Wine yeast species show strong inter- and intra-specific variability in their sensitivity to ultraviolet radiation.

While the trend in winemaking is toward reducing the inputs and especially sulphites utilization, emerging technologies for the preservation of wine is a relevant topic for the industry. Amongst yeast spoilage in wine, Brettanomyces bruxellensis is undoubtedly the most feared. In this study, UV-C treatment is investigated. This non-thermal technique is widely used for food preservation. A first approach was conducted using a drop-platted system to compare the sensitivity of various strains to UV-C surface treatment. 147 strains distributed amongst fourteen yeast species related to wine environment were assessed for six UV-C doses. An important variability in UV-C response was observed at the interspecific level. Interestingly, cellar resident species, which are mainly associated with wine spoilage, shows higher sensitivity to UV-C than vineyard-resident species. A focus on B. bruxellensis species with 104 screened strains highlighted an important effect of the UV-C, with intra-specific variation. This intra-specific variation was confirmed on 6 strains in liquid red wine by using a home-made pilot. 6624 J.L-1 was enough for a reduction of 5 log10 of magnitude for 5 upon 6 strains. These results highlight the potential of UV-C utilization against wine yeast spoiler at cellar scale.

An optimized yeast display strategy for efficient scFv antibody selection using ribosomal skipping system and thermo resistant yeast.

OBJECTIVES: Establish a method to restrict unexpected fragments including stop codons in scFv library and generate a thermo resistant strain for screening of thermal stable scFv sequences. RESULTS: Here, we have constructed a T2A-Leu2 system for selection of yeast surface display libraries that blocks amplification of "stop codon" plasmids within the library, thereby increasing the quality of the library and efficiency of the selection screen. Also, we generated a temperature-resistant yeast strain, TR1, and validated its combined use with T2A-Leu2 for efficient screening. Thus, we developed a general approach for a fast and efficient screening of scFv libraries using a ribosomal skipping system and thermo-resistant yeast. CONCLUSIONS: The method highlights the utility of the T2A-Leu2-based ribosomal skipping strategy for increasing the quality of the input library for selection, along with an optimized selection protocol based on thermo-resistant yeast cells.

Ultraviolet-C resistance of selected spoilage yeasts in orange juice.

This study determined the ultraviolet-C (UV-C) dose necessary to reduce 90% population (DUV-C) of 17 spoilage yeasts and their composited inoculum in orange juice (pH 3.71, 11.60 °Brix, 0.55% citric acid, 2.46% w/v insoluble solids). Growth parameters of all test yeasts were first established to standardize the growth stage of the cells prior to harvesting and eventual UV-C challenge studies. Approximately 4-5 log CFU/ml cells in the mid-stationary growth phase (30.3 t0 39.9 h, 25 °C) were suspended in 4 ml turbulent flowing juice and subjected to UV-C irradiation at an incident surface irradiance of 3.64-4.97 mW/cm2. The inactivation rates of each yeast and their composited inoculum were determined using 2 methods namely, the linear regression and Baranyi and Roberts (1994) model-fitting. Results showed that the yeasts exhibited either log-linear or biphasic inactivation behavior with downward concavity or inactivation lag. Regardless of the method of determination, Cryptococcus albidus (LJY1) exhibited the significantly greatest (p < 0.05) UV-C resistance with DUV-C values of 1924.31 and 2174.63 mJ/cm2. On the other hand, Candida parapsilosis was determined to be least resistant with a DUV-C values of 245.83 and 357.88 mJ/cm2. Majority of the DUV-C values determined from the model-fitting were greater than those calculated from linear regression. However, only those determined for the composited inoculum were significantly different. The results of this study address knowledge gaps pertinent to the UV-C resistance of less studied spoilage yeast, and help in better understanding the utility of this non-thermal food processing technology.

Survival of Extremophilic Yeasts in the Stratospheric Environment during Balloon Flights and in Laboratory Simulations.

