Table 1.
Siderophores described among E. coli strains used in this study.
Figure 1.
LC-MS metabolite profiling reveals differential product formation based on media iron content.
Depicted are total ion chromatograms of conditioned culture media following 18 hour culture of E. coli strain rUTI2 in M63 media containing 1 µM (“iron poor”, top trace) or 100 µM ferric chloride (“iron rich”, top trace). Mass spectrometric analysis confirms that the majority of differentially expressed peaks in the iron poor supernatant derive from siderophores.
Table 2.
Siderophore production in previously sequenced or genotyped strains.
Figure 2.
Identity of E. coli siderophore peaks is confirmed by stable isotope substitution.
E. coli strain rUTI2 was grown in M63 media (top trace), with 15N-ammonium as the nitrogen source (middle trace), and with 13C-glycerol as the carbon source (bottom trace). The 13C-labeled forms are used as internal standards for mass spectrometric quantification in this study.
Figure 3.
Siderophore production by the model uropathogen UTI89 and selected mutants.
A) MS/MS chromatograms showing siderophore production in wild type UTI89 and strains with deletions in specific siderophore biosynthesis genes (ΔentB, ΔiroB, ΔybtS, ΔentB/ΔybtS). Chromatograms are shown at the retention times for enterobactin (m/z 670), salmochelin (m/z 1012), and ferric yersiniabactin (m/z 535). For each column, the vertical scale is a fixed fraction of the corresponding 13C internal standard peak height, allowing comparison between samples. B) Chrome azurol S (CAS) plate upon which UTI89 strains have been streaked for overnight growth. A yellow halo is produced around siderophore-secreting bacteria. In UTI89, a double mutant (ΔentB/ΔybtS) is required to specifically abolish siderophore production.
Table 3.
Patient characteristics for paired strain study.
Table 4.
Patient PFGE types, siderophore expression, and genotypes related to source and UTI event.
Figure 4.
Differences in siderophore production between single-patient urinary and rectal isolates.
Each data point is the difference in siderophore production between coincident urinary and rectal strains recovered from an individual with UTI. These differences were determined by stable isotope dilution mass spectrometry and expressed in reference strain equivalents. The median and interquartile range is depicted for each siderophore. *Medians differs significantly (p<0.05) from zero.
Figure 5.
In addition to directing salmochelin biosynthesis, the iroA cluster also increases production of linear enterobactin, a candidate virulence-associated siderophore.
A) Cyclic enterobactin is converted into a linear form by ester bond hydrolysis. B) Clinical isolates that produce salmochelin (+salmochelin) exhibit greater enterobactin linearization than salmochelin nonproducers (-salmochelin). C) Removal of the entire iroA cluster (iroBCDEN) significantly reduced enterobactin linearization. A similar reduction in enterobactin linearization is seen in an iroE mutant. Enterobactin linearization in MG1655, a K12 strain without the iroA cluster, was comparable to that seen in these mutants. These findings support the esterase IroE as an enterobactin linearizing enzyme in vivo.
Figure 6.
Biosynthesis of siderophores associated with recurrent UTI depends on chorismic acid utilization.
Table 5.
Prevalence of detectable siderophore expression among different PFGE types in this study according to source.