The Streptomycin-Treated Mouse Intestine Selects \u3cem\u3eEscherichia coli envZ\u3c/em\u3e Missense Mutants That Interact with Dense and Diverse Intestinal Microbiota

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 ΔflhD, 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 ΔflhD and grows ca. 15% slower in vitro in mouse cecal mucus and on several sugars present in mucus compared to E. coli MG1655 ΔflhD but grows 30% faster on galactose. Moreover, E. coli MG1655 mot-1 and E. coli MG1655 ΔflhD 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 ΔflhD. 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

<i>E. coli</i> strains and plasmids.

<p><i>E. coli</i> strains and plasmids.</p

Role of catecholate siderophores in gram-negative bacterial colonization of the mouse gut

We investigated the importance of the production of catecholate siderophores, and the utilization of their iron (III) complexes, to colonization of the mouse intestinal tract by Escherichia coli. First, a ΔtonB strain was completely unable to colonize mice. Next, we compared wild type E. coli MG1655 to its derivatives carrying site-directed mutations of genes for enterobactin synthesis (ΔentA::Cm; strain CAT0), ferric catecholate transport (Δfiu, ΔfepA, Δcir, ΔfecA::Cm; CAT4), or both (Δfiu, ΔfepA, ΔfecA, Δcir, ΔentA::Cm; CAT40) during colonization of the mouse gut. Competitions between wild type and mutant strains over a 2-week period in vivo showed impairment of all the genetically engineered bacteria relative to MG1655. CAT0, CAT4 and CAT40 colonized mice 10[superscript 1]-, 10[superscript 5]-, and 10[superscript 2]-fold less efficiently, respectively, than MG1655. Unexpectedly, the additional inability of CAT40 to synthesize enterobactin resulted in a 1000-fold better colonization efficiency relative to CAT4. Analyses of gut mucus showed that CAT4 hyperexcreted enterobactin in vivo, effectively rendering the catecholate transport-deficient strain iron-starved. The results demonstrate that, contrary to prior reports, iron acquisition via catecholate siderophores plays a fundamental role in bacterial colonization of the murine intestinal tract

SDS-PAGE of OM fractions.

<p>The prototypic <i>E. coli</i> strain MG1655 and its derivatives CAT4 (<i>Δfiu, ΔfepA, Δcir, ΔfecA::Cm</i>) and CAT40 (<i>Δfiu, ΔfepA, Δcir, ΔfecA, ΔentA::Cm</i>) were grown in LB broth, subcultured at 1% into iron-deficient MOPS minimal media at 37°C and grown to late log phase. The bacteria were collected by centrifugation, lysed in a French pressure cell and their OM fractions were purified, resolved by SDS-PAGE, and the gels were stained with coomassie blue R. Fiu, FepA and Cir are seen in MG1655 (Lane 1), but absent from CAT4 (lane 2) and CAT40 (lane 3). Molecular weight standards were included in lane 4. FecA, which is inducible by growth in the presence of citrate, is not visible in this experiment, but its absence was verified by PCR.</p

Enterobactin quantification in vivo.

<p>Mice were inoculated on day 0 and on day 12 caecal mucus from mice that were uncolonized or orally inoculated with <i>E. coli</i> strains MG1655, CAT4 or CAT40 was collected and diluted into 5 mM NaHPO₄, pH 6.9. Samples from five individual mice in each experimental group were consolidated, the solution was clarified by centrifugation, 100 uL of each supernatant was mixed with 10 µCi of ⁵⁹FeCl₃, the samples were incubated on ice for an hour and then chromatographed on Sephadex LH20 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#s4" target="_blank">Materials and Methods</a>). We chromatographed authentic FeEnt with the mucus as an internal marker, and determined and plotted the absorbances at 280 nm and 495 nm, and the radioactivity of each fraction. The black and red dashed lines show the absorbances of the eluted fractions at 280 nm and 495 nm, respectively: the blue line depicts their radioactivity.</p

Summary of enterobactin production in vivo.

<p>The amount of FeEnt produced by the colonizing bacteria was standardized in relation to the amount of protein in the mucosal samples (CPM/A₂₈₀ nm). The plotted data therefore depicts the relative amount of enterobactin production by each of the strains on day 12 after inoculation. This experiment was performed only once, but each data point represents the mean values from pooled extracts of 5 animals. Despite the fact that the cell numbers of <i>E. coli</i> CAT4 were 4-logs lower than those of MG1655 at this time, we found more ⁵⁹FeEnt in the former strain's gut mucus. This finding, that CAT4 hyperexcretes enterobactin in vivo, corroborated previous findings <i>in vitro </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Cox1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Young1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Young2" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Seiffert1" target="_blank">[61]</a>. Even though they were present at 10,000-fold lower abundance (a finding that was substantiated by statistical analysis of three colonization experiments), CAT4 cells secreted as much or more enterobactin as wild type bacteria. This was validated by the background control, uncolonized mice treated with streptomycin, that established a baseline for the detection of FeEnt in the mice.</p

Colonization Competition between <i>E. coli</i> MG1655 and CAT0, CAT4 or CAT40.

<p>In each trial three streptomycin-treated mice were simultaneously fed with a mutant strain and wild type parent MG1655, which are both streptomycin resistant. If even a slight advantage exists between the two strains, the conditions of the large intestine select for the preferred strain, which dominates within a few days. If neither strain has an advantage, then the two strains co-colonize at almost equal levels. Fecal plate counts determined the relative colonizing abilities (the log difference in CFU/g of feces). Three-log differences or greater between the mutant and wild-type strain indicates in a major colonization defect. A 1.5 to 3 log difference shows a significant colonization defect, and a 1 to 1.5 log difference denotes a minor defect. Log differences less than 1 are not significant. In these experiments pairs of bacteria were orally inoculated into mice on day 0, and their presence in feces was monitored for 15 days. The plotted data represents the mean of two or more independent trials; error bars represent standard deviations of the means. <i>E. coli</i> MG1655 out-competed CAT0, CAT4 and CAT40 for colonization. Unexpectedly, CAT40 showed 1000-fold better persistence than CAT4, and maintained colonization at almost the same level as MG1655 for the first week.</p