Regulation of formation of the intracellular β-gaiactosidase activity ofAspergillus nidulans

Abstract

The regulation of formation of the single intracellular beta-galactosidase activity of Aspergillus nidulans was investigated. beta-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose or D-galactose. L-Arabinose, and -- with lower efficacy -- D-xylose also induced beta-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in beta-galactosidase activity. In contrast, in cultures growing on D-galactose, addition of glucose decreased the activity of beta-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not of D-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of beta-galactosidase and partially interfered with the induction of beta-galactosidase by D-galactose, L-arabinose, and D-xylose. D-Galactose phosphorylation was not necessary for beta-galactosidase induction, since induction by D-galactose occurred in an A. nidulans mutant defective in galactose kinase, and by the non-metabolizable D-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced beta-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible by D-galactose, whereas it was still inducible by L-arabinose. We conclude that biosynthesis of the intracellular beta-galactosidase of A. nidulans is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced by D-galactose and L-arabinose in independent ways.

D-galactose catabolism inPenicillium chrysogenum: Expression analysis of the structural genes of the Leloir pathway

“…The fresh cell-free extract was centrifuged at 8.500 × g (5 min, 4 °C), and the supernatant immediately used to assay galactokinase activity. The assay was described earlier in [13,14]. In short, the concentration of galactose-1-phosphate in a reaction mixture containing 10 mM ATP, 20 mM D-galactose, 10 mM MgSO 4 , and 0.7 ml cell-free extract in a 0.1 M phosphate buffer (pH 7.6) was monitored with time, using High Performance Liquid Chromatography (HPLC) using a calibration curve of the phosphorylated sugar made in the same buffer.…”

Lactose and D-galactose catabolism in the filamentous fungusAspergillus nidulans

“…The time-profile of activity essentially parallel growth and lactose consumption, and decline after exhaustion of the carbon source. The ß-galactosidase activity of mycelia growing on lactose can further raised by addition of galactose, indicating that enzyme formation is not maximal during growth on lactose [3,6].…”

“…The first step in lactose metabolism is its transport by a lactose permease, followed by a hydrolysis to glucose and galactose by an intracellular ß-galactosidase [3]. D-glucose catabolism proceeds via either the glycolytic or the oxidative pentose phosphate pathway, while D-Galactose degradation was known to occur exclusively via the Leloir-pathway.…”

“…The time-profile of activity essentially parallel growth and lactose consumption, and decline after exhaustion of the carbon source. The ß-galactosidase activity of mycelia growing on lactose can further raised by addition of galactose, indicating that enzyme formation is not maximal during growth on lactose [3,6].The formation of ß-galactosidase on lactose or D-galactose, but not on glucose or glycerol suggests that its biosynthesis may be under carbon catabolite control. Addition of glucose to cultures growing on lactose indeed immediately decreases ß-galactosidase activity.…”

Induction of the gal Pathway and Cellulase Genes Involves No Transcriptional Inducer Function of the Galactokinase in Hypocrea jecorina

“…The resulting homogenate was centrifuged at 10.000 ϫ g for 20 min at 4°C. The supernatant (average protein concentration 10 -15 mg ml Ϫ1 ) was used as a cell extract for measuring intracellular galactokinase activity as described previously (19). Specific activities are reported as nanokatal/mg protein, which was determined by the Bio-Rad protein assay (Bio-Rad Laboratories).…”