The high-altitude atmosphere is a harsh environment with extremely low temperatures, low pressure, and high UV irradiation. For this reason, it has been proposed as an analogue for Mars, presenting deleterious factors similar to those on the surface of that planet. We evaluated the survival of extremophilic UV-resistant yeasts isolated from a high-elevation area in the Atacama Desert under stratospheric conditions. As biological controls, intrinsically resistant Bacillus subtilis spores were used. Experiments were performed in two independent stratospheric balloon flights and with an environmental simulation chamber. The three following different conditions were evaluated: (i) desiccation, (ii) desiccation plus exposure to stratospheric low pressure and temperature, and (3) desiccation plus exposure to the full stratospheric environment (UV, low pressure, and temperature). Two strains, Naganishia (Cryptococcus) friedmannii 16LV2 and Exophiala sp. strain 15LV1, survived full exposures to the stratosphere in larger numbers than did B. subtilis spores. Holtermanniella watticus (also known as Holtermanniella wattica) 16LV1, however, suffered a substantial loss in viability upon desiccation and did not survive the stratospheric UV exposure. The remarkable resilience of N. friedmannii and Exophiala sp. 15LV1 under the extreme Mars-like conditions of the stratosphere confirms its potential as a eukaryotic model for astrobiology. Additionally, our results with N. friedmannii strengthen the recent hypothesis that yeasts belonging to the Naganishia genus are fit for aerial dispersion, which might account for the observed abundance of this species in high-elevation soils.IMPORTANCE Studies of eukaryotic microorganisms under conditions of astrobiological relevance, as well as the aerial dispersion potential of extremophilic yeasts, are still lacking in the literature compared to works with bacteria. Using stratospheric balloon flights and a simulation chamber, we demonstrate that yeasts isolated from an extreme environment are capable of surviving all stressors found in the stratosphere, including intense UV irradiation, scoring an even higher survival than B. subtilis spores. Notably, the yeast N. friedmannii, which displayed one of the highest tolerances to the stratospheric environment in the experiments, was recently proposed to be adapted to airborne transportation, although such a hypothesis had not yet been tested. Our results strengthen such an assumption and can help explain the observed distribution and ecology of this particular yeast species.

UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage.

Tolerance to Ultraviolet Radiation of Psychrotolerant Yeasts and Analysis of Their Carotenoid, Mycosporine, and Ergosterol Content.

Yeasts colonizing the Antarctic region are exposed to a high ultraviolet radiation evolving mechanisms to minimize the UV radiation damages, such as the production of UV-absorbing or antioxidant compounds like carotenoid pigments and mycosporines. Ergosterol has also been suggested to play a role in this response. These compounds are also economically attractive for several industries such as pharmaceutical and food, leading to a continuous search for biological sources of them. In this work, the UV-C radiation tolerance of yeast species isolated from the sub-Antarctic region and their production of carotenoids, mycosporines, and ergosterol were evaluated. Dioszegia sp., Leuconeurospora sp. (T27Cd2), Rhodotorula laryngis, Rhodotorula mucilaginosa, and Cryptococcus gastricus showed the highest UV-C radiation tolerance. The yeasts with the highest content of carotenoids were Dioszegia sp. (OHK torulene), Rh. laryngis (torulene and lycopene), Rh. mucilaginosa, (torulene, gamma carotene, and lycopene), and Cr. gastricus (2-gamma carotene). Probable mycosporine molecules and biosynthesis intermediates were found in Rh. laryngis, Dioszegia sp., Mrakia sp., Le. creatinivora, and Leuconeurospora sp. (T27Cd2). Ergosterol was the only sterol detected in all yeasts, and M. robertii and Le. creatinivora showed amounts higher than 4 mg g−1. Although there was not a well-defined relation between UV-C tolerance and the production of these three kinds of compounds, the majority of the yeasts with lower amounts of carotenoids showed lower UV-C tolerance. Dioszegia sp., M. robertii, and Le. creatinivora were the greatest producers of carotenoids, ergosterol, and mycosporines, respectively, representing good candidates for future studies intended to increase their production for large-scale applications.

UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes.

The cytotoxicity of UV light-induced DNA lesions results from their interference with transcription and replication. DNA lesions arrest elongating RNA polymerases, an event that triggers transcription-coupled nucleotide excision repair. Since arrested RNA polymerases reduce the accessibility of repair factors to DNA lesions, they might be displaced. The fate of arrested RNA polymerases-II at DNA lesions has been extensively studied, yielding partially contradictory results. Considerably less is known about RNA polymerases-I that transcribe nucleosomes-depleted rRNA genes at very high rate. To investigate the fate of arrested RNA polymerases-I at DNA lesions, chromatin-immunoprecipitation, electron microscopy, transcription run-on, psoralen-cross-linking and chromatin-endogenous cleavage were employed. We found that RNA polymerases-I density increased at the 5'-end of the gene, likely due to continued transcription initiation followed by elongation and pausing/release at the first DNA lesion. Most RNA polymerases-I dissociated downstream of the first DNA lesion, concomitant with chromatin closing that resulted from deposition of nucleosomes. Although nucleosomes were deposited, the high mobility group-box Hmo1 (component of actively transcribed rRNA genes) remained associated. After repair of DNA lesions, Hmo1 containing chromatin might help to restore transcription elongation and reopening of rRNA genes chromatin.

Optically transparent polymer devices for in situ assessment of cell electroporation.

In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 µl was quantified in situ as a function of applied voltage from 10 to 90 V in a series of ~2-ms pulses across 0.5-mm electrode spacing. The electric field at the interface and device current was calculated using a model that takes into account bulk screening of the transient pulse. The voltage dependence of the number of electroporated cells could be explained using a stochastic model for the electroporation kinetics, and the free energy for pore formation was found to be 45.6 ± 0.5 kT at room temperature. With this device, the optimum electroporation conditions can be quickly determined by monitoring the uptake of PI marker dye in situ under the application of millisecond voltage pulses. The electroporation efficiency was also quantified using an ex situ fluorescence-assisted cell sorter, and the morphology of cultured cells was evaluated after the pulsing experiment. Importantly, the efficacy of the developed device was tested independently using two cell lines (C2C12 mouse myoblast cells and yeast cells) as well as in three different electroporation buffers (phosphate buffer saline, electroporation buffer and 10% glycerol).

Study of the inactivation of spoilage microorganisms in apple juice by pulsed light and ultrasound.

The aim of this study was to evaluate the effect of ultrasound (US) (600 W, 20 kHz and 95.2 µm wave amplitude; 10 or 30 min at 20, 30 or 44 ± 1 °C) and pulsed light (PL) (Xenon lamp; 3 pulses/s; 0.1 m distance; 2.4 J/cm(2)-71.6 J/cm(2); initial temperature 2, 30, 44 ± 1 °C) on the inactivation of Alicyclobacillus acidoterrestris ATCC 49025 spores and Saccharomyces cerevisiae KE162 inoculated in commercial (pH: 3.5; 12.5 °Brix) and natural squeezed (pH: 3.4; 11.8 °Brix) apple juices. Inactivation depended on treatment time, temperature, microorganism and matrix. Combination of these technologies led up to 3.0 log cycles of spore reduction in commercial apple juice and 2.0 log cycles in natural juice; while for S. cerevisiae, 6.4 and 5.8 log cycles of reduction were achieved in commercial and natural apple juices, respectively. In natural apple juice, the combination of US + 60 s PL at the highest temperature build-up (56 ± 1 °C) was the most effective treatment for both strains. In commercial apple juice, US did not contribute to further inactivation of spores, but significantly reduced yeast population. Certain combinations of US + PL kept on good microbial stability under refrigerated conditions for 15 days.

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

Effect of photostimulation through red LED light radiation on natural fermentation of table olives: An innovative case study with Negrinha the Freixo variety.

In this study, for the first time, red LED light radiation was applied to the fermentation process of table olives using the Negrinha de Freixo variety. Photostimulation using LED light emission (630 ± 10 nm) is proposed to shorten and speed up this stage and reduce time to market. Several physical-chemical characteristics and microorganisms (total microbial count of mesophilic aerobic, molds, yeasts, and lactic acid bacteria) and their sequence during fermentation were monitored. The fermentation occurred for 122 days, with two irradiation periods for red LED light. The nutritional composition and sensory analysis were performed at the end of the process. Fermentation under red LED light increased the viable yeast and lactic acid bacteria (LAB) cell counts and decreased the total phenolics in olives. Even though significant differences were observed in some color parameters, the hue values were of the same order of magnitude and similar for both samples. Furthermore, the red LED light did not play a relevant change in the texture profile, preventing the softening of the fruit pulp. Similarly, LED light did not modify the existing type of microflora but increased species abundance, resulting in desirable properties and activities. The species identified were yeasts - Candida boidinii, Pichia membranifaciens, and Saccharomyces cerevisiae, and bacteria - Lactobacillus plantarum and Leuconostoc mesenteroides, being the fermentative process dominated by S. cerevisiae and L. plantarum. At the end of fermentation (122 days), the irradiated olives showed less bitterness and acidity, higher hardness, and lower negative sensory attributes than non-irradiated. Thus, the results of this study indicate that red LED light application can be an innovative technology for table olives production.

