Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems

The herbicidally active compound N-2-(5-chloro-pyridyl)-aminomethylene-bisphosphonic acid acts by inhibiting both glutamine and aromatic amino acid biosynthesis.

The effect of the herbicidally active compound N-2-(5-chloro-pyridyl)aminomethylene bisphosphonic acid (Cl-pyr-AMBPA), previously found in vitro to inhibit the activity of the first enzyme in the shikimate pathway 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, was investigated in vivo on suspension cultured cells of Nicotiana plumbaginifolia Viviani. Amino acid pool measurement showed an actual reduction of tyrosine, tryptophan and phenylalanine level following the addition of the compound to the growth medium. However, an even stronger effect was noticed for other amino acids, mainly glutamine. When the activity of the enzymes involved in the glutamate cycle was measured in the presence of Cl-pyr-AMBPA, glutamate synthase was unaffected, while glutamine synthetase was significantly inhibited. Contrary to the herbicide phosphinothricin, the inhibitor bound reversibly to the enzyme. Kinetic analysis accounted for an inhibition of uncompetitive type with respect to ammonium, glutamate and ATP, withKivalues of 113, 97 and 39 M, respectively. Only the exogenous supply of a mixture of glutamine and aromatic amino acids relieved cell growth inhibition, suggesting that the phytotoxic properties of Cl-pyr-AMBPA are due to inhibition of key enzymes in both the corresponding pathways

N-pyridyl-aminomethylene-bisphosphonic acids inhibit the first enzyme in the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase.

A series of seven structurally diverse N -substituted aminomethylene bisphosphonic acids exhibiting remarkable herbicidal activity was found to inhibit the activity of the first enzyme in the prechorismate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, partially purified from Nicotiana plumbaginifolia suspension cultured cells. At millimolar concentrations all compounds inhibited the Co 2+ -dependent, cytosol-localized enzyme form. However, the addition of excess divalent cations to the reaction mixture was able to completely relieve their effect, suggesting that the chelating properties of these compounds could account for their inhibitory effect. In contrast, only five compounds of seven reduced significantly the activity of the plastidial and Mn 2+ -stimulated isoform. The inhibition brought about by N -2-(6-methyl-pyridyl)- and N -2-(5-chloro-pyridyl)-aminomethylene bisphosphonic acid could not be relieved by raising the manganese concentration in the assay mixture. A kinetic analysis showed that the latter compound inhibits enzyme activity uncompetitively with respect to phospho enol pyruvate, competitively with respect to the other substrate, erythrose-4-phosphate. As manganese is a V max stimulator of the plastidial enzyme, this ruled out the possibility of an inhibition simply based upon metal chelation

The herbicidally active compound N-2-(6-methyl-pyridyl)-aminomethylene bisphosphonic acid inhibits in vivo aromatic biosynthesis.

The effect of N-2-(6-methyl-pyridyl)-aminomethylene bisphosphonic acid (M-pyr-AMBPA), a compound previously shown to exhibit herbicidal properties on whole plants and to inhibit in vitro activity of the first enzyme in the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, was investigated on Nicotiana plumbaginifolia suspension cultured cells and compared to that of the herbicide glyphosate. The addition of M-pyr-AMBPA from 10-4 to 10-3 M was found to cause a severe cell growth reduction. Kinetic analysis of partially purified DAHP synthase accounted for non-competitive inhibition type with respect to both phospho-enol-pyruvate and erythrose-4-phosphate, with KI values of 0.43 and 0.62 mM, respectively. Amino acid pool measurements of cells grown in the presence of sublethal doses of M-pyr-AMBPA pointed to an actual reduction of free aromatic amino acids, showing that DAHP synthase inhibition takes place in vivo, and suggesting that the interference of this aminophosphonate with plant aromatic biosynthesis may account for a large part of its phytotoxicity. However, exogenous supply of a mixture of phenylalanine, tyrosine and tryptophan failed to achieve full reversal of cell growth inhibition, yet the occurrence of other target(s) cannot be ruled out

A metal-independent hydrolase from a Penicillium oxalicum strain able to use phosphonoacetic acid as the only phosphorus source.

A Penicillium oxalicum strain was capable of the phosphate-sensitive utilization of phosphonoacetic acid as the sole source of phosphorus. A carbon-to-phosphorus bond-cleavage enzyme yielding acetic acid and inorganic phosphate was detected and characterized in extracts from cells grown on this phosphonate. Contrary to bacterial phosphonoacetate hydrolases, the fungal enzyme neither required nor was stimulated by divalent cations