The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity

Extremophiles - A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC),...     

Glucose kinases from Streptomyces peucetius var. caesius

Glucose kinases (Glks) are enzymes of the glycolytic pathway involved in glucose phosphorylation. These enzymes can use various phosphoryl donors such as ATP, ADP, and polyphosphate. In several streptomycetes, ATP-glucose kinase (ATP-Glk) has been widely studied and regarded as the main glucose phosphorylating enzyme and is likely a regulatory protein in carbon catabolite repression. In cell extracts from the doxorubicin overproducing strain Streptomyces peucetius var. caesius, grown in glucose, a polyphosphate-dependent Glk (Pp-Glk) was detected by zymogram. Maximum activity was observed during the stationary growth phase (48 h) of cells grown in 100 mM glucose. No activity was detected when 20 mM glutamate was used as the only carbon source, supporting a role for glucose in inducing this enzyme. Contrary to wild-type strains of Streptomyces coelicolor, Streptomyces lividans, and Streptomyces thermocarboxydus K-155, S. peucetius var. caesius produced 1.8 times more Pp-Glk than ATP-Glk. In addition, this microorganism produced five and four times more Pp-Glk and anthracyclines, respectively, than its wild-type S. peucetius parent strain, supporting a role for this enzyme in antibiotic production in the overproducer strain. A cloned 726-bp DNA fragment from S. peucetius var. caesius encoded a putative Pp-Glk, with amino acid identities between 83 and 87 % to orthologous sequences from the above-cited streptomycetes. The cloned fragment showed the polyphosphate-binding sequences GXDIGGXXIK, TXGTGIGSA, and KEX₍₄₎SWXXWA. Sequences for the Zn-binding motif were not detected in this fragment, suggesting that Pp-Glk is not related to the Glk ROK family of proteins.     

Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.Mannitol has been described as one of the main compatible solutes in fungi (20) and may play a role as a storage carbon source (3) or a protectant against a variety of stresses (10, 16, 20, 22). Mannitol metabolism in fungi has been the subject of study for decades. It was proposed to exist in the form of a cyclic pathway, the mannitol cycle (9). This cycle consists of four steps enabling the conversion of fructose into mannitol and back to fructose (Fig. 1). The main role proposed for this cycle was regenerating NADPH (9, 10). Subsequently, many studies have questioned the existence of a mannitol cycle (reviewed in reference 20), and it has been shown that a mannitol cycle is not involved in NADPH regeneration in Stagonospora nodorum (19), Aspergillus niger (16), and Alternaria alternata (21). However, all enzymes of the cycle were detected in both sporulating and nonsporulating mycelia in A. niger (16), suggesting that a cycle could operate in this fungus. Fungi are able to use mannitol as a sole carbon source but do so in various ways (7).Open in a separate windowFig. 1.Putative mannitol cycle in fungi as proposed by Hult and Gatenbeck (9). HXK, hexokinase (EC 2.7.1.1); MTD, mannitol dehydrogenase (EC 1.1.1.138); MPD, mannitol-1-phosphate dehydrogenase (EC 1.1.1.17); MPP, mannitol-1-phosphate phosphatase (EC 3.1.3.22).d-Mannitol plays an important role in germination of Aspergillus conidia. In A. niger (23) and Aspergillus oryzae (8), mannitol accumulates in conidiospores and is utilized during the initial stages of germination. Production of mannitol appears to be largely dependent on mannitol-1-phosphate dehydrogenase (MPD) while mannitol dehydrogenase (MTD) contributes to a lesser extent (16, 19, 20).In this study we demonstrate that MTD and MPD as well as the expression of the corresponding genes (mtdA and mpdA) are spatially separated in colonies of A. niger. This demonstrates that a mannitol cycle does not exist in this fungus and shows that spores express specific genes that are involved in germination.