Moniliella zaluziensis sp. nov., a black yeast related to fruit trees of the Rosaceae family.

Our previous studies focused on the diversity of yeasts related to the aboveground parts of fruit trees, as well as the soil adjacent to these trees, located in the south-west of Slovakia. During these studies, we isolated two Moniliella strains: CCY 11-1-1T from the blossoms of a peach tree (Prunus persica) and CCY 11-1-2 from the soil adjacent to a pear tree (Pyrus communis), both found in the Malé Záluzie locality. The sequences of the D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region were identical in both strains. They differed only by two nucleotide substitutions in the segment of the gene of translation elongation factor one alpha (TEF-1α). Phylogenetic analysis demonstrated that strains CCY 11-1-1T and CCY 11-1-2 formed a separate species in the clade of insect-associated members of the genus Moniliella. The strains differed from the closest species Moniliella oedocephalis by 23 nucleotide substitutions and 12 indels in the D1/D2 domain, more than 6 % in the ITS region (31 nt and 25 indels) and by 44 nt in the segment of TEF-1α. Therefore, these two strains are recognized as belonging to a novel species, for which we have proposed the name Moniliella zaluziensis sp. nov., derived from the locality of their origin, Malé Záluzie. The type strain of M. zaluziensis sp. nov. is CCY 11-1-1T.

The UPC2 gene in Kluyveromyces lactis stress adaptation.

KlUpc2p, a transcription factor belonging to the fungal binuclear cluster family, is an important regulator of ergosterol biosynthesis and azole drug resistance in Kluyveromyces lactis. In this work, we show that the absence of KlUpc2p generates Rag- phenotype and modulates the K. lactis susceptibility to oxidants and calcofuor white. The KlUPC2 deletion leads to increased expression of KlMGA2 gene, encoding an important regulator of hypoxic and lipid biosynthetic genes in K. lactis and also KlHOG1 gene. The absence of KlUpc2p does not lead to statistically significant changes in glycerol, corroborating the expression of KlGPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase, that is similar in both the deletion mutant and the parental wild-type strain. Increased sensitivity of Klupc2 mutant cells to brefeldin A accompanied with significant increase in KlARF2 gene expression point to the involvement of KlUpc2p in intracellular signaling. Our observations highlight the connections between ergosterol and fatty acid metabolism to modulate membrane properties and point to the possible involvement of KlUpc2p in K. lactis oxidative stress response.

Effect of N-acetyl chito-oligosaccharides on the biosynthesis and properties of chitin in Saccharomyces cerevisiae.

Chitin exists in yeast cells both as free and bound in a complex with ß-1,3/ß-1,6-glucan. The formation of covalent links between chitin and ß-glucans is catalyzed by the enzymes Crh1 and Crh2, acting as transglycosylases. We found that N-acetyl-chito-oligosaccharides, as well as laminarioligosaccharides, the respective products of partial hydrolysis of chitin, and ß-1,3-glucan, interfered with reactions catalyzed by Crh1p and Crh2p in vitro. However, the N-acetyl-chito-oligosaccharides did not influence the growth rate of the yeast, neither did they affect the yeast phenotype, but they prolonged the lag phase. Inhibition of Crh1 and Crh2 in vivo with oligosaccharides derived from chitin leads to an increase of alkali-soluble chitin and a decrease in the amount of chitin linked to ß-glucans. In addition, yeast cells growing in the presence of N-acetyl-D-chito-oligosaccharides accumulated more chitin than control cells.

Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.