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

Abstract

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).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 invol...

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

“…Such is the case with cmp1 (conidial multidomain protein 1; TR_72379, TV_10277, and TA_323283), expression of which is strongly upregulated during conidiation and is considered specific for conidiophores and conidia (1085). Also, lxr1 (TR_74194, TV_110629, and TA_297930) was used as a marker gene for conidiation because its homolog is only expressed during formation of conidiospores in A. niger (1086) and T. reesei, where its gene product also accumulates in conidiospores (1087). However, microarray data revealed only a moderate increase of lxr1 over the course of conidiation (352).…”

Expression of Biomass-Degrading Enzymes Is a Major Event during Conidium Development in Trichoderma reesei

“…To induce asexual sporulation in T. reesei, the fungus was grown on malt extract (MEX; Merck, Darmstadt, Germany) agar plates (11-cm diameter) on a layer of cellophane to facilitate removal of mycelia. The formation of spores was followed microscopically and by measuring the activity of a marker enzyme of conidiation, the D-mannitol dehydrogenase LXR1 (3,35). Samples of at least two biological replicates, consisting of at least 3 independent plates, were taken at appropriate time points (see Results).…”

The effect of natamycin on the transcriptome of conidia of Aspergillus niger

“…Gene An07g08720, which has strong similarity to trehalose-phosphate synthase and the acid trehalase encoding gene (An01g01540) were 4.7- and 3-fold up-regulated respectively, in the treated conidia (Table 6). Mannitol-synthesising and degrading enzymes (see also Ruijter et al 2003, Aguilar-Osorio et al 2010) were also up-regulated in the presence of the anti-fungal (5.4-fold for mpdA , An02g05830 and 4.8-fold for mtdA , An15g05450). Moreover, a gene with similarity to a mannitol transporter (An02g06710) was 42 times up-regulated in the presence of natamycin.…”