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The ability of Hypocrea jecorina (Trichoderma reesei) to grow on lactose strongly depends on the formation of an extracellular glycoside hydrolase (GH) family 35 [beta]-galactosidase, encoded by the bga1 gene. Previous studies, using batch or transfer cultures of pregrown cells, had shown that bga1 is induced by lactose and D-galactose, but to a lesser extent by galactitol. To test whether the induction level is influenced by the different growth rates attainable on these carbon sources, bga1 expression was compared in carbon-limited chemostat cultivations at defined dilution (= specific growth) rates. The data showed that bga1 expression by lactose, D-galactose and galactitol positively correlated with the dilution rate, and that galactitol and D-galactose induced the highest activities of [beta]-galactosidase at comparable growth rates. To know more about the actual inducer for [beta]-galactosidase formation, its expression in H. jecorina strains impaired in the first steps of the two D-galactose-degrading pathways was compared. Induction by D-galactose and galactitol was still found in strains deleted in the galactokinase-encoding gene gal1, which is responsible for the first step of the Leloir pathway of D-galactose catabolism. However, in a strain deleted in the aldose/D-xylose reductase gene xyl1, which performs the reduction of D-galactose to galactitol in a recently identified second pathway, induction by D-galactose, but not by galactitol, was impaired. On the other hand, induction by D-galactose and galactitol was not affected in an L-arabinitol 4-dehydrogenase (lad1)-deleted strain which is impaired in the subsequent step of galactitol degradation. These results indicate that galactitol is the actual inducer of Bga1 formation during growth on D-galactose in H. jecorina.

Lactose (1,4-O-[beta]-D-galactopyranosyl-D-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on D-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that D-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with D-galactose, lactose, galactitol and D-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that D-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0.015 [h.sup.-1]) but not at higher dilution rates. At the same dilution rate, growth on D-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on D-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0.015 [h.sup.-1] than D-galactose and induced cellulases even at growth rates up to 0.042 [h.sup.-1]. Results of chemostats with an equimolar mixture of D-galactose and D-glucose essentially mimicked the behaviour on D-galactose alone, whereas an equimolar mixture of D-galactose and galactitol, the first intermediate of a recently described second pathway of D-galactose catabolism, led to cellulase induction at D=0.030 [h.sup.-1]. It is concluded that D-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.

Research was conducted to analyze phase variation in the symbiotic bacterium, Xenorhabdus nematophilus, which is found in the intestine of entomopathogenic nematodes. Several phenotypic characteristics of the different strains of the X nematophilus bacterium were analyzed to provide a more thoroughly defined phase variation. Results reveal the existence of phenotypic traits, the lack of dye binding and swarming, the inability to produce antibiotic and protease activity and the absence of hemolysis can be used to define phase variant forms of X nematophilus.

Carbon catabolite repression by the CreA-transcriptional repressor is widespread in filamentous fungi, but the mechanism by which glucose triggers carbon catabolite repression is still poorly understood. We investigated the hypothesis that the growth rate on glucose may control CreA-dependent carbon catabolite repression by using glucose-limited chemostat cultures and the intracellular β-galactosidase activity of Aspergillus nidulans, which is repressed by glucose, as a model system. Chemostat cultures at four different dilution rates (D=0.095, 0.068, 0.045 and 0.015 h-1) showed that formation of β-galactosidase activity is repressed at the two highest Ds, but increasingly derepressed at the lower Ds, the activity at 0.015 h-1 equalling that in derepressed batch cultures. Chemostat cultures with the carbon catabolite derepressed A. nidulans mutant strain creAΔ4 revealed a dilution-rate independent constant β-galactosidase activity of the same range as that found in the wild-type strain at D=0.015 h-1. Two other enzymes – isocitrate lyase, which is almost absent on glucose due to a CreA-independent mechanism; and galactokinase, which is formed constitutively and independent of CreA – were measured as controls. They were formed at constant activity at each dilution rate, both in the wild-type strain as well as in the carbon catabolite derepressed mutant strain. We conclude that the growth rate on glucose is a determinant of carbon catabolite repression in A. nidulans, and that below a certain growth rate carbon catabolite derepression occurs. [Copyright &y& Elsevier]