Germán Larriba

Cork Taint of Wines: Role of the Filamentous Fungi Isolated from Cork in the Formation of 2,4,6-Trichloroanisole by O Methylation of 2,4,6-Trichlorophenol

ABSTRACT Cork taint is a musty or moldy off-odor in wine mainly caused by 2,4,6-trichloroanisole (2,4,6-TCA). We examined the role of 14 fungal strains isolated from cork samples in the production of 2,4,6-TCA by O methylation of 2,4,6-trichlorophenol (2,4,6-TCP). The fungal strains isolated belong to the genera Penicillium (four isolates); Trichoderma (two isolates); and Acremonium, Chrysonilia, Cladosporium, Fusarium, Mortierella, Mucor, Paecilomyces, and Verticillium (one isolate each). Eleven of these strains could produce 2,4,6-TCA when they were grown directly on cork in the presence of 2,4,6-TCP. The highest levels of bioconversion were carried out by the Trichoderma and Fusarium strains. One strain of Trichoderma longibrachiatum could also efficiently produce 2,4,6-TCA in liquid medium. However, no detectable levels of 2,4,6-TCA production by this strain could be detected on cork when putative precursors other than 2,4,6-TCP, including several anisoles, dichlorophenols, trichlorophenols, or other highly chlorinated compounds, were tested. Time course expression studies with liquid cultures showed that the formation of 2,4,6-TCA was not affected by a high concentration of glucose (2% or 111 mM) or by ammonium salts at concentrations up to 60 mM. In T. longibrachiatum the O methylation of 2,4,6-TCP was catalyzed by a mycelium-associated S-adenosyl-l-methionine (SAM)-dependent methyltransferase that was strongly induced by 2,4,6-TCP. The reaction was inhibited by S-adenosyl-l-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings increase our understanding of the mechanism underlying the origin of 2,4,6-TCA on cork, which is poorly understood despite its great economic importance for the wine industry, and they could also help us improve our knowledge about the biodegradation and detoxification processes associated with chlorinated phenols.

Rad52 depletion in Candida albicans triggers both the DNA‐damage checkpoint and filamentation accompanied by but independent of expression of hypha‐specific genes

We have analysed the effect of RAD52 deletion in several aspects of the cell biology of Candida albicans. Cultures of rad52Δ strains exhibited slow growth and contained abundant cells with a filamentous morphology. Filamentation with polarization of actin patches was accompanied by the induction of the hypha‐specific genes (HSG) ECE1, HWP1 and HGC1. However, filament formation occurred in the absence of the transcription factors Efg1 and Cph1, even though disruption of EFG1 prevented expression of HSG. Therefore, expression of HSG genes accompanies but is dispensable for rad52Δ filamentation. However, deletion of adenylate cyclase severely impaired filamentation, this effect being largely reverted by the addition of exogenous cAMP. Filaments resembled elongated pseudohyphae, but some of them looked like true hyphae. Following depletion of Rad52, many cells arrested at the G2/M phase of the cell cycle with a single nucleus suggesting the early induction of the DNA‐damage checkpoint. Filaments formed later, preferentially from G2/M cells. The filamentation process was accompanied by the uncoupling of several landmark events of the cell cycle and was partially dependent on the action of the cell cycle modulator Swe1. Hyphae were still induced by serum, but a large number of rad52 cells myceliated in G2/M.

Homologous recombination in Candida albicans: role of CaRad52p in DNA repair, integration of linear DNA fragments and telomere length

Chromosomal rearrangements are common in both clinical isolates and spontaneous mutants of Candida albicans. It appears that many of these rearrangements are caused by translocations around the major sequence repeat (MSR) that is present in all chromosomes except chromosome 3, suggesting that homologous recombination (HR) may play an important role in the survival of this organism. In order to gain information on these processes, we have cloned the homologue of RAD52, which in Saccharomyces cerevisiae is the only gene required for all HR events. CaRAD52 complemented poorly a rad52 mutant of S. cerevisiae. Two null Carad52Δ/Carad52Δ mutants were constructed by sequential deletion of both alleles and two reconstituted strains were obtained by reintegration of the gene. Characterization of these mutants indicated that HR plays an essential role in the repair of DNA lesions caused by both UV light and the radiomimetic compound methyl‐methane‐sulphonate (MMS), whereas the non‐homologous end‐joining pathway (NHEJ) is used only in the absence of Rad52p or after extensive DNA damage. Repair by HR is more efficient in exponentially growing than in stationary cells, probably because a larger number of cells are in late S or G2 phases of the cell cycle (and therefore, can use a sister chromatid as a substrate for recombinational repair), whereas stationary phase cells are mainly in G0 or G1, and only can be repaired using the chromosomal homologue. In addition, CaRad52p  is  absolutely  required  for  the  integration of linear DNA with long flanking homologous sequences. Finally, the absence of CaRad52p results in the lengthening of telomeres, even in the presence of an active telomerase, an observation not described in any other organism. This raises the possibility that both telomerase and homologous recombination may function simultaneously at C. albicans telomeres.

Degradation of vanillic acid and production of guaiacol by microorganisms isolated from cork samples

The presence of guaiacol in cork stoppers is responsible for some cases of cork taint causing unpleasant alterations to wine. We have performed a characterization of the cork-associated microbiota by isolating 55 different microorganisms: eight yeast, 14 filamentous fungi or molds, 13 actinomycetes and 20 non-filamentous bacteria. A screening for degradation of vanillic acid and guaiacol production showed that none of the filamentous fungi could achieve any of these processes. By contrast, five of the eight yeast strains isolated were able to degrade vanillic acid, although it was not converted to guaiacol. Guaiacol production was only detected in four bacterial strains: one isolate of Bacillus subtilis and three actinomycetes, Streptomyces sp. A3, Streptomyces sp. A5 and Streptomyces sp. A13, were able to accumulate this compound in both liquid media and cultures over cork. These results suggest that guaiacol-mediated cork taint should be attributed to the degradative action of vanillic acid by bacterial strains growing on cork.

