Gottfried J. Feistner

On the collision-activated fragmentation of proferrioxamines: Evidence for a succinimide-mediated mechanism

The fragmentation mechanism of acyclic proferrioxamines has been studied by tandem mass spectrometry in a triple stage quadrupole mass analyzer by using activation in the collision cell as well as in the high pressure region prior to the first mass analyzer. The data suggest that proferrioxamines fragment preferentially at the hydroxamate bonds via cyclic rearrangement to succinimide derivatives. This pattern was observed most clearly for the peracetyl derivatives, in which the influence of terminal functional groups was masked. Free amino or carboxylic acid functions may modify this basic fragmentation pattern. Using hydrogen-deuterium exchange, we also were able to show that the hydrogen atoms that are “recruited” in the formation of ammonium ions are “acidic” ones from elsewhere in the molecule or the matrix. At the same time, this rules out that they originate from “activated” methylene groups, as previously proposed

Proferrioxamine profiles of Erwinia herbicola and related bacteria

Two strains of Erwinia herbicola effective in the biocontrol of E. amylovora, the etiological agent of fire blight, were screened for proferrioxamine siderophores by on-line liquid chromatography-electrospray mass spectrometry (LC-MS). Type strains of E. herbicola and Pantoea species were included in this study for taxonomic comparisons. Proferrioxamine profiles similar to that previously described for E. amylovora, including tri- and tetrameric hydroxamates and diaminopropane-containing proferrioxamines, were observed for P. agglomerans, but not for other E. herbicola-like species. Biocontrol activity was not correlated with proferrioxamine synthesis. The results of this study are consistent with the notion that some, but not all, biocontrol strains may inhibit E. amylovora via competition for iron. Further studies into the link between biocontrol of fire blight and siderophores are thus warranted. This study also revealed limitations of standard nutrient utilization and fatty acid profile analyses for the differentiation of P. agglomerans, P. dispersa and other E. herbicola-like species from each other. Given these limitations, LC-MS may become a much needed additional diagnostic tool for the identification of E. herbicola-like strains at the species level.

Proferrioxamine synthesis in Erwinia amylovora in response to precursor or hydroxylysine feeding: metabolic profiling with liquid chromatography-electrospray mass spectrometry.

Metabolic profiling by capillary liquid chromatography-electrospray mass spectrometry was used to monitor shifts in the proferrioxamine profiles of Erwinia amylovora in response to externally supplied potential proferrioxamine precursors, selected stable-isotope-labeled precursors and atypical precursors. Based on the qualitative and quantitative shifts in the proferrioxamine profiles, lysine and arginine are unambiguous, and agmatine, ornithine, diaminobutyric acid and the corresponding C3–5 diamines are highly likely precursors for proferrioxamine biosynthesis in E. amylovora. 5-Hydroxylysine (Hyl), a recently discovered growth inhibitor for E. amylovora, suppresses proferrioxamine production. The Hyl-induced growth inhibition can be reversed by basic amino acids. The basic amino acids also partly restore proferrioxamine synthesis.

Suggestion for a new, semirational nomenclature for the free chelators of ferrioxamines

The author proposes that the free chelators of ferrioxamines should be referred to as proferrioxamines (pFOs) and the corresponding structural and regulatory genes as pfoA, pfoB, pfoC, etc. He furthermore suggests that individual proferrioxamines should be distinguished by subscripts that indicate the number and sizes of the constituting diamines, and, where applicable, a cyclic structure or a C-terminal acetic acid substitution. Modifications to the basic structures can be accomodated by standard nomenclature rules. The new semirational nomenclature features the best of the old alphabetical and the Chemical Abstracts nomenclatures; it is structure-based and thus chemically informative but still quite concise.

Proferrorosamines and phytopathogenicity in Erwinia spp.

