Regulation of the cellulolytic system in Trichoderma reesei by sophorose: induction of cellulase and repression of beta-glucosidase.

Sophorose has two regulatory roles in the production of cellulase enzymes in Trichoderma reesei: beta-glucosidase repression and cellulase induction. Sophorose also is hydrolyzed by the mycelial-associated beta-glucosidase. Repression of beta-glucosidase reduces sophorose hydrolysis and thus may increase cellulase induction.

Induction of cellulolytic enzymes in Trichoderma reesei by sophorose.

Sophorose (2-O-beta-glucopyranosyl-D-glucose) induces carboxymethyl cellulase in Trichoderma reesei QM6a mycelium with 1.5 to 2 h. The induction response to sophorose concentration, although complicated by the metabolism of sophorose, shows saturation kinetics. Most of the cellulase appears after most of the sophorose has been taken up, but the presence of an inducer is required to maintain cellulase synthesis because enzyme production ceases after separation of the mycelium from the induction medium. Cellulase appears simultaneously in the medium and in the mycelium, and no appreciable levels accumulate in the mycelium. Response to pH suggest either that synthesis and secretion of the enzyme are closely associated or concurrent events affected by surface interactions with the medium. Effects of temperature and pH on cellulase induction by sophorose are similar to those reported for induction by cellulose. The kinetics of absorption by mycelium differs from that of other beta-linked saccharides and glucose, the uptake of sophorose being much slower. Under our cultural conditions, sophorose appears to induce an incomplete array of cellulase enzymes, as indicated by enzymatic and electrophoretic studies.

Endogenous metabolism of fungus spores: stimulation by physical and chemical means.

Endogenous respiration of spores of the fungus Myrothecium verrucaria can be stimulated up to over-10 fold by diverse chemicals or by physical treatments. Greatest effects were caused by azide (12-fold at 250 mum) and by 2,4-dinitrophenol (7-fold at 300 mum). Marked stimulation was also caused by 10 mum silver (5-fold), 30 mum pentachlorophenol (6-fold), 10 mum carbonyl cyanide m-chlorophenyl hydrazone (4.5-fold) and 10 mum merthiolate (4-fold). Physical treatments such as heat (50 C), freezing, and sonication at sublethal levels were also stimulatory. Stimulation by azide or dinitrophenol was much greater in young than in old spores, whereas response to other chemicals and to freezing was relatively unaffected by spore age. In older spores the effect of azide was no greater than some other inhibitors. During incubation with azide, the endogenous trehalose reserves decreased and changes in free amino acids occurred, both increases and decreases. Thus anabolic as well as catabolic changes occur as evidenced also by the germination of a few (up to 5%) spores. The mechanisms of stimulation must be varied and complex. Permeability changes in the membrane confining endogenous reserves are proposed as a common initial cause. Additional changes in characteristics of membranes of other subcellular particles, as well as enzymic phenomena such as uncoupling of oxidative phosphorylation, are presumably involved in instances where greater stimulation occurs. The data are consistent with the hypothesis that dormancy in these spores results from separation of substrates from metabolic enzymes and more specifically that metabolites are sequestered rather than enzymes.

Crypticity of Myrothecium verrucaria spores to maltose and induction of transport by maltulose, a common maltose contaminant.

Spores of the fungus Myrothecium verrucaria are cryptic to maltose and isomaltose. Induction of a transport system can be effected by several sugars whose order of effectiveness is: turanose > maltulose > sucrose > d-arabinose, d-fructose, nigerose, maltotriulose, kestose > melezitose, raffinose, nystose, and stachyose. The transport system is not specific to maltose and isomaltose, and it is apparently identical to an induced trehalose permease described previously. Induction of the permease is markedly influenced by spore age-older spores being more responsive. Pure maltose is not absorbed by spores. Absorption of commercial reagent-grade maltose is due to permease induction by maltulose as an impurity. Maltulose contamination of maltose was demonstrated by charcoal column chromatography and comparison of its physical, chemical, and permease-inductive properties with those of authentic maltulose. Maltose accumulates temporarily in spores after absorption and then decreases, although no conversion to glucose can be detected. Although spores contain small quantities of maltase, metabolism of maltose may be via some nonhydrolytic pathway.

Constitutive and induced trehalose transport mechanisms in spores of the fungus Myrothecium verrucaria.

Trehalose is absorbed by two distinct systems-one constitutive, the other induced by turanose and to a lesser extent by nigerose but not by trehalose. The constitutive system is apparently mediated by a surface trehalase; the induced system has the characteristics of a permease. The specificity of the induced system is apparently limited to the alpha glucosyl-glucose or glucosyl-fructose linkage, because absorption of kojibiose, nigerose, maltose, isomaltose, turanose, sucrose, and melezitose, in addition to that of trehalose, was increased. Absorption of beta-linked or of galactose-containing disaccharides was not increased. The constitutive and induced trehalose-absorbing systems differ in their activity, specificity, lability to acid treatment, effects of substrate concentration, and pH optima. Both systems require oxygen, and no marked differential effects of inhibitors were observed. The activity of the induced system is proportional to log turanose concentration (from about 1 to 300 mug/ml), and is an approximate linear function of time of exposure (from about 1 to 50 min). Accumulation of trehalose occurred against a concentration gradient in both systems but particularly in the induced. No leakage was observed. The activity of the induced system declined slowly upon removal of the inducer. Accumulated trehalose is metabolized after activation by azide as are the endogenous trehalose reserves. The accumulated trehalose appears to enter the endogenous trehalose pool found in these spores, although some data suggest it may be more accessible. Respiratory data indicate that absorbed trehalose is available for metabolism while in transit from the external membrane to the internal pool.