Carbon-phosphorus lyase activity in permeabilized cells of Arthrobacter sp. GLP-1

AbstractArthrobacter sp. GLP-1 is capable of using the phosphonate herbicide glyphosate (N-[phosphonomethyl]glycine) as its sole source of phosphorus. The formation of sarcosine from glyphosate by a carbon-phosphorus (CP) lyase activity could be demonstrated in vivo, as well as in situ in cells permeabilized by 2% dimethylsulfoxide, when sarcosine oxidase was inhibited by acetate. The apparent Km of the CP lyase for glyphosate was found to be 61 μM, and a V of 2.2 pmol · s−1 · mg−1 soluble protein was determined. Uptake of glyphosate into the cells and CP lyase activity represent distinct processes

Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase

Inorganic polyphosphate (iPoP)—linear chains of up to hundreds of phosphate residues—is ubiquitous in nature and appears to be involved in many different cellular processes. In Saccharomyces cerevisiae, iPoP has been detected in high concentrations, especially after transfer of phosphate-deprived cells to a high-phosphate medium. Here, the dynamics of iPoP synthesis in yeast as a function of the growth phase as well as glucose and phosphate availability have been investigated. To address this question, a simple, fast and novel method for the quantification of iPoP from yeast was developed. Both the iPoP content during growth and the iPoP "overplus” were highest towards the end of the exponential phase, when glucose became limiting. Accumulation of iPoP during growth required excess of free phosphate, while the iPoP "overplus” was only observed after the shift from low- to high-phosphate medium. The newly developed iPoP quantification method and the knowledge about the dynamics of iPoP content during growth made it possible to define specific growth conditions for the analysis of iPoP levels. These experimental procedures will be essential for the large-scale analysis of various mutant strains or the comparison of different growth condition

The 33 kDa Protein of the Oxygen-Evolving Complex: a Multi-Gene Family in Tomato

A cDNA was isolated by chance from tomato which had a high similarity to a cDNA clone from potato known to code for the 33 kDa protein of the oxygen-evolving complex [van Spanje et al. (1991) Plant Mol. Biol. 17: 157]. The sequence of a previously described partial cDNA clone from tomato [Ko et al. (1990) Plant Mol. Biol. 14: 217] which has also a high similarity but is not identical to the sequence described here indicates that tomato contains at least two genes coding for 33 kDa proteins per haploid genome. This conclusion is supported by Southern blot analysis. The tissue specific expression of the corresponding genes is describe

Biochemical basis for glyphosate-tolerance in a bacterium and a plant tissue culture

AbstractThe broad spectrum herbicide glyphosate (N-[phosphonomethyl]-glycine) is an inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid-3-phosphate (EPSP)-synthase of both bacterial and higher plant origin. Aerobacter aerogenes, as well as cultured cells of the higher plant Corydalis sempervirens, adapted to growth in the presence of up to 10 mM glyphosate, exhibited a 10–30-fold increase in their EPSP-synthase activity, and excreted (A. aerogenes) or accumulated (C. sempervirens) massive amounts of shikimic acid-3-phosphate and/or shikimic acid

Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism

BACKGROUND: Inorganic polyphosphate (poly P) occurs universally in all organisms from bacteria to man. It functions, for example, as a phosphate and energy store, and is involved in the activation and regulation of proteins. Despite its ubiquitous occurrence and important functions, it is unclear how poly P is synthesized or how poly P metabolism is regulated in higher eukaryotes. This work describes a systematic analysis of poly P levels in yeast knockout strains mutated in almost every non-essential gene. RESULTS: After three consecutive screens, 255 genes (almost 4% of the yeast genome) were found to be involved in the maintenance of normal poly P content. Many of these genes encoded proteins functioning in the cytoplasm, the vacuole or in transport and transcription. Besides reduced poly P content, many strains also exhibited reduced total phosphate content, showed altered ATP and glycogen levels and were disturbed in the secretion of acid phosphatase. CONCLUSION: Cellular energy and phosphate homeostasis is suggested to result from the equilibrium between poly P, ATP and free phosphate within the cell. Poly P serves as a buffer for both ATP and free phosphate levels and is, therefore, the least essential and consequently most variable component in this network. However, strains with reduced poly P levels are not only affected in their ATP and phosphate content, but also in other components that depend on ATP or free phosphate content, such as glycogen or secreted phosphatase activity