High-Performance Liquid Chromatography Analyses of Pyoverdin Siderophores Differentiate among Phytopathogenic Fluorescent Pseudomonas Species

Abstract

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine. The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin. The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two ␤-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based. The peptide chain influenced the chelation of iron more in atypical pyoverdins. Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.

Yersiniabactin Production by Pseudomonas syringae and Escherichia coli , and Description of a Second Yersiniabactin Locus Evolutionary Group

“…It is the second time that the solid-liquid technique in petri dishes (10) has proved to be an efficient way of producing siderophores, and it probably has potential for other genera and metabolites. The HPLC method for detecting YBT in the culture medium is similar to that described for PVDs (12) and comparable to the HPLC methods used to detect salicylic acid-based siderophores in concentrated supernatant extracts (2,26,51,52,68,76). The method seems to be useful because YBT production appears to be more widely dispersed in the environment than previously thought and because it can be adapted for every environmental microorganism without having to grow potentially human-pathogenic indicator strains.…”

“…Indeed, a diversifying selection occurred in the genes encoding the Pseudomonas aeruginosa PVD peptide chain (79), and numerous siderophore outer membrane receptors are present in the genomes of P. aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and P. syringae (19,50). P. syringae, Pseudomonas viridiflava, and Pseudomonas ficuserectae produce the same PVD with a stability constant for iron of 10 25 which is not incorporated by other pseudomonads, except for Pseudomonas cichorii (10,11,12,13,17,40,53). This PVD is not essential for growth and virulence of P. syringae pv.…”

Phenazine-1-Carboxylic Acid Promotes Bacterial Biofilm Development via Ferrous Iron Acquisition

“…In summary, filtrates with Fe(III)-chelated pyoverdine(s) at pH 5.0 were prepared for HPLC by adding FeCl 3 into the samples, followed by filtration (0.2-m-pore-size filter) and pH adjustment. Analysis was carried out in a gradient profiling with solvent A as water-17 mM NaOH-acetic acid at pH 5.0 and solvent B as acetonitrile (solvent B) (9). For samples with pyoverdine being released at concentrations higher than 0.1 M, a single Fe(III)-pyoverdine peak was detected at 403 nm.…”

Salicylic Acid, Yersiniabactin, and Pyoverdin Production by the Model PhytopathogenPseudomonas syringaepv. tomato DC3000: Synthesis, Regulation, and Impact on Tomato andArabidopsisHost Plants

“…A fraction was collected around a dominant A385 peak and submitted for LC-MS. For Pvd-Fe 3ϩ analysis, 100 M FeCl 3 was added to supernatant from a low-iron wild-type DC3000 stationary-phase culture, and the supernatant was then concentrated 10-fold by evaporation with heating and vacuum. The sample was then analyzed by HPLC method 1 (14). A fraction was collected around the dominant A400 peak and submitted for LC-MS. For all Ybt and Pvd experiments, a Prevail C 18 5u 150-by 4.6-mm (Alltech) column maintained at 27°C was used on a Shimadzu SCL-10AVP series HPLC system equipped with a Shimadzu SPD-10AVP photodiode array detector and a Shimadzu RF-10AXL fluorescence detector.…”

“…tomato DC3000 likely synthesizes the yellow-green fluorescent siderophore Pvd, based on the presence of the required biosynthetic genes in its genome (11) and the synthesis of Pvd by other P. syringae pv. tomato strains (13,14). Therefore, we first wanted to determine whether DC3000 produces Pvd under iron-limiting conditions.…”