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Review

Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii

Tikam Chand Dakal, Lisa Solieri, Paolo Giudici

Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42122, Reggio Emilia, Italy

a b s t r a c ta r t i c l e i n f o

Article history:Received 14 November 2013

Received in revised form 18 April 2014Accepted 4 May 2014Available online 25 May 2014

Keywords: Zygosaccharomyces rouxiiSpoilage yeastOsmotoleranceHalotoleranceGlycerol accumulation and retentionCation homeostasis

The osmotolerantand halotolerant food yeast Zygosaccharomyces rouxii is known for itsability to grow andsurvivein the face of stresscaused by high concentrations of non-ionic (sugars and polyols)and ionic (mainly Na + cations)solutes.This abilitydetermines thesuccessof fermentationon high osmolarity food matricesand leadsto spoilage of high sugar and high salt foods.The knowledgeaboutthe genes,the metabolicpathways, and theregulatory circuitsshaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation con-trol, optimizationof food starter culture,andprevention of food spoilage. Thisreviewsummarizes recent insights onthe mechanisms used by Z. rouxii and other osmo and halotolerant food yeasts to endure salts and sugars stresses.Using the information gathered from S. cerevisiae as guide, we highlight how these non-conventional yeasts inte-grate general and osmoticum-speci c adaptive responses under sugar and salts stresses, including regulation of Na+ and K+ - uxes across the plasma membrane,modulation of cellwallproperties, compatible osmolyte produc-tion andaccumulation, andstress signallingpathways.We suggesthowan integrated andsystem-basedknowledgeon these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control infood, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms.

2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. A matter of nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1413. Osmotolerant and halotolerant yeasts in food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1424. Gene circuits and metabolic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

4.1. Cell wall and plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1434.2. Cation homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4.2.1. Na+ inward and outward movements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1444.2.2. K+ inward and outward movements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

4.3. Sugar transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144.4. Production and accumulation of osmolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

4.4.1. Glycerol metabolic pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1474.4.2. Glycerol biosynthesis in non-stressed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484.4.3. Glycerol biosynthesis in osmo-stressed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484.4.4. Glycerol retention and active transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484.4.5. Other compatible solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

5. Signal transduction and cis/trans -acting regulatory factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505.1. High osmolarity glycerol (HOG) pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505.2. Calcineurin/Crz1 pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515.3. Ras-cAMP signalling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

6. Non genetic regulation of osmostress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1526.1. Chromatin-mediated mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

International Journal of Food Microbiology 185 (2014) 140 157

Abbreviations: CDRE, calcineurin dependent response element; CNV, copy number variation; CWI, cell wall integrity; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate;HOG, high-osmolarity glycerol; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase;P-Hog1, phosphorylated Hog 1; STRE, stress responsive element; SWI/SNF complex, switch/sucrose non-fermenting complex. Corresponding author. Tel.: +39 0522522057; fax +39 0522522027.

E-mail address: [email protected] (P. Giudici).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.05.015

0168-1605/ 2014 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

International Journal of Food Microbiology

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6.2. Phenotypic heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17. Food exploitation and biotechnological perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1528. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

The high concentrations of ionic (mainly Na + ) and non-ionic (mainlysugars andpolyols)solutes reduce water activity(a w ) infood and are twoof the major abiotic stressors, both limiting the yeast growth. High exter-nal osmolarity has been used forcenturies for food preservation,becauseit causeswater out ow from thecell and results ina higher intracellularconcentration of ions andmetabolitesandin an eventual arrest of cellu-lar activity. The yeast ability to cope with these environmental insultsdetermines both the success of certain food and beverage fermentationand the thriving of food spoilage.

Since the sequencing of strainS288c ( Goffeau et al., 1996 ), impressiveadvances in genomics, proteomics, and systems biology have madeS. cerevisiae the paradigm for understanding these osmo-adaptivemechanisms, which have been exhaustively summarized by several re-views ( Nevoigt and Stahl, 1997; Hohmann, 2002; Ario et al., 2010;Khn and Klipp, 2012 ). As a result, the S. cerevisiae response to highexternal solute concentrations has been described as a system-levelcoordination between the extracellular environment and the geneticmake-up inside the cell. The following interconnected modules are in-volved: (i) receiving information fromexternalenvironment (sensing);(ii) conducting it to the inside (signal transduction); (iii) integrating itwith internal genetic information in order to mount an appropriate re-sponse (effector processes) ( de Nadal et al., 2011 ). This system-levelknowledge has been exploited in food industry to improve yeast fer-mentations on highly salty and sugary matrices or to decrease thefood spoilage by sugar and salt resistant-yeast species. However, asbeing moderately halotolerant and osmotolerant, S. cerevisiae could beinappropriate to describe the yeast response to hypersaline andhyperosmotic food.