Quantitative analysis of fitness and genetic interactions in yeast on a genome scale.

Global quantitative analysis of genetic interactions is a powerful approach for deciphering the roles of genes and mapping functional relationships among pathways. Using colony size as a proxy for fitness, we developed a method for measuring fitness-based genetic interactions from high-density arrays of yeast double mutants generated by synthetic genetic array (SGA) analysis. We identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate single- and double-mutant fitness measurements, which rival the accuracy of other high-resolution studies. We applied the SGA score to examine the relationship between physical and genetic interaction networks, and we found that positive genetic interactions connect across functionally distinct protein complexes revealing a network of genetic suppression among loss-of-function alleles.

Occurrence and low pressure ultraviolet inactivation of yeasts in real water sources.

Low pressure ultraviolet photolysis proved to be an efficient treatment to achieve inactivation of different yeast species (Candida sp., Cryptococcus carnescens, Metschnikowia viticola/Candida kofuensis, Rhodosporidium babjevae, Rhodotorula minuta, and Rhodotorula mucilaginosa) isolated from water sources with very different compositions. The sensitivity degree of various yeast isolates to UV treatment varied among different genera. Species isolated from surface water gained additional photoprotective resistance as a defence mechanism to be able to survive under constant sunlight conditions compared to the groundwater isolates. Yeasts were found to be more resistant to UV treatment than E. coli, Cryptosporidium, and Giardia.

Hot water, UV-C and superatmospheric oxygen packaging as hurdle techniques for maintaining overall quality of fresh-cut pomegranate arils.

BACKGROUND: In recent years there has been increasing consumer pressure to avoid the use of agrochemicals such as chlorine on fresh plant products for extending their shelf life. The combined use of eco-sustainable techniques may be an alternative. The effect of hot water (HW), ultraviolet-C (UV-C) light and high oxygen packaging (HO) on the overall quality of fresh-cut pomegranate arils stored under modified atmosphere packaging (MAP) for up to 14 days at 5 °C was studied. RESULTS: Arils extracted manually, washed with chlorine (100 µL L⁻¹ NaClO, pH 6.5, 5 °C water), rinsed and drained were exposed to single or combined (double and triple) hurdle techniques. The HW treatment consisted of a 30 s immersion in water at 55 °C followed by air surface drying. A 4.54 kJ m⁻² dose was used for the UV-C treatment before packaging. Active MAP with initial 90 kPa O2 was used in the HO treatment. The respiration rate remained relatively constant throughout shelf life, with no differences among treatments. CO2 accumulation was higher within HO packages. HW induced a slight reduction in total soluble solids, while no changes in titratable acidity were found. HO-treated arils had the highest phenolic content, while the lowest was found in HW-treated arils. The lowest antioxidant activity was found in HW + UV-C + HO and the highest in UV-C + HO and HO treatments. HW alone or in combination with UV-C and HO inhibited mesophile, mould and yeast growth, while UV-C + HO was most effective for controlling yeast and mould growth. CONCLUSION: UV-C and HO either alone or in combination are promising techniques to preserve the quality of pomegranate arils for up to 14 days at 5 °C.

Efficiency of selected mutagens in generating Xanthophyllomyces dendrorhous strains hyperproducing astaxanthin.

Astaxanthin is a xanthophill pigment with commercial application in the aquaculture, pharmaceutical, food and cosmetic industries. The red yeast Xanthophyllomyces dendrorhous is one of the most promising microorganisms for its industrial production. However, astaxanthin content in wild yeast strains is low. Pigment production by X. dendrorhous can be improved by mutagenesis. The aim of the study was to assess the efficiency of four mutagens: UV radiation, benomyl, ethyl methanesulfonate and ethidium bromide in generating asthaxanthin-hyperproducing strains of the yeast Xanthophyllomyces dendrorhous DSM 5626. Mutations with benomyl, ethidium bromide and UV radiation generated a group of hyperpigmented mutants exhibiting increases up to 100% in astaxanthin content. Ethyl methanesulfonate turned out to be useless in this respect.

