In this work the bio-availability of amino acids for the root-colonizing Pseudomonas fluorescens strain WCS365 in the tomato rhizosphere was studied. The amino acid composition of axenically collected tomato root exudate was determined. The results show that aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino acid components. The concentrations of individual amino acids in the rhizosphere of gnotobiotically grown tomato plants were estimated and considered to be too low to support growth of rhizosphere micro-organisms to numbers usually found in the tomato rhizosphere. To test this experimentally, mutants of P. fluorescens WCS365 auxotrophic for the amino acids leucine, arginine, histidine, isoleucine plus valine, and tryptophan were isolated after mutagenesis with Tn5lacZ. Root tip colonization of these mutants was measured after inoculation of germinated tomato seeds and subsequent growth in a gnotobiotic quartz sand system (M. Simons, A. J. van der Bij, I. Brand, L. A. de Weger, C. A. Wijffelman, and B. J. J. Lugtenberg. 1996. Gnotobiotic system for studying rhizo-sphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 9:600–607). In contrast to the wild-type strain, none of the five amino acid auxotrophs tested was able to colonize the tomato root tip, neither alone nor after co-inoculation with the wild-type strain. However, addition of the appropriate amino acid to the system restored colonization by the auxotrophic mutants, usually to wild-type levels. Analysis of the root base showed that cells of auxotrophic mutants were still present there. The results show that, although amino acids are present in root exudate, the bio-availability of the tested amino acids is too low to support root tip colonization by auxotrophic mutants of P. fluorescens strain WCS365. The genes that are required for amino acid synthesis are therefore necessary for root colonization. Moreover, these compounds apparently play no major role as nutrients in the tomato rhizosphere.

Introduction Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed. Objectives To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions. Methods We present a double-labelling LC–MS approach with a ¹³C-labeled yeast cellular extract as complex surrogate matrix, and ¹³C¹⁵N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates. Results While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions. Conclusions Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study. Graphical abstract

Transport of solutes between the cytosol and the vacuolar lumen is of crucial importance for various functions of vacuoles, including ion homeostasis; detoxification; storage of different molecules such as amino acids, phosphate, and calcium ions; and proteolysis. To identify proteins that catalyze solute transport across the vacuolar membrane, the membrane proteome of purified Saccharomyces cerevisiae vacuoles was analyzed. Subtractive proteomics was used to distinguish contaminants from true vacuolar proteins by comparing the relative abundances of proteins in pure and crude preparations. A robust statistical analysis combining enrichment ranking with the double boundary iterative group analysis revealed that 148 proteins were significantly enriched in the pure vacuolar preparations. Among these proteins were well characterized vacuolar proteins, such as the subunits of the vacuolar H⁺-ATPase, but also proteins that had not previously been assigned to a cellular location, many of which are likely novel vacuolar membrane transporters, e.g. for nucleosides and oligopeptides. Although the majority of contaminating proteins from other organelles were depleted from the pure vacuolar membranes, some proteins annotated to reside in other cellular locations were enriched along with the vacuolar proteins. In many cases the enrichment of these proteins is biologically relevant, and we discuss that a large group is involved in membrane fusion and protein trafficking to vacuoles and may have multiple localizations. Other proteins are degraded in vacuoles, and in some cases database annotations are likely to be incomplete or incorrect. Our work provides a wealth of information on vacuolar biology and a solid basis for further characterization of vacuolar functions.

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B₂) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. (2000) Anal. Chem. 72, 5655–5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (K_d for riboflavin is 0.6 nm), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure.