Dinuclear [(V^VO(putrebactin))₂(μ-OCH₃)₂] Formed in Solution as Established from LC-MS Measurements Using ⁵⁰V‑Enriched V₂O₅

Analysis of 1:1 solutions of V­(V) and the macrocyclic dihydroxamic acid siderophore putrebactin (pbH₂) in 1:1 H₂O/CH₃OH using triple quadrupole liquid chromatography–mass spectrometry (LC-MS-QQQ) (pH ≈ 4) showed two well-resolved peaks (<i>t</i>_R(1) 10.85 min; <i>t</i>_R(2) 14.27 min) using simultaneous detection modes (absorbance, 450 nm; selective ion monitoring, <i>m</i>/<i>z</i> 437) characteristic of the previously identified oxidoV­(V) complex [V^VO­(pb)]⁺ ([M]⁺, <i>m</i>/<i>z</i>_calc 437.1). Peak 1 gave mass spectrometry (MS) signals consistent with [V^VO­(pb)]⁺, together with [V^VO­(pb)­(OH)] and the dinuclear complexes [(V^VO­(pb))₂(μ-OH)]⁺ and [(V^VO­(pb))₂(μ-OH)₂]. Peak 2 gave MS signals consistent with [V^VO­(pb)]⁺, together with [V^VO­(pb)­(OCH₃)] and the dinuclear complexes [(V^VO­(pb))₂(μ-OCH₃)]⁺ and [(V^VO­(pb))₂(μ-OCH₃)₂]. This analysis showed that two groups of V­(V)/pbH₂ complexes with water- or methanol-derived ancillary ligands were resolved by liquid chromatography (LC). The detection of [V^VO­(pb)]⁺ in both peaks could be accounted for by its production from dissociation (peak 1: [(V^VO­(pb))₂(μ-OH)]⁺ → [V^VO­(pb)]⁺ + [V^VO­(pb)­(OH)]; peak 2: [(V^VO­(pb))₂(μ-OCH₃)]⁺ → [V^VO­(pb)]⁺ + [V^VO­(pb)­(OCH₃)]). The assignment of the signal at <i>m</i>/<i>z</i>_obs 959.2 (100%) as the dinuclear complex [(V^VO­(pb))₂(μ-OCH₃)₂] ([M + Na⁺]⁺, <i>m</i>/<i>z</i>_calc 959.3) and not an ion cluster of mononuclear [V^VO­(pb)­(OCH₃)] ({2­[M] + Na⁺}⁺, <i>m</i>/<i>z</i>_calc 959.3) was made unequivocal by the use of ⁵⁰V-enriched V₂O₅, which gave a signal with an isotope pattern comprising the sum of the patterns of the three constituent ⁵¹V–⁵¹V, ⁵¹V–⁵⁰V, and ⁵⁰V–⁵⁰V species. Coordination of methoxide was confirmed upon the replacement of CH₃OH with CD₃OD, which generated [(V^VO­(pb))₂(μ-OCD₃)₂] ([M + Na⁺]⁺, <i>m</i>/<i>z</i>_calc 965.3, <i>m</i>/<i>z</i>_obs 965.3). Analysis of 1:1 solutions of Mo­(VI) and pbH₂ showed a single peak in the LC (<i>t</i>_R 16.04 min), which gave MS signals that were characterized as mononuclear [Mo^VI(O)₂(pb)] ([M + Na⁺]⁺, <i>m</i>/<i>z</i>_calc 523.1, <i>m</i>/<i>z</i>_obs 523.1) and dinuclear [(Mo^VIO­(pb))₂(μ-O)₂] ([M + Na⁺]⁺, <i>m</i>/<i>z</i>_calc 1019.1, <i>m</i>/<i>z</i>_obs 1019.2). The steric and electronic effects of the <i>cis</i>-dioxido group(s) in [Mo^VI(O)₂(pb)] mitigated coordination of solvent-derived ancillary ligands. The work highlights the value of using isotopically enriched metal ion sources and deuterated solvents to deconvolute metal/siderophore solution speciation. The results have relevance for an improved understanding of the coordination chemistry of pbH₂ and other marine siderophores in V­(V)- and Mo­(VI)-rich surface ocean waters

Proteomics of <i>Pseudomonas aeruginosa</i> Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire <i>P. aeruginosa</i> from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and <i>S</i>-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (<i>pchDFG</i> and <i>fptA</i>) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R

Proteomics of <i>Pseudomonas aeruginosa</i> Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire <i>P. aeruginosa</i> from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and <i>S</i>-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (<i>pchDFG</i> and <i>fptA</i>) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R