Zygosaccharomyces rouxii is the osmotolerant and halotolerant yeastmost phylogenetically related to S. cerevisiae and inhabits a varietyof highly sugary and salty food, where it carries out fermentation ordetermines food spoilage. It belongs to the genus Zygosaccharomyces ,which includes the highest number of salt and sugar-tolerant yeasts.The majority of these species are osmotolerant (positive growth athigh sugar concentration up to 60 70% glucose), whereas only a fewareboth highlyosmoand halotolerant ( Table 1 ). Recently, thecompletegenome sequences of Z. rouxii (Souciet et al., 2009 ) and other highlyosmo and halotolerant yeasts, such as Millerozyma farinosa (formerlyPichia sorbitophila ) ( Louis et al., 2012), Debaryomyces hansenii (Kumaret al., 2012 ), and Zygosaccharomyces bailii (Galeote et al., 2013 ), havebecome available. Furthermore, omics tools and genetic manipulation

protocols have been recently employed to analyze the relationshipsof osmostress phenotype to genetic and molecular determinants(Prybilova et al., 2007a,b; Watanabe et al., 2010 ). From these intenseefforts, the yeast osmostress adaptation emerges as a complex mecha-nism that integrates genes, regulatory networks, and signalling path-ways, and that differs depending upon the species and the osmoticumin the surrounding medium. Comparison of species with differentsugar and salt tolerance highlighted how yeasts exploit different strate-gies to survive under osmotic and salt stress ( Ramos et al., 2011 ). Forexample, Z. rouxii resembles S. cerevisiae in extruding Na + cations outof the cell or driving them into the vacuole ( Ramos, 1999 ), while thehalotolerant yeast Debaryomyces hansenii is a sodium includer, whichaccumulates intracellularly Na + without getting intoxicated ( Ramos,1999 ). Beyond the species-speci c strategies, other osmostress re-

sponses, such as the osmolytes accumulation, are ubiquitous among

yeasts to avoid out ow of cellular water in low a w environments(Nevoigt and Sthal, 1997; Lages et al., 1999; Silva-Graa and Lucas,2003 ). Another emerging issue concerns how salt and sugars elicit dis-tinct or partial overlapping responses in yeasts. Whereas sugars andpolyols modifyosmoticpressure, salts induce alterations both in osmot-ic pressure and ion homeostasis. The result is that partially differentmechanisms become operational in response to sugar and salts. Sincehalo and osmotolerance could be paired and unpaired phenotypes in Z. rouxii and relatives, these yeasts are very attractive models fordeciphering genetic circuits and functional pathways underlyinghalotolerance and osmotolerance.

Here, we review recent insights on the mechanisms that governhalotolerance and osmotolerance in Z. rouxii and compare them tothose active in S. cerevisiae and in other osmo and halotolerant foodyeasts atgenetic,metabolic, signalling,andepigenetic level. Furthermore,we highlight how these yeasts can achieve generic and osmoticum-speci c responses to sugar and salt stresses. Finally, we point out howthe understanding of osmostress responsive mechanisms can advan-tage microbial fermentation and food quality.

2. A matter of nomenclature

Tolerance to high ionic and non-ionic solute concentrations is aspeci c cellular adaptability to sudden and severe uctuations in wateravailability anda tendency of cells to restore or maintain normalphysiol-ogy, morphology and biological functions ( Yancey, 2005; Klipp et al.,2005 ). Microbial growth under high externalosmolarity is frequently de-scribed in terms of a w that is the chemical potential of free water in solu-tion. Microorganisms able to colonize food with high osmolarity and,consequently, low a w , were collectively indicated as xerotolerant (no ab-solute requirement of low a w ), and xerophilic ( lovers of low a w ) ( PittandHocking,2009 ) (Table1 ). A moreappropriatemicrobial classi cationwould consider the kind of osmoticum and include the following catego-ries: osmophilic, absoluterequirement fornon-ionic solutes andability togrow up to solute concentrations approaching saturation; osmotolerant,no absolute requirement of non-ionic solutes for viability and ability totolerate a wide range of osmolarity, from hypo-osmotic to hyper-osmotic solutions; osmosensitive, sensitive to excess concentration of non-ionic solutes; halophilic, absolute requirement for high salt andability to grow up to salt concentrations approaching saturation;halotolerant, no absolute requirement of salt for viability and ability totolerate a wide range of salinity, from hypo-saline to hyper-saline solu-tion; and halosensitive, sensitive to excess concentration of salt.