Characterization of an inducible chlorophenol O-methyltransferase from Trichoderma longibrachiatum involved in the formation of chloroanisoles and determination of its role in cork taint of wines.

ABSTRACT A novel S-adenosyl-l-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of Trichoderma longibrachiatum CECT 20431. The enzyme could be identified in partially purified protein preparations by direct photolabeling with [methyl-3H]SAM, and this reaction was prevented by previous incubation with S-adenosylhomocysteine. Gel filtration indicated that the Mr was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500. The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28°C, with a pI of 4.9. The Km values for 2,4,6-trichlorophenol and SAM were 135.9 ± 12.8 and 284.1 ± 35.1 μM, respectively. S-Adenosylhomocysteine acted as a competitive inhibitor, with a Ki of 378.9 ± 45.4 μM. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu2+, Hg2+, Zn2+, and Ag+, and to a lesser extent by p-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents. The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures. Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated.

A Search for Hyperglycosylation Signals in Yeast Glycoproteins

N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13–14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200–300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of α-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection.

A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with Mr of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.

Chromosome instability and unusual features of some widely used strains of Candida albicans

Electrophoretic karyotyping of the Candida albicans revealed a different migration pattern of ChR in three different stocks of the sequencing strain SC5314. In one stock, the high instability of ChR size prevented the migration of ChR as a compact band; ChR appeared, instead, as a smear. In some stocks, ChR and/or Ch1 ploidy diminished, suggesting mixed populations of disomic and monosomic cells. Similarly, some stocks of widely used derivatives CAI4 and BWP17 contained smearing of ChR. In addition, the most manipulated strain in the lineage of SC5314, the last derivative, BWP17, acquired an increase in the size of Ch7b and revealed an unusual property. BWP17 did not tolerate a well‐established procedure of telomere‐mediated fragmentation of a chromosome; the remaining intact homologue always duplicated. We suggest that some stocks of SC5314 are unstable and that BWP17 may not be appropriate for general studies. Instead of BWP17 or CAI4, we recommend using for general research CAF4‐2, which is a relatively stable Ura− derivative, and which has been successfully used for more than a decade in our laboratory. Copyright

Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoproteins.

We have carried out a screen of 622 deletion strains generated during the EUROFAN B0 project to identify non‐essential genes related to the mannosylphosphate content of the cell wall. By examining the affinity of the deletants for the cationic dye alcian blue and the ion exchanger QAE‐Sephadex, we have selected 50 strains. On the basis on their reactivity (blue colour intensity) in the alcian blue assay, mutants with a lower phosphate content than wild‐type cells were then arranged in groups defined by previously characterized mutants, as follows: group I (mnn6), group II (between mnn6 and mnn9) and group III (mnn9). Similarly, strains that behaved like mnn1 (i.e. a blue colour deeper than wild‐type) were included in group VI. To confirm the association between the phenotype and a specific mutation, strains were complemented with clones or subjected to tetrad analysis. Selected strains were further tested for extracellular invertase and exoglucanase. Within groups I, II and III, we found some genes known to be involved in oligosaccharide biosynthesis (ALG9, ALG12, HOC1), secretion (BRE5, COD4/COG5, VPS53), transcription (YOL072w/THP1, ELP2, STB1, SNF11), cell polarity (SEP7, RDG1), mitochondrial function (YFH1), cell metabolism, as well as orphan genes. Within group VI, we found genes involved in environmentally regulated transduction pathways (PAL2 and RIM20) as well as others with miscellaneous or unknown functions. We conclude that mannosylphosphorylation is severely impaired in some deletants deficient in specific glycosylation/secretion processes, but many other different pathways may also modulate the amount of mannosylphosphate in the cell wall. Copyright

Inhibition of yeast exoglucanases by glucosidase inhibitors

Castanospermine, 1-deoxynojirimycin, and N-methyl-1-deoxynojirimycin, three well-characterized inhibitors of the glucosidases involved in the processing of N-linked oligosaccharides, did not affect the biosynthesis or the secretion of exoglucanases (EC 3.2.1.58) from Saccharomyces cerevisiae and Candida albicans but inhibited the activity itself. Regardless of the substrate used, laminarin or p-nitrophenyl beta-D-glucoside (pNPG), all three inhibitors proved to act in a competitive manner. Castanospermine was the most potent inhibitor, with Ki values ranging from 0.16 to 0.5 microM for three different purified yeast exoglucanases. The inhibition caused by 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin was poorer, but still significant. By contrast, the glucosidase inhibitors did not show any action on a partially purified endoglucanase (EC 3.2.1.39) Candida albicans. A purified exoglucanase from Basidiomycete QM 806, which was specific for laminarin, was unaffected by castanospermine but it was still inhibited in an uncompetitive manner by 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin. The presence of castanospermine in the culture medium of growing yeasts did not have any effect on yeast growth in spite of the fact that, under the conditions used, the external exoglucanase was fully inhibited. None of the yeast exoglucanases hydrolyzed the glucan synthesized in vitro by membrane preparations derived from either yeast. These results support the concept that yeast exoglucanases are glucosidases that also attack laminarin, rather than glucanases capable of attacking pNPG.