Yersiniabactin production by Pseudomonas syringae and Escherichia coli, and description of a second yersiniabactin locus evolutionary group.

The siderophore and virulence factor yersiniabactin is produced by Pseudomonas syringae. Yersiniabactin was originally detected by high-pressure liquid chromatography (HPLC); commonly used PCR tests proved ineffective. Yersiniabactin production in P. syringae correlated with the possession of irp1 located in a predicted yersiniabactin locus. Three similarly divergent yersiniabactin locus groups were determined: the Yersinia pestis group, the P. syringae group, and the Photorhabdus luminescens group; yersiniabactin locus organization is similar in P. syringae and P. luminescens. In P. syringae pv. tomato DC3000, the locus has a high GC content (63.4% compared with 58.4% for the chromosome and 60.1% and 60.7% for adjacent regions) but it lacks high-pathogenicity-island features, such as the insertion in a tRNA locus, the integrase, and insertion sequence elements. In P. syringae pv. tomato DC3000 and pv. phaseolicola 1448A, the locus lies between homologues of Psyr_2284 and Psyr_2285 of P. syringae pv. syringae B728a, which lacks the locus. Among tested pseudomonads, a PCR test specific to two yersiniabactin locus groups detected a locus in genospecies 3, 7, and 8 of P. syringae, and DNA hybridization within P. syringae also detected a locus in the pathovars phaseolicola and glycinea. The PCR and HPLC methods enabled analysis of nonpathogenic Escherichia coli. HPLC-proven yersiniabactin-producing E. coli lacked modifications found in irp1 and irp2 in the human pathogen CFT073, and it is not clear whether CFT073 produces yersiniabactin. The study provides clues about the evolution and dispersion of yersiniabactin genes. It describes methods to detect and study yersiniabactin producers, even where genes have evolved.

Identification of ethylsuccinylcarnitine present in some human urines.

Ethylsuccinylcarnitine, a previously undescribed acylcarnitine, was identified in urines obtained from 81% of adult volunteers. Its chemical structure was obtained by 1H nuclear magnetic resonance spectroscopy of the urinary purified compound and confirmed by its chemical synthesis. Its urinary excretion followed a circadian rhythm with a maximum occurring between 8 p.m. and 1 a.m. Excretion of this compound was enhanced after a load of L-carnitine and in this case, total 24-h urinary excretion may raise up to about 75 microM. It was observed that the day after adryamicine treatment, the compound was no more excreted. This molecule was absent or in trace amounts in urines obtained from few adults as well as in urines obtained from young subjects. In the positive urines, we detected an unknown organic acid whose excretion was almost parallel to that of ethylsuccinylcarnitine.

Key fragmentation patterns of aporphine alkaloids by electrospray ionization with multistage mass spectrometry.

This work reports a detailed study of the fragmentations of aporphine alkaloids by electrospray ionization with multistage mass spectrometry (ESI-MS(n)) in positive mode. In a first step the loss of the amino group and its substituent is observed. Further steps display the loss of the peripheral groups. Losses of methanol and CO are observed if an OH is vicinal to an OCH(3) on the aromatic ring. Otherwise the spectra show radical losses of CH(3)* or CH(3)O* as the main fragmentations. If a methylenedioxy group is present losses of formaldehyde followed by CO are observed. These fragmentations yield important information on the structures of aporphines.

High-performance liquid chromatography analyses of pyoverdin siderophores differentiate among phytopathogenic fluorescent Pseudomonas Species.

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 beta-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.