Production of the siderophore enterobactin: Use of four different fermentation systems and identification of the compound by HPLC

Abstract

The article describes four different fermentation procedures for Escherichia coli AN311, a producer of enterobactin. A regular rotary shaker culture with a biphasic system consisting of an agar layer (as a reservoir for feeding processes) and a layer of liquid medium, 2.4 L and 10 L batch cultures, and a novel dialysis membrane fermentor were used. With the use of this latter fermentor type, the production of enterobactin could be increased by a factor of about 9.5, while growth increased by a factor of 12 compared to the other systems. For the rapid and reliable quantification of the concentration and purity of enterobactin an analytical and preparative high-performance liquid chromatography (HPLC) method was established. The degradation compounds of this siderophore were detected by diodearray and bioassays. A comparison of total catechol production as well as the distribution between enterobactin and its degradation compounds is given.

Role of Catecholate Siderophores in Gram-Negative Bacterial Colonization of the Mouse Gut

“…Despite the fact that the cell numbers of E. coli CAT4 were 4-logs lower than those of MG1655 at this time, we found more 59 FeEnt in the former strain's gut mucus. This finding, that CAT4 hyperexcretes enterobactin in vivo, corroborated previous findings in vitro [31], [32], [60], [61]. 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.…”

“…Each reported data point derived from the pooled gut lavage samples of 5 mice, and therefore represents an intrinsic average of five individuals, which provides a high degree of reliability. In light of these considerations, and the fact that the observed catecholate levels in vivo recapitulated extensive previously published data [31], [32], [60], [61], we did not see the need to statistically re-validate a well-known phenomenon by the involvement of 25 additional animals.…”

A Metabolite Produced by Gut Microbes Represses Phage Infections in Vibrio cholerae

“…E. coli and other enterobactin producing bacteria are commonly present in the gut microbiome, albeit at low concentrations . In fact, their levels can increase following V. cholerae infection. , In pure culture, strains of E. coli have produced greater than 200 μM enterobactin (which readily hydrolyzes into LinEnt) . Furthermore, enterobactin has been observed in animal guts .…”

HPLC separation of enterobactin and linear 2,3-dihydroxybenzoylserine derivatives: a study on mutants ofEscherichia coli defective in regulation (fur), esterase (fes) and transport (fepA)

“…Trifluoroactic acid guarantees a continuous acidity (pH 3) over the whole gradient and it can easily be removed by evaporation or lyphilization. While in earlier studies additional bioassays were used for the identification of HPLC peaks (Seiffert et al 1993). the present investigation makes use of electrospray mass spectrometry which proved to be especially well suited for the analysis of DHBS compounds because the molecules are preferentially split at the ester bonds.…”

“…Therefore, analytical HPLC is the method of choice tofollow enterobactin production. Enterobactin and derivatives from culture fluids have previously been identified spectroscopically (Scarrow et al 1991, Berner et al 1991 or by bioassays (Seiffert et al 1993). However, the relative quantities of hydrolysis products of enterobactin esterase (Brickrnan and McIntosh 1992) have never been determined in detail.…”