Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin

Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages

List of Chemical Shifts for Corynebactin.

<p>The group assignment is as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034591#pone-0034591-g005" target="_blank">Fig. 5</a>-C. All shifts are referenced to internal DSS.</p

Analysis of corynebactin structure by NMR spectroscopy.

<p>A. ¹H-¹H DQF-COSY spectrum of corynebactin dissolved in 90% H₂O at pH 4.0. Dashed lines illustrate the correlations between the protons in the central lysine core moiety B. Overlay of the ¹H-¹³C HSQC Spectrum of corynebactin dissolved in D₂O at pH 6.0 (black) with the ¹H-¹³C HMBC (red). The inset shows an expansion of the region indicated by the dashed box.</p

Quantitative bioassay for corynebactin.

<p>A. Varying concentrations of corynebactin (expressed as EDDA equivalents) were added to wells containing 25 µM FeCl₃. Growth of <i>C. diphtheriae</i> C7(β) Δ<i>ciuE</i> was stimulated by corynebactin, and the growing bacteria reduced the triphenyltetrazolium chloride indicator dye and turned red. No visible bacterial growth was present around the control well without corynebactin. The figure shows the results of a representative bioassay. B. Within the range from 6.75 µM to 108 µM EDDA equivalents, the average diameter of the growth stimulation zone for <i>C. diphtheriae</i> C7(β) Δ<i>ciuE</i>, based on triplicate samples, was directly proportional to the log₂ of the concentration of the corynebactin sample in the well.</p

Comparison of structures of corynebactin, staphyloferrin A, and rhizoferrin.

<p>A. Structure of corynebactin determined by NMR spectroscopy. B. The published structure of staphyloferrin. C. The published structure of rhizoferrin.</p

Analysis of corynebactin by electrospray ionization mass spectra (negative ion mode).

<p>A. A representative mass spectrum for corynebactin. The average <i>m/z</i> value for corynebactin was 493.138 based on the average of five separate mass spectra. Additional peaks at 475.28, 500.31, 501. 28, and 546.31 correspond to the following ions: [M-H-H₂O]⁻, [M-H+Fe²⁺-2H-HCOOH]⁻, [M-H+Fe³⁺-3H-HCOOH]⁻, and [M-H+Fe³⁺-3H]⁻, respectively.</p