Prospects for the study of biological systems with high power sources of terahertz radiation.

The emergence of intense sources of terahertz radiation based on lasers and electron accelerators has considerable potential for research on biological systems. This perspective gives a brief survey of theoretical work and the results of experiments on biological molecules and more complex biological systems. Evidence is accumulating that terahertz radiation influences biological systems and this needs to be clarified in order to establish safe levels of human exposure to this radiation. The use of strong sources of terahertz radiation may contribute to the resolution of controversies over the mechanism of biological organization. However the potential of these sources will only be realized if they are accompanied by the development of sophisticated pump-probe and multidimensional experimental techniques and by the study of biological systems in the controlled environments necessary for their maintenance and viability.

A cassette of N-terminal amino acids of histone H2B are required for efficient cell survival, DNA repair and Swi/Snf binding in UV irradiated yeast.

The highly charged histone N-terminal domains are engaged in inter- and intra-nucleosomal interactions, and contain a host of sites used for posttranslational modification. We have studied the effect of deleting residues 30-37 from the N-terminal domain of histone H2B in yeast cells, on nucleotide excision repair (NER) following UV irradiation, as these cells are quite sensitive to UV. We find that H2B Delta30-37 cells exhibit reduced NER efficiency at three specific chromatin loci: the transcriptionally active, RPB2 locus; the transcriptionally silenced, nucleosome-loaded HML locus; and the transcriptionally repressed, non-silenced, GAL10 locus. Nuclease digestion studies indicate that H2B Delta30-37 chromatin has increased nucleosome accessibility and/or nucleosome mobility. In addition, H2B Delta30-37 mutants acquire more DNA damage, compared to wt cells, following the same dose of UV radiation. Reducing the level of damage in H2B Delta30-37 cells to match that of wt cells restores the NER rate to wt levels in the RPB2 and GAL10 loci, but NER efficiency remains low in the silenced HML locus. Interestingly, recruitment of Snf5 to the HML locus is reduced in H2B Delta30-37 cells and more transient following UV irradiation. This may reflect a lower binding affinity of the SWI/SNF complex to H2B Delta30-37 nucleosomes.

Effects of ultraviolet light and ultrasound on microbial quality and aroma-active components of milk.

BACKGROUND: Heat treatment is the most common way to extend the shelf life of milk. However, alternative technologies such as ultraviolet (UV) light and ultrasound (US), which are non-thermal methods for processing milk, have been developed to replace heat treatment. These technologies do not have any adverse effects on the quality of milk. The major purpose of this study was to investigate the effects of UV and US on different micro-organism groups and aroma compounds in milk. RESULTS: Heat pasteurisation at 65 °C for 30 min was used as thermal control treatment. The growth of total coliform group bacteria, Escherichia coli and Staphylococcus spp. was completely reduced by UV treatment. Application of US was not sufficient to reduce the numbers of yeasts and moulds. In neutral/basic fractions, 3-methylthiophene (plastic), hexanal (grass) and 1-hexen-3-one (floral) were major volatiles in milk samples. CONCLUSION: UV had a major effect on total coliforms, E. coli and Staphylococcus spp., but US was not as effective as UV and heat treatment in reducing certain groups of micro-organisms. No major differences were observed in terms of aroma-active compounds and flavour of milk following the different treatments. However, some new volatiles and change in rheological properties were generated by UV and US treatments.

Lights on and action! Controlling microbial gene expression by light.

Light-mediated control of gene expression and thus of any protein function and metabolic process in living microbes is a rapidly developing field of research in the areas of functional genomics, systems biology, and biotechnology. The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes in a noninvasive and spatiotemporal fashion. This mini review describes recently developed strategies to generate photo-sensitive expression systems in bacteria and yeast. Naturally occurring and artificial photoswitches consisting of light-sensitive input domains derived from different photoreceptors and regulatory output domains are presented and individual properties of light-controlled expression systems are discussed.