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

Role of the Pseudomonas aeruginosa PlcH Tat Signal Peptide in Protein Secretion, Transcription, and Cross-Species Tat Secretion System Compatibility

The secretion of PlcH and its homolog PlcN of Pseudomonas aeruginosa through the inner membrane depends upon a functional twin arginine translocase (Tat) system and a Tat signal sequence. Conserved twin arginine (Arg) residues within the Tat signal sequence consensus motif (S/TRRxFLK) are considered essential for the secretion of Tat substrates, but some exceptions (e.g., Lys and Arg) to the twin Arg residues in this motif have been noted. The roles of all three Arg residues within the PlcH RRRTFLK consensus motif were examined. Data are presented which indicate that Arg-9 and Arg-10 are essential for PlcH secretion across the inner membrane, but the mutation of Arg-8 (e.g., to Ala or Ser) had no observable effect on the localization of PlcH. In the signal sequence of PlcH and in all of its homologs in other bacteria, there are basic amino acid residues (Arg, Lys, and Gln) immediately adjacent to the signal peptidase cleavage site (Ala-X-Ala) that are not seen in Sec-dependent signal sequences. The mutation of these basic residues to Ala caused slightly decreased levels of extracellular PlcH, but normal localization was still observed. Deletion of the entire Tat signal sequence of PlcH not only resulted in the absence of detectable extracellular PlcH activity and protein but also caused a substantial decrease in the detectable level of plcH mRNA. Finally, data are presented which indicate that P. aeruginosa PlcH exhibits cross-species compatibility with the Escherichia coli Tat secretion machinery, but only when the E. coli Tat machinery is expressed in a P. aeruginosa host

Plasmids used in this study.

<p>Plasmids used in this study.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Phagocytosis of wild type and isogenic alanine racemase deficient mutant strains of <i>B. pseudomallei</i> and <i>B. mallei</i> by murine macrophages.

<p>Immediately after the phagocytosis phase of macrophage killing assays, the macrophages were fixed and examined by transmission electron microscopy for the presence of intracellular bacteria. A) Wild type <i>B. pseudomallei</i> K96243; B) Isogenic alanine racemase deficient <i>B. pseudomallei</i> K96243; C) Wild type <i>B. mallei</i> ATCC 23344; D) Isogenic alanine racemase deficient <i>B. mallei</i> ATCC 23344.</p

Alanine racemase deficient mutants of <i>B. pseudomallei</i> and <i>B. mallei</i> grew normally in LB broth with 10 mM d-alanine, but they exhibited growth arrest and rapidly lost viability in LB broth without d-alanine.

<p>In contrast, the isogenic parental strains of <i>B. pseudomallei</i> and <i>B. mallei</i> grew normally in LB broth either with or without added d-alanine. In each panel, a closed square (▪) represents the wild type strain grown in the presence of d-alanine; an open square (□) represents the wild type strain grown without d-alanine; a closed circle (•) represents the alanine racemase deficient mutant grown in the present of d-alanine; and an open circle (○) represents the alanine racemase deficient mutant grown without d-alanine. Panel A: Turbidity of cultures of wild type and mutant strains of <i>B. pseudomallei</i> K96243. Panel B: Viability of bacteria in cultures of wild type and mutant strains of <i>B. pseudomallei</i> K96243. Panel C: Turbidity of cultures of wild type and mutant strains of <i>B. mallei</i> ATCC 23344. Panel D: Viability of bacteria in cultures of wild type and mutant strains of <i>B. mallei</i> ATCC 23344. Results with wild type and mutant strains of <i>B. pseudomallei</i> 1026b (not shown) were similar to those obtained with wild type and mutant strains of <i>B. pseudomallei</i> K96243.</p

Survival of wild type and isogenic alanine racemase deficient mutant strains of <i>B. mallei</i> within murine macrophages.

<p>Murine peritoneal periodate-elicited macrophages were infected in vitro with wild type or alanine racemase deficient mutant strains of <i>B. mallei</i>. After 2 h of phagocytosis in d-alanine-supplemented RPMI⁺ medium, the macrophages were washed with prewarmed RPMI⁺ medium containing 6 µg/ml gentamicin with or without 5 mM d-alanine. The surviving intracellular bacteria were enumerated after 4 h of culture. The % survival was enumerated by (cfu t_n/cfu t₀)100.</p