Proferrorosamine A (pFR A) of the plant pathogenic bacterium Erwinia rhapontici was shown to inhibit growth of wheat and cress seedlings at the ≥ 10 ppm level. When the seeds were continuously exposed to 100 ppm pFR A, the germination of cress and wheat seeds was inhibited up to 90% and 80%, respectively. The inhibition could be reversed through addition of equimolar amounts of ferrous iron, which indicates that the strong iron chelating capability of pFR A is responsible for the observed effect. The Fe(II) in the corresponding iron complex, ferrorosamine A, was found to be remarkably resistant towards oxidation by hydrogen peroxide and therefore redox-cycling in the Haber-Weiss cycle. It is thus conceivable that pFR A may also attenuate the generation of reactive hydroxyl radicals during the resistant and wound reaction. The apparent correlation between proferrorosamine production and virulence in erwiniae was further corroborated through the analysis of Erwinia persicinus, a newly described species. Using electrospray ionization mass spectrometry, E. persicinus was shown to produce pFR A and pFR B, and preliminary evidence for the phytopathogenicity of E. persicinus was found in cress. Inhibition of wheat seedlings by E. persicinus could not be demonstrated, but this may be due to technical difficulties or different host specificities. Taken together, our results indicate that the phytopathogenicity of E. rhapontici and E. persicinus may, as least in part, be due to the release of proferrorosamines.

Revised primary structures of rat pituitary γ-lipotrophin and β- endorphin

In-depth investigations, by high performance liquid chromatographic purification, radio-immunoassay, mass spectrometry, tandem mass spectrometry, Edman sequencing and limited C-terminal ladder sequencing, were prompted by mass spectrometric charting experiments which suggested that the amino acid sequences for rat γ-lipotrophin and β-endorphin require revision. The results for γ-lipotrophin identify a histidine for glutamine substitution at position 12, and heterogeneity in the expressed protein presumably due to partial dehydration. Partial dehydration for acidic joining peptide, previously reported by Toney et al was corroborated. The results for β-endorphin confirm the presence of alanine at position 26 and provide no evidence for the expression of multiple forms of the hormone.

Application of capillary liquid chromatography — electrospray mass spectrometry to identify major siderophores of Erwinia amylovora as proferrioxamines and their potential role in virulence

A large number of hydroxamate siderophores of Erwinia amylovora have been identified using capillary liquid chromatography — electrospray mass spectrometry in combination with ferric ion complexation studies. Specifically, we have identified the trihydroxamate siderophores, proferrioxamines E, D2, X1 X2, and novel X7, the corresponding open chain proferrioxamines G1, G2a G2b, and G1t (a truncated G1), novel tetrahydroxamate siderophores, T1-T3, as well as a partially characterized siderophore T4. Production of the siderophores was achieved in a minimal medium with iron concentrations equal to or less than 10-6 M. No qualitative differences in the siderophore pattern were found between E. amylovora strains from different parts of the world or between dihydrophenylalanine-producing and -non-producing strains. Proferrioxamines may be important virulence factors of E. amylovora and proferrioxamine biosynthesis may offer targets for new fire blight control agents. Before launching a new program to develop specific inhibitors of proferrioxamine biosynthesis, it is desirable to confirm that E. amylovora mutants solely defective in proferrioxamine biosynthesis are indeed avirulent. Sneath et al. previously reported on mutants Ea321T140, Ea321T145, and Ea321T160 that were avirulent as well as siderophore-deficient and could partly be complemented by cosmid pCPP610 [Acta Horticult. 273, 255 (1990)]. It was not conclusively shown, however, that avirulence was the result of blocked siderophore biosynthesis and not mutations in regulatory genes with the siderophore deficiencies merely being pleiotropic effects. According to our mass spectrometric analysis, only Ea321T160 has a clear proferrioxamine deficiency that can be reversed by complementation with cosmid pCPP610. Ea321T140 is still capable of expressing large amounts of proferrioxamines, whereas pCPP610 does not restore proferrioxamine production in Ea321T145. Traces of proferrioxamines are also found with Ea321T145 and Ea321T160. These results suggest that, other than previously thought, the mutations have affected regulatory rather than biosynthetic genes and that pCPP610 does not contain all the regulatory and biosynthetic genes that are necessary for proferrioxamine production. The question whether fire blight can be controlled via interference with proferrioxamine biosynthesis thus remains open.

Charting of Rat Pituitary Peptides by Plasma Desorption and Electrospray Ionization Mass. Spectrometry

Previously, we demonstrated that it is feasible to use mass spectrometry for the charting of the most abundant peptides in bovine posterior/intermediate pituitary and pointed out that this opens the possibility to use mass spectrometry to study the regulation of entire peptide/protein profiles in response to drug treatment, endogenous regulators, stress, and disease [1]. Andrews et al. have shown similarly good results for charting catfish pancreatic peptides and suggested the use of mass spectrometry to investigate alterations of peptide profiles between different tissues and species [2]. The present state of the art of peptide/protein charting by mass spectrometry has recently been reviewed [3].