than 100 billion bacteria per g of contents, a microbial community dominated by anaerobes with essentially no aerobes (2,38,57,68). It is becoming increasingly clear that facultative anaerobes consume oxygen and thereby modify their host environment. For example, kidney infection caused by uropathogenic Escherichia coli results in local ischemia (47). The facultatively anaerobic enteric pathogen Shigella flexneri responds to oxygen in the gut by modulating virulence gene expression (45). It has been postulated that the importance of Salmonella motility for chemotaxis through the mucus layer (61) may be due to aerotaxis (44). That E. coli requires cytochrome bd oxidase to gain a competitive advantage implies that the colon is not the anaerobic environment that many consider it to be (30). Here, we explore the possibility that efficient oxygen scavenging by E. coli in the intestine causes it to depend also on anaerobic respiration.To maximize their energy efficie...

High-affinity RNA ligands were generated against intact 30S ribosomes, S1-depleted 30S ribosomes, and purified ribosomal protein S1. Sequence analysis indicated two classes of ligand: unstructured RNAs containing a Shine-Dalgarno sequence and structured RNAs containing a pseudoknot. The Shine-Dalgarno-containing ligands were generated against S1-depleted 30S ribosomes but, surprisingly, not against intact 30S ribosomes or ribosomal protein S1. In contrast, pseudoknot-containing ligands were generated against intact ribosomes as well as purified S1 protein. The two classes of ligand exhibited specificity for their respective targets, as well as conserved sequence and secondary structure reminiscent of naturally occurring, cis-acting mRNA elements.

The ribosomal mRNA track was investigated by toeprinting 30S ribosomes, in the presence or absence of tRNA, using a variety of different ribosome-binding sites. We found that: (1) the ribosome, by itself, recognizes the mRNA translational initiation site; (2) the ribosomal mRNA track makes extensive contact with mRNA independent of tRNA and the start codon; (3) ribosome-mRNA complexes are less stable than complexes containing tRNA; and (4) toeprinting can be used to analyze the contour of the ribosomal mRNA track, yielding information on its "height" as well as its "length" dimension. Examination of several ribosome-binding sites, including those containing very stable secondary structure, indicated that the "height" of the mRNA track is quite roomy, while the nucleotide distance between the site of Shine-Dalgarno annealing, the P site, and the 3'-edge of the mRNA track is fixed. The data suggest a mechanism for tethering regulatory elements to the ribosome during translation.

In Escherichia coli the unusual amino acid selenocysteine is incorporated cotranslationally at an in-frame UGA codon. Incorporation of selenocysteine relies, in part, on the interaction between a specialized elongation factor, the SELB protein, and a cis-acting element within the mRNA. Boundary and toeprint experiments illustrate that the SELB-GTP-Sec-tRNA^''' ternary complex binds to the selenoprotein encoding mRNAs fdhF and fdnG, serving to increase the concentration of SELB and Sec-tRNA**'' on these mRNAs in vivo. Moreover, toeprint experiments indicate that SELB recognizes the ribosome-bound message and that, upon binding, SELB may protrude out of the ribosomal-mRNA track so as to approach the large ribosomal subunit. The results place the mRNA-bound SELB-GTP-Sec-tRNA^^'' ternary complex at the selenocysteine codon (as expected) and suggest a mechanism to explain the specificity of selenocysteine insertion. Cis-acting mRNA regulatory elements can tether protein factors to the translation complex during protein synthesis.