Abstract
Under iron limitation, the plant pathogen Erwinia chrysanthemi produces the catechol-type siderophore chrysobactin, which acts as a virulence factor. It can also use enterobactin as a xenosiderophore. We began this work by sequencing the 5'-upstream region of the fct-cbsCEBA operon, which encodes the ferric chrysobactin receptor and proteins involved in synthesis of the catechol moiety. We identified a new iron-regulated gene (cbsH) transcribed divergently relative to the fct gene, the translated sequence of which is 45.6% identical to that of Escherichia coli ferric enterobactin esterase. Insertions within this gene interrupt the chrysobactin biosynthetic pathway by exerting a polar effect on a downstream gene with some sequence identity to the E. coli enterobactin synthase gene. These mutations had no effect on the ability of the bacterium to obtain iron from enterobactin, showing that a functional cbsH gene is not required for iron removal from ferric enterobactin in E. chrysanthemi. The cbsH-negative mutants were less able to utilize ferric chrysobactin, and this effect was not caused by a defect in transport per se. In a nonpolar cbsH-negative mutant, chrysobactin accumulated intracellularly. These defects were rescued by the cbsH gene supplied on a plasmid. The amino acid sequence of the CbsH protein revealed characteristics of the S9 prolyl oligopeptidase family. Ferric chrysobactin hydrolysis was detected in cell extracts from a cbsH-positive strain that was inhibited by diisopropyl fluorophosphate. These data are consistent with the fact that chrysobactin is a d-lysyl-l-serine derivative. Mössbauer spectroscopy of whole cells at various states of (57)Fe-labeled chrysobactin uptake showed that this enzyme is not required for iron removal from chrysobactin in vivo. The CbsH protein may therefore be regarded as a peptidase that prevents the bacterial cells from being intracellularly iron-depleted by chrysobactin.
Highlights
Under iron limitation, the plant pathogen Erwinia chrysanthemi produces the catechol-type siderophore chrysobactin, which acts as a virulence factor
As ferric enterobactin esterase is an essential component of the enterobactin-mediated iron transport pathway in E. coli, we investigated whether the L2 cbsH-19 mutant could use ferric enterobactin as an iron source (Table II)
We report the functional analysis of a new gene that belongs to the chrysobactin-dependent iron transport gene cluster of E. chrysanthemi strain 3937
Summary
Quantitative Determination of Ferric Chrysobactin in Cell Lysates—A culture (grown exponentially in L broth) of the strain to be studied was diluted 1:40 in 20 ml of Tris medium supplemented with glucose and 5 M FeCl3. The concentrations of ferric chrysobactin in cell lysates and in culture supernatants equivalent to 5 ϫ 108 colony-forming units was determined spectrophotometrically. Assay for Ferric Enterobactin Esterase Activity—For ferric enterobactin esterase activity, cell extracts from the bacterial strains tested were prepared as described above. Transport Experiments—An overnight culture in L broth of the strain to be studied was diluted 1:40 in Tris medium supplemented with glucose and incubated with shaking until the required absorbance at 600 nm was reached. Cultures of strains L2 cbsE-1 and L2 cbsH-19 were grown in Tris medium supplemented with glucose for 12 h. Isomer shift (␦), quadrupole splitting (⌬EQ), and percentage of the total absorption area were obtained by least-squares fits of lorentzian lines to the experimental spectra
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