fslE is necessary for siderophore-mediated iron acquisition in Francisella tularensis Schu S4

Abstract

Strains of Francisella tularensis secrete a siderophore in response to iron limitation. Siderophore production is dependent on fslA, the first gene in an operon that appears to encode biosynthetic and export functions for the siderophore. Transcription of the operon is induced under conditions of iron limitation. The fsl genes lie adjacent to the fur homolog on the chromosome, and there is a canonical Fur box sequence in the promoter region of fslA. We generated a Deltafur mutant of the Schu S4 strain of F. tularensis tularensis and determined that siderophore production was now constitutive and no longer regulated by iron levels. Quantitative reverse transcriptase PCR analysis with RNA from Schu S4 and the mutant strain showed that Fur represses transcription of fslA under iron-replete conditions. We determined that fslE (locus FTT0025 in the Schu S4 genome), located downstream of the siderophore biosynthetic genes, is also under Fur regulation and is transcribed as part of the fslABCDEF operon. We generated a defined in-frame deletion of fslE and found that the mutant was defective for growth under iron limitation. Using a plate-based growth assay, we found that the mutant was able to secrete a siderophore but was defective in utilization of the siderophore. FslE belongs to a family of proteins that has no known homologs outside of the Francisella species, and the fslE gene product has been previously localized to the outer membrane of F. tularensis strains. Our data suggest that FslE may function as the siderophore receptor in F. tularensis.

FIG. 1.

Characterization of Δfur mutant. (A) PCR analysis of Δfur mutant. The fur gene is depicted with flanking sequences and locations of primers used in PCR analysis. Shown beneath is the extent of sequences carried on the deletion plasmid construct. The primer pairs as shown were used to generate PCR products from Schu S4, GR201 (deletion plasmid integrant derivative), GR203 (Δfur), and GR204 (fur⁺ derivative). “M” indicates lanes with the 1-kb DNA ladder (Invitrogen). (B) Siderophore activity in Δfur mutant and complementation. Schu S4 and GR203 and transformants harboring the control vector pFNLTP6 or the fur⁺ plasmid pGIR461 were grown overnight in iron-replete CDM, and the specific CAS activities of culture supernatants were determined. Assays were carried out in triplicate, and the averages with standard deviations are represented.

FIG. 2.

The fur-fsl region and transcriptional analysis of fsl genes. (A) Representation of the fur-fsl region of the chromosome. The locations of primers used in RT-PCR are shown. (B) qPCR analysis of fslA and fslE expression in the Δfur mutant. cDNAs prepared from Schu S4 and from GR203 were tested for fslA and fslE expression relative to that of trpB as an internal standard. Reactions were run in triplicate, and results are represented as averages with standard deviations. Note that the y axis for fslA expression is a log scale. (C) RT-PCR to delineate the fsl operon. cDNA prepared from GR203 was used in PCR with primer pairs to detect transcription across genetic loci as follows: 109-112 for fur and fslA, 110-112 for fslA, 137-132 for fslAB, 182-119 for fslBC, 138-136 for fslCD, 178-165 for fslDE, 184-211 for fslEF, and 204-201 for fslF and FTT0023c. “−” represents negative controls, where reverse transcriptase was left out of the cDNA reaction; “+” indicates reactions with cDNA; and “g” represents reactions with gDNA as a template. “M” represents the 1-kb DNA ladder (Invitrogen).

FIG. 3.

Western blotting with FslE antibodies. Whole-cell lysates normalized for cell density were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and probed with polyclonal antiserum raised to recombinant FslE. The locations of the prestained standards run on the gel are indicated. The lysates are denoted by lanes: 1, GR203 (Δfur) grown in iron-replete CDM; 2, Schu S4 grown in iron-replete CDM; 3, Schu S4 grown in iron-limiting CDM; 4, GR211 (ΔfslE) grown in iron-limiting CDM; 5, vector (pFNLTP6gro-GFP)-transformed GR211 grown in iron-limiting CDM; 6, fslE+ plasmid (pGIR469)-transformed GR211 grown in iron-limiting CDM.

FIG. 4.

PCR analysis of ΔfslE mutant. The fslE gene is depicted with flanking sequences and locations of primers used in PCR analysis of gDNA from Schu S4 and GR211. Lane M shows the 1-kb DNA ladder (Invitrogen). Schu S4 yields a band of 1.669 kb, including the full-length fslE and indicated by the arrowhead, while the deletion mutant yields a band of 218 bp.

FIG. 5.

Growth deficiency of ΔfslE mutant under iron limitation. (A) Growth on iron-replete and iron-deficient agar plates. Cultures of Schu S4 and GR211 in iron-replete CDM were washed in che-CDM−Fe and resuspended to an optical density of 1.0. Tenfold serial dilutions were made in che-CDM−Fe and spotted on an iron-replete or iron-limiting agar plate. Growth was assessed after 3 days on the rich plate and 4 days on the iron-limiting plate. (B) Growth in liquid culture. Washed cells were inoculated into iron-replete (high Fe³⁺) or iron-limiting (low Fe³⁺) che-CDM and growth followed over a period of 56 h. Cultures were grown in triplicate, and the means and standard deviations of one representative experiment are shown in the growth plot. (C) Complementation of ΔfslE mutant in trans. GR211 cells transformed with either control vector pFNLTP6gro-GFP or the fslE⁺ plasmid pGIR469 were washed and inoculated into iron-replete (hi Fe³⁺) or iron-limiting (lo Fe³⁺) che-CDM. The growth of cultures in duplicate was monitored over 48 h, and the results at the 48-h time point are shown for one representative experiment.

FIG. 6.

Siderophore production and utilization by ΔfslE mutant. Iron-limiting CDM−Fe plates were seeded with Schu S4 (A and B), the ΔfslE strain, GR211 (C and D), or GR211 harboring plasmid vector pFNLTP6groGFP (E) or the fslE⁺ plasmid, pGIR469 (F). Washed cells of LVS, GR7 (siderophore-deficient derivative of LVS), Schu S4, and GR211 were spotted on the plates as indicated. A filter paper spotted with 10 μl of 20-mg/ml FeSO₄ was placed on plate D. Experiments were repeated at least two independent times with similar results; plates from a representative experiment are shown.