Heteroaromatic monothiocarboxylic acids from Pseudomonas spp.

Pyridine derivatives substituted with monothiocarboxylic acid groups are the unique metabolites of certain Pseudomonas species. Pyridine-2,6-di-(monothiocarboxylic acid) 1a was found during a screening program for antibiotically active bacterial metabolites due to its ability to complex Fe3+. The structure of this complex, its redox behavior and the biogenesis of the ligand molecule were studied in detail. This lead to the discovery of a new class of natural products, viz. acylsulfenic acid derivatives. Interest in la was revived shortly when complexes with other metals were studied as models for sulfur-containing enzymes. It could also be shown that a quinoline monothiocarboxylic acid derivative acted as an alternative siderophore for Pseudomonas fluorescens. But a real renaissance was observed only when the role of la in the degradation of CCl4 by Pseudomonas stutzeri became evident.

Fluorescent Pseudomonas mainly produce the dihydro form of pyoverdine at low specific growth rate.

AIMS: To analyse the influence of cell growth rate and iron concentration on the production of pyoverdines (PVDs) and of their reduced dihydro forms by three fluorescent Pseudomonas strains (P. putida BTP16, P. fluorescens BTP7 and P. aeruginosa 7NSK2). METHODS: PVD and dihydropyoverdine (DHPVD) productions were determined by LC ESI-MS and spectrophotometry during batch and chemostat culture at different dilution rates. SIGNIFICANCE: The relatively high PVD-to-DHPVD ratio (0.57) observed in pH-controlled batch cultures suggested that a base-catalysed chemical oxidation of the dihydroform is not the prime mechanism involved in generating PVDs. Interestingly, in chemostat cultures the PVD-to-DHPVD ratio was significantly reduced at low specific growth rate. Our results suggest that the oxidation of DHPVD to PVD is catalysed by an iron-dependent enzymatic reaction rather than a chemical oxidation.

Chalconoid and stilbenoid glycosides from Guibourtia tessmanii.

Phytochemical studies on the stem bark of Guibourtia tessmanii yielded a dihydrochalcone glucoside, 2',4-dihydroxy-4'-methoxy-6'-O-beta-glucopyranoside dihydrochalcone and a new stilbene glycoside, 3,5-dimethoxy-4'-O-(beta-rhamnopyranosyl-(1-->6)-beta- glucopyranoside) stilbene besides the known pterostilbene. Their structures were established on the basis of one and two dimensional NMR spectroscopic techniques, FABMS and chemical evidence.

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.

The structure of the pyoverdin isolated from various Pseudomonas syringae pathovars.

From seven different pathovars of Pseudomonas syringae representing various genetic subgroups, and one strain of Pseudomonas viridiflava the same pyoverdin siderophore (1) was isolated, probably identical with the pyoverdin whose amino acid composition (but not their sequence) had been reported before. 1 is the first pyoverdin where two of the ligands for Fe3+ are beta-hydroxy Asp units. Its remarkably high complexing constant for Fe3+ at pH 5 as compared with other pyoverdins offers a definite advantage in plant infection. The structure elucidation of 1 will be described and the taxonomical implications regarding pyoverdins with different structures ascribed previously to P. syringae strains will be discussed.

The pyoverdins of Pseudomonas sp. 96-312 and 96-318.

The structures of the pyoverdins isolated from the Pseudomonas spp. 96-312 and 96-318 were elucidated by spectroscopic and degradation techniques. As observed before for Pseudomonas spp. producing pyoverdins with a C-terminal cyclopeptidic substructure, the two strains can recognize to some extent structurally different pyoverdins as long as they have also a similar cyclopeptidic C-terminus.

A fast atom bombardment and matrix-assisted laser desorption/ionization mass spectrometry study of doubly charged porphyrins.

In this mass spectrometry (MS) study of doubly charged porphyrin salts, fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) MS techniques are utilized to examine several unique ionic species. The predominant transformation of preformed doubly charged ions in the desorption/ionization mechanism of FAB and MALDI is the result of deprotonation reactions to form singly charged ions of the type [M(2+) - H(+)](+) and of one-electron reductions to form radical cations [M(2+) + e(-)](+.). The dependence of this phenomenon and the formation of a number of additional ionic species on the different matrices and the FAB-matrix additive benzoquinone is examined. The significant analogous behavior of doubly charged porphyrins in FAB- and MALDI-MS leads to the conclusion that one-electron reductions are of distinct relevance in the desorption/ionization mechanism of MALDI.

Ferribactins as the biosynthetic precursors of the Pseudomonas siderophores pyoverdins.

By a feeding experiment with a 15N-labelled precursor it is shown that ferribactins are transformed into pyoverdins and thus are their biogenetic precursors.

An isopyoverdin from Pseudomonas putida CFML 90-44.

From Pseudomonas putida CFML 90-44 an isopyoverdin was isolated. Its structure could be elucidated by chemical degradation and spectroscopic data.

The structure of a pyoverdine produced by a Pseudomonas tolaasii-like isolate.

Cultures of Agaricus bisporus, the most extensively cultivated mushroom, can be infected severely by Pseudomonas tolaasii. This pathogen is characterized by the so-called white line reaction, a precipitate formed on agar plates between its colonies and those of P. reactans, both belonging to the collective species P. fluorescens. A recent study has shown that a group of P. tolaasii isolates can be subdivided into two groups or 'siderovars', based on the pyoverdines they produce (Munsch et al. 2000). One group of strains is characterized by the pyoverdine described by Demange et al. (1990). A representative of the second group (strain Ps3a) was found to produce the same pyoverdine as a strain which had been classified before as P. aureofaciens. However, based mainly on 16S rRNA gene sequence comparisons and REP-PCR generated fingerprints, the two strains are not identical. They are also distinguishable from the P. tolaasii type strain.

Further isoflavonoid metabolites from Millettia griffoniana (Bail).

Three new isoflavonoids, griffonianone A (1), B (2) and C (4) were isolated from the root bark of Millettia griffoniana, along with the known maximaisoflavone G (5) and 7-hydroxy-6-methoxy-3',4'-methylenedioxyisoflavone (6). Their structures were assigned on the basis of spectroscopic data and chemical transformations.