Most food yeasts can develop well at a w values around 0.95 0.90. Acut-off of a w b 0.70 has been frequently used to delineate osmotolerantand halotolerant yeasts. In the past, yeasts isolated from sugary andsalty food with a w lower than 0.70 were referred to as osmophilic and halophilic (Tokuoka, 1993 ). For instance, Debaryomyces hansenii hasbeen described as halophilic yeast based on the ability to grow at 1.0 Mof salt with growth rate and nal biomass close to the values obtainedwithout salt ( Almagro et al., 2000; Gonzlez-Hernndez et al., 2004;Aggarwal and Mondal, 2009 ). Other yeasts were classi ed as halophilicor osmophilic, such as M. farinosa (formerly P. sorbitophila ) (Rodriguesde Miranda et al., 1980 ), Candida etchellsii (formerly Candidahalonitratophila ), Candida versatilis (Barnett et al., 2000 ), and the blackyeast Hortea werneckii (Gunde-Cimerman et al., 2000 ). However,differentlyfrom halophilic andosmophilicbacteria, noneof these yeasts

satis es the true de nition of osmophily or halophily, because they

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tolerate high osmotic conditions, without requiring high sugar orsalt amounts for their growth ( Silva-Graa et al., 2003 ). Similarly, theterms osmotolerant and halotolerant have been improperly used assynonymous, but they should be reserved for yeasts that are able tolive at high sugar and ionic solutes (mainly Na + ) concentrations,respectively ( Onishi, 1963; Tokuoka et al., 1992; Tokuoka, 1993;

Lages et al., 1999; Mare ova and Sychrov, 2003; Pribylova et al.,2007a,b ). Since different solutes elicit distinct yeast stress responses,osmotolerant and halotolerant phenotypes have been de ned inrelation to the yeast ability to grow up to 55 65% (w/v) sugar (lowerthan ~0.88 a w ) or at 15 25% (w/v) salt concentrations (correspondingto 0.92 0.85 a w range), respectively ( Deak, 2006, 2007; Kurtzamnet al., 2011 ). Based on these assays, tolerance to sugar and salt couldbe differentially distributed among yeasts species. Table 1 shows theclassi cation of representative yeast species into four classes accordingto their degree of sugar and salt tolerance.

3. Osmotolerant and halotolerant yeasts in food

Osmotolerant and halotolerant yeasts have a pivotal role in food

fermentation and spoilage ( Fleet, 1992 ) and several of them belong to

the genus Zygosaccharomyces . The genus comprises 7 species ( Jamesand Stratford, 2011 ) frequently isolated from highly sugary (honey, jams, syrups, fruit-juices, fruit juice concentrates, chocolate candies,and concentrated grape must) and salty food (soy sauce and misopaste) ( Dek and Beuchat, 1993; Tokuoka, 1993; Solieri et al., 2006,2013a,b; Martorellet al., 2007; Suezawa et al., 2008 ). Zygosaccharomyces

sapae and Z. rouxii are the main biocatalysts of alcoholic fermentation inhigh sugar and/or salt fermented food, such as traditional balsamic vin-egar and miso, respectively ( Suezawa et al., 2008; Solieri and Giudici,2008;Solieri et al., 2013a ). Furthermore, Z. rouxiiand Zygosaccharomycesbailii are involved in alcoholic fermentation during kombuchaproduction. Zygosaccharomyces species have been also recognized asone of the main spoilage yeasts in food industry due to their tolerancesto salt, sugar, and weak acid preservatives ( Pitt and Hocking, 2009;Fleet, 2011 ). In food manufacture, the yeast spoilage of products causessevere economic loss and affects a variety of processed foods, includingbread, cereals, spices, dairy products such as cheese, spreads (marga-rine), dressings, fondant,chocolate, fermented sauces (soy), soft drinks,fruits, jams, and high-sugar fruit syrups ( Stratford, 2006 ). In particular,Z. bailii and Zygosaccharomyces lentus represent the most important

Zygosaccharomyces from the point of view of weak acid preservative

Table 1Proposal for a classi cation of representative yeast species according to their halotolerance and osmotolerance behaviors.

Category De nition Species name 1 D-glucose %(w/v)

NaCl (M); % (w/v) Food spoilage2 References 3

50 60

Moderately osmotolerantand moderatelyhalotolerant

Lack of growthat N 50% (w/v)D glucose;lack of growthat N 2.0M NaCl

Saccharomyces cerevisiae b 1.70; 10% Soft drink, fruit juice Onishi, 1963Schizosaccharomyces pombe

1.00; 5.8% Cheese, fruit (rarely) Lages et al., 1999;Barnett et al., 2000

Zygosaccharomyces orentinus( Zygotorulaspora orentina )

+ 1.00; 5.8% Wine Lages et al., 1999;Barnett et al., 2000

Candida glabrata + 1.70; 10% Juice concentrate Pitt and Hocking, 2009Osmotolerant and

moderatelyhalotolerant

Growth at N 50%(w/v) D glu-cose;lack of growthat N 2.0M NaCl

Candida tropicalis + 1.7 2.0; 10-11.7% Fruit juice Barnett et al., 2000;Deak, 2007;Pitt and Hocking, 2009

Zygosaccharomycesmellis

+ + 1.70; 10% Juice concentrate,honey

Kurtzman et al., 2011

Zygosaccharomycessapae

+ + 2.0; 11.7% n Solieri et al., 2014

Zygosaccharomyces bailii + w 1.0 2.0; 5.8-11.7% Juice, sauces, ciders,wines

Lages et al., 1999;Barnett et al., 2000

Zygosaccharomycesbisporus

+ + 1.0 2.0; 5.8-11.7% Soft drink, wine James and Stratford, 2003

Moderately osmotolerantand halotolerant

Lack of growthat N 50% (w/v)D glucose;growth atN 2.0M NaCl

Candida parapsilosis + 3.0; 17.5% Dairy food Pitt and Hocking, 2009Pichia membranifaciens + 3.00; 17.5% Bread, fermented milk,

oliveLages et al., 1999;Barnett et al., 2000

Issatchenkia orientalis(Pichia kudriavzevii )

+ 2.0; 11.7% Olives, pickles andsauces (rarely)

Lages et al., 1999;Barnett et al., 2000

Osmotolerant andhalotolerant

Growth at N 50%(w/v) D glu-cose;growth atN 2.0M NaCl

Pichia sorbitophila(Millerozyma farinosa )

+ + 3.0 4.0; 17.5-23.4% Beer, sake, soy sauce,Mash of rice vinegar

Lages and Lucas, 1995

Zygosaccharomycesrouxii(allodiploid strains)

+ + 3.0; 17.5% Juice concentrate, honey, jams, confectionery,dried fruits, soy sauce

Lages et al., 1999;Solieri et al., 2014

Candida magnoliae + + 3.0; 17.5% Sugary food Barnett et al., 2000 :Martorell et al., 2007

Pichia guillermondii(Meyerozyma guilliermondii )

+ + 3.0; 17.5% Olive, salt meat Butinar et al., 2005

Hortaea werneckii + n 5.20; 30.8% Salt sh Butinar et al., 2005;Lenassi et al., 2011

Debaryomyces hansenii(Candida famata )

+ + 3.0 4.0; 17.5-23.4% Olive Barnett et al., 2000;Lages et al., 1999

Candida halophila(Candida versatilis ) + + 4.0

5.0; 23.4-29.1% Cheese brines Barnett et al., 2000;Silva-Graa andLucas, 2003

1 Species names are reported according to the corresponding reference; current names are reported in bracket.2 Information about food spoilage was retrived from Pitt and Hocking, 2009 .3 References used for growth data; , variable trait; n, not reported; w, weak.

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resistance ( Thomas and Davenport, 1985; Steels et al., 1999; 2002 ),displayingan unusuallyhigh resistance to the small number of acids ap-proved for use as food preservatives (primarily sorbic, benzoic, aceticand propionic acids). On the other hand, Z. rouxiiand Z. melliscan toler-ate low a w with different sensitivity to ionic and non-ionic solutes. Zygosaccharomyces rouxii is well-known for osmo and halotoleranceand survives up to 0.80 a w in presence of ionic solutes (salt) and upto 0.65 a w in presence of non-ionic solutes (sugars), while Z. mellis

survives at low a w values only when the solutes are sugars ( Stratford,2006 ). Moreover, inter-strain differences in halotolerance have beenreported within Z. rouxii, with the strains isolated from high-salt foodstolerating better NaCl than those isolated from high-sugar foods(Pribylova et al., 2007b; Solieri et al., 2014 ). The highly salt-tolerantstrains are allodiploid strains arising from putative outcrossing events,or aneuploid strains which differ from haploid Z. rouxii for karyotype,ploidy level and copy number variation (CNV) of housekeeping genes(Solieri et al., 2008, 2013b ). This nding suggests that osmo andhalotolerance are two distinct physiological phenotypes.

Food yeasts other than Zygosaccharomyces species have beendescribed as being osmotolerant and/or halotolerant. Millerozyma farinosa (formerly P. sorbitophila ) is a ubiquitous, halotolerant yeastfound mainly in food (alcoholic beverages like beer and sake, soysauce, miso, mash of rice vinegar etc.) and it is known for the ability togrow in more than 4.0 M NaCl ( Lages and Lucas, 1995; Silva-Graaand Lucas, 2003; Silva-Graa et al., 2003 ) and to tolerate up to 70%glucitol ( Rodriques de Miranda et al., 1980 ). Debaryomyces hanseniiwas originally isolated from saline environments, such as seawaterand concentrated brines, and it is also associated with cheese andmeat processing. It can tolerate salinity levels up to 4.0 M NaCl andsurvive in high-sugar products with a w values as low as 0.62 (asreviewed by Aggarwal and Mondal, 2009 ). The strongly halotolerantyeast-like fungus H. werneckii was found on salty food and other low-water-activity substrates for its ability to grow, albeit extremely slowly,in a nearly saturated salt solution (5.2 M NaCl), or completely withoutsalt, with a broad growth optimum from 1.0 3.0 M NaCl. In addition, agroup of poorly studied osmotolerant species have been associatedwith spoilage of sugary food and with insects, including Candidadavenportii (Stratfordet al.,2002 ), Candidastellata and Candidamagnoliae(Rosa et al., 2003). Schizosaccharomyces pombe is an osmotolerant,preservative-resistant yeast, but it is rarely associated to food spoilagedue to its salt-sensitivity ( Pitt and Hocking, 2009 ).

4. Gene circuits and metabolic pathways

In presence of high extra-cellular solute concentrations, yeast cellexperiences three main physiological alterations: changes in physicaland chemical structure of the cell wall and plasma membrane; increaseof intracellular solute/ion toxicity; and alterations in the osmoticpressure and cell volume. Therefore, three systems enable yeasts tocounteract stress challenges andto restore osmoticbalance:a) regulationof morphological and structural properties of the cell wall and plasma

membrane; b) modulation of transport systems; c) production, accu-mulation and retention of metabolically compatible osmolytes.

4.1. Cell wall and plasma membrane

Morphological and structural properties of the cell wall and plasmamembrane are important factors affecting the yeast osmo andhalotolerance. By reshaping their integrity and uidity, yeast cell estab-lishes a balance by which the force driving water across the osmoticgradient into the cell is counteracted by turgor pressure against theplasma membrane and cell wall ( Klis et al., 2006; Levin, 2011 ).

The yeast cell wall is a rigid skeleton formed by four classes of macromolecules interconnected by covalent bonds: the mannosylatedcell wall proteins called mannoproteins, 1,3- -D-glucan, 1,6- -D-

glucan and chitin (a polymer of GlcNAc) ( Klis et al., 2006 ). Early studies

on cell wall composition suggested that Z. rouxii decreases cell wallmannans in presence of salt ( Hamada et al., 1984; Hosono, 1992 ).These studies rstly suggested that cell wall rigidity and integrity havebeen implicated in tolerance to salt-induced stress in Z. rouxii. Strain-speci c differences have been also described in the internal layer of -D-glucan and cell wall mannans. In particular, Z. rouxii strains havinga more rigid cell wall tend to be less halotolerant than those having amore exible and elastic cell wall ( Pribylova et al., 2007b ). Although

these variations are congruent with the possible involvement of cellwall mannansin salt tolerance,more evidences arerequired to reinforcesuch speculations. Recently, some highly salt-tolerant Z. rouxii strainshave been found to possess an increased copy number of FLO11 genethat encodes a glycophosphatidylinositol-anchored cell surface glyco-protein. This copy number ampli cation affects positively the cell wallhydrophobicity and enables strains with a higher copy number of FLO11 to exhibit a tness advantage compared to a reference strainunder osmostress static culture conditions ( Watanabe et al., 2013 ).Interestingly, in S. cerevisiae FLO11 is responsible for lamentation