Metabolism of polyamines and basic amino acids inErwinia amylovora: Application of liquid chromatography/electrospray mass spectrometry to proferrioxamine precursor feeding and inhibition studies

Abstract

Erwinia amylovora, the etiological agent of fire blight, produces a family of proferrioxamine siderophores, which may be essential for the pathogen to establish itself in its hosts. If so, then control of fire blight may perhaps be possible via interference with proferrioxamine biosynthesis. Proof of this hypothesis requires prior knowledge of the corresponding biosynthetic pathways in E. amylovora. As a first step towards understanding proferrioxamine biosynthesis, it was of interest to investigate the ability of the fire blight bacterium to utilize various potential biosynthetic pathways for diamines. Feeding of lysine, ornithine and diaminobutyric acid gave rise to highly elevated levels of cadaverine, putrescine and diaminopropane, respectively, indicating that the corresponding decarboxylase activities are all present in E. amylovora. The conclusion for lysine decarboxylase was confirmed with (15N2)lysine, which was converted to (15N2)cadaverine. Arginine did not increase putrescine levels substantially, but (13C6)arginine nevertheless gave rise to (13C4)putrescine while suppressing excretion of non-labeled putrescine. A serendipitous result of this study was the finding that the growth of E. amylovora can be inhibited with 5-hydroxylysine and 1,4-diamino-2-butanone. The mechanism of inhibition appears complex and is not yet understood. For 5-hydroxylysine, preliminary investigations point to a competitive inhibition of lysine decarboxylase. However, the growth inhibition cannot be reversed by providing cadaverine, the decarboxylation product of lysine.

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

“…The same holds true for the profiles observed after feeding methionine or either one of the two isomeric acetylspermidines. These observations are consistent with my previously reported finding (Feistner 1994d) that spermidine and its acetyl derivatives are not appreciably degraded by E. amylovora and that in the case of spermine acetylated rather than free diamines are the final degradation products. Escherichia coli, a closely related bacterium, also does not degrade spermidine (Tabor & Dobbs 1970), whereas some bacteria outside the family Enterobacteriaceae are able to do so (Razin et al 1959, Bachrach et al 1960, Bachrach 1962.…”

“…0I E. amylovora EA1430 was exclusively used for this study and was grown as described (Feistner 1994d). The analysis of the spent culture supernatants for basic amino acids and polyamines suggests that these precursors are not used as carbon sources Profi'rrioxamine biosynthesis in E. amylovora by E. am.vlovora, at least not in the presence of sucrose, since large amounts of the labeled and unlabeled amino acids and their decarboxylation products were detected (Feistner 1994d).…”

“…Methanolic extracts of the dried culture supernatants were prepared as described (Feistner 1994d). The tinal extracts were l ml aqueous solutions, except for the methionine feeding study where the corresponding extract had to be dissolved in 2 ml because of the poor solubility of methionine.…”

“…Similar relative increases for pFO,,55r pFO445~ and pFO444~ were also observed after feeding agmatine and arginine. It is interesting to note that while the proferrioxamine profile shifts induced by ornithine and arginine were in the same ballpark, the biosynthetic intermediate putrescine accumulated to high levels in the growth medium only in the feeding study with ornithine (Feistner 1994d). …”

“…A single or all of these pathways may be active in E. amylovora. Thus far, evidence has only been gathered for the primary decarboxylation pathway (Feistner 1994d and this study). The other biosynthetic pathways remain hypothetical.…”