Biocontrol Activity of the Local Strain of Metschnikowia pulcherrima on Different Postharvest Pathogens.

The strains of the yeast Metschnikowia pulcherrima have strong biocontrol activity against various microorganisms. Biocontrol activity of M. pulcherrima largely depends on its iron immobilizing pigment pulcherrimin. Biocontrol activity of pulcherrimin producing strain, M. pulcherrima UMY15, isolated from local vineyards, was tested on different molds that cause food spoilage. M. pulcherrima UMY15 was a very effective biocontrol agent against Penicillium roqueforti, P. italicum, P. expansum, and Aspergillus oryzae in in-vitro plate tests. However, the inhibitory activity of M. pulcherrima UMY15 was less effective on Fusarium sp. and A. niger species in biocontrol assays. In addition, M. pulcherrima UMY15 strain completely inhibited the germination and mycelia growth of A. oryzae, A. parasiticus, and Fusarium sp. spores on artificial wounds of apples when they coinoculated with M. pulcherrima UMY15. Moreover, when coinoculated, M. pulcherrima UMY15 strain also inhibited the growth of P. roqueforti, P. italicum, P. expansum, A. oryzae, Fusarium sp., and Rhizopus sp. in grape juice, indicating that M. pulcherrima UMY15 can be used as a very effective biocontrol yeast against various species of postharvest pathogens, including Penicillium, Aspergillus, Fusarium, and Rhizopus.

Glucose signalling pathway controls the programmed ribosomal frameshift efficiency in retroviral-like element Ty3 in Saccharomyces cerevisiae.

Ty3 elements of S. cerevisiae contain two overlapping coding regions, GAG3 and POL3, which are functional homologues of retroviral gag and pol genes, respectively. Pol3 is translated as a Gag3-Pol3 fusion protein dependent on a +1 programmed frameshift at a site with the overlap between the two genes. We show that the Ty3 frameshift frequency varies up to 10-fold in S. cerevisiae cells depending on carbon source. Frameshift efficiency is significantly lower in cells growing on glucose as carbon source than in cells growing on poor alternative carbon sources (glycerol/lactate or galactose). Our results indicate that Ty3 programmed ribosomal frameshift efficiency in response to glucose signalling requires two protein kinases: Snf1p and cAMP-dependent protein kinase A (PKA). Increased frameshifting on alternative carbon sources also appears to require cytoplasmic localization of Snf1p, mediated by the Sip2p protein. In addition to the two required protein kinases, our results implicate that Stm1p, a ribosome-associated protein involved in nutrient sensing, is essential for the carbon source-dependent regulation of Ty3 frameshifting. These data indicate that Ty3 programmed ribosomal frameshift is not a constitutive process but that it is regulated in response to the glucose-signalling pathway.

Effect of different stresses on trehalose levels in Rhizopus oryzae.

Rhizopus oryzae accumulates and degrades trehalose in response to environmental stress conditions such as heat, osmotic stress, nitrogen starvation and pH. When heat stress was applied to R. oryzae, the trehalose content of the cells was increased from 0.9 to 4.8 mg/g dw and when the stress was relieved it decreased back to 1.1 mg/g dw. Under osmotic stress and nitrogen starvation, trehalose content was increased by 3.5 and 3 fold, respectively. The decrease in external pH increased trehalose level up to 2.6 mg/g dw from 1.1 mg/g dw. Increased trehalose level decreased back to normal after pH stress was relieved. These results indicate that trehalose accumulates and acts as a stress protectant in R. oryzae.

Glucose signaling pathway and growth conditions regulate gene expression in retrotransposon Ty2.

Gene expression in the yeast retrotransposon Ty2 is regulated at transcriptional and translational levels. In this study, we have shown that the transcription of Ty2 is partially dependent on the membrane-bound glucose sensors Gpr1p and Mth1p in Saccharomyces cerevisiae. Transcription of Ty2 decreased approx. 3-fold in the gpr1, mth1 yeast mutant. Moreover, our results revealed that the transcription of Ty2 fluctuates during the growth stages of S. cerevisae. Both transcription and the frameshift rate of Ty2 rapidly dropped when the stationary stage yeast cells were inoculated into fresh medium. There was an instant activation of Ty2 transcription and a high level expression during the entire logarithmic stage of yeast growth. However, the transcription of Ty2 decreased 2-fold when the yeast cultures entered the stationary stage. The frameshift rate in Ty2 also varied depending on the growth conditions. The highest frameshift level was observed during the mid-logarithmic stage. It decreased up to 2-fold during the stationary stage. Furthermore, we have found that the frameshift rate of Ty2 diminished at least 5-fold in slowly growing yeasts. These results indicate that the transcription and the frameshift efficiency are coordinately regulated in the retrotransposon Ty2 depending on the growth conditions of S. cerevisiae.

Isolation and characterization of new Metschnikowia pulcherrima strains as producers of the antimicrobial pigment pulcherrimin.

Metschnikowia pulcherrima is a highly effective biocontrol yeast due to its pigment pulcherrimin that accumulates in the cells and in the growth medium. Three different strains of M. pulcherrima were isolated from local grapes. The yeast isolates were characterized on the basis of their biochemical, physiological and ITS1-5.8 s rDNA-ITS2 region. Based on the obtained results, the M. pulcherrima isolates were identified as new strains of M. pulcherrima. Strong antagonistic activities of the M. pulcherrima strains on the human pathogens Proteus vulgaris, Escherichia coli, Candida albicans, Candida parapsilosis, Candida krusei, and Trichosporon mucoides were determined. In addition, antagonistic effects of these M. pulcherrima strains were also tested against Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Trichoderma spp., Paecilomyces spp., and Bipolaris spp. and it was shown that the three different strains of M. pulcherrima also have an antagonistic effect on the growth of these fungal species at different extents. This study showed that all three strains of M. pulcherrima produce the same amount of the pigment pulcherrimin, but their antimicrobial activities on different microorganisms show important variations.

Glucose signaling controls the transcription of retrotransposon Ty2-917 in Saccharomyces cerevisiae.

We have analyzed the effects of glucose signaling on the transcription in the yeast retrotransposon Ty2-917. Growth of Saccharomyces cerevisiae in non-fermentable carbon sources such as glycerol, lactate, or ethanol resulted in a dramatic decrease in the transcription of Ty2-917. However, when the yeast cells were transferred to a fermentable growth medium, Ty2-917 transcription is activated by 13-fold. Nonetheless, it appears that the activation of Ty2 transcription requires high levels of glucose since low levels of glucose or 2-deoxyglucose were not sufficient for the activation of Ty2 transcription. In addition, we have shown that glucose induction of Ty2 transcription may require the transcription factor Gcr1p since the glucose induced transcription level of Ty2 is much lower in a gcr1 mutant yeast strain than the GCR1+ strain. These results demonstrate that glucose signaling activates the transcription in the retroviral-like element Ty2-917.

Isolation and characterization of glucose derepressed invertase mutants from Schizosaccharomyces pombe.

We have isolated 14 different Schizosaccharomyces pombe mutants that synthesize invertase enzyme constitutively. Analyses of invertase activities revealed that the degrees of resistance to glucose repression were not similar among different complementation groups. One of the complementation groups appeared to be associated with functional and/or regulatory defects in hexose transport. Another complementation group appeared to be specific for the regulation of the inv1 gene alone, implying that these mutations might be associated with different genes acting on the glucose sensing and signaling pathway. In addition, we found that the wild-type level glucose uptake is essential for the full-level repression of inv1 expression.

Non-histone proteins Nhp6A and Nhp6B are required for the regulated expression of SUC2 gene of Saccharomyces cerevisiae.

Transcription of the SUC2 gene that encodes invertase enzyme is controlled by glucose repression and derepression mechanisms in Saccharomyces cerevisiae. Several regulatory factors such as Mig1p complex, Gcr1p, Hxk2p, nucleosomes, and the Snf1p kinase complex have been identified as the regulators of SUC2 transcription. The results presented in this study indicate that the non-histone proteins Nhp6A and Nhp6B were also required for the regulated expression of SUC2 gene. Expression of the SUC2 gene reduced to one-fiftieth-one-tenth in the Deltanhp6A Deltanhp6B double mutant strain depending on the growth conditions. Moreover, SUC2 expression and invertase synthesis became constitutive after long-term derepression, and decreased to a low level in Deltanhp6A Deltanhp6B double deletion mutant. A time course analysis of the invertase synthesis revealed that both the repression and derepression rates were very slow in the Deltanhp6A Deltanhp6B double mutant yeast. These results indicate that the architectural transcription factors Nhp6A and Nhp6B play a very critical role in the regulation of SUC2 gene expression.

The GCR2 gene is required for the transcriptional activation of retrotransposon Ty2-917 in Saccharomyces cerevisiae.

Ty2 retrotransposons are the mobile genetic elements of the yeast Saccharomyces cerevisiae. Transcriptional regulation of Ty2-917 requires a complex set of cis acting elements which are located both upstream and downstream of the transcription initiation site. Previously, the glycolysis regulatory protein Gcr1p has been identified as the major transcriptional regulator of Ty2-917. Gcr1p is a DNA binding transcription factor that requires Gcr2p for its functions. In this study, the effect of Gcr2p on the transcriptional regulation of Ty2-917 was analyzed. The result of this study indicates that Ty2-917 transcription decreases 24-fold in gcr2 mutant yeast cells. In addition, Ty2 enhancer element dependent transcriptional activation of a heterolog promoter also decreases at a significant level. These results showed that Gcr2p is essential for the high level transcription of Ty2-917.