We present genetic evidence for the enzymes 4-aminobutyrate : 2-oxoglutarate aminotransferase (EC 2.6.1.19) and succinate-semialdehyde dehydrogenase [NAD(P)+] (EC 1.2.1.16) constituting the functional pathway for the utilization of 4-aminobutyric acid as a nitrogen source by Saccharomyces cerevisiae. We show that the pathway is induced by 4-aminobutyric acid and that the presence of the pathway enzymes probably requires the integrity of a positive control element.In Pseudomonas~uorescens [l ,2] GABA utilization occursStep (1) is catalyzed by a GABA transaminase and step (2) by a succinate semialdehyde dehydrogenase. These enzyme activities have also been detected in the yeasts Torulopsis utilis and Saccharomyces cerevisiae and the latter is able to use GABA as sole nitrogen source [3]. In order to establish this route genetically as the functional pathway in GABA utilization as a nitrogen source and to study the regulation of GABA catabolism, we have isolated and studied mutants unable to use GABA as sole source of nitrogen. via the following reactions: MATERIALS AND METHODSStrains used in our work were wild type C1278b and strain 2512c, derived from the former but which has lost the general amino-acid permease activity (Gap) as a result of a mutation in the gap gene [4].Media. The standard minimal medium M (without nitrogen source) is described in reference [5]. Nitrogen sources were added to a final concentration of 0.1 %.Genetic techniques. Mutagenesis by ethylmethane sulfonate was carried out according to [6], using strain 2512c. Standard genetic techniques are described in [7] and [8].Preparation of cell extracts. 500 ml cultures were inoculated and grown at 29 T. Exponentially growing cultures were harvested at a cell density of about 0.35 x lo7 cells/ ml, centrifuged, washed and resuspended in 2ml of 0.1 M potassium phosphate buffer at pH 8.2 containing 0.2 mM EDTA and 0.05 mM dithiothreitol. Extracts were made with a French press. Centrifuged extracts were passed through a Sephadex G-25 column pre-conditioned with the same buffer. Partial purification of succinate semialdehyde dehydrogenaseIn certain assays it was necessary to use GABA-transaminase-free succinate semialdehyde dehydrogenase. The purification was carried out in the following manner: cell extracts were treated with 1 YO neutralized protamine sulfate (0.15 g/l g protein) for 20 min at 0°C under gentle stirring. The homogenate was centrifuged and the supernatant solution was treated with increasing amounts of ammonium sulfate. Both GABA transaminase and SSA dehydrogenase were contained in the 60 -80% ammonium sulfate fraction. This fraction was dialyzed against 20 mM potassium phosphate buffer at pH 7.5 with protectors. The separation of the two enzymes was carried out on a 5 x 5 0 m m column (Pharmacia FPLC system) filled with Pharmacia polyanionic resin Polyanion SI-HR5/5 no. 17.0549.01 and eluted with a 20-200 mM potassium phosphate buffer, pH 7.5 with protectors. Under these conditions the GABA transaminase activity elutes with the exclus...
Rhizobium meliloti grows on fructose as sole carbon source. Following nitrosoguanidine mutagenesis, a mutant of R. meliloti M5N1 was isolated as unable to grow on fructose. Enzyme assays with cell‐free extracts showed it to lack significative phosphoglucose isomerase activity. Other enzymes were present at low levels. Both fructose and fructose 6‐phosphate were accumulated within this mutant. The in vitro inhibition of fructokinase by fructose 6‐phosphate was show. Symbiotic properties remained unaffected in the mutant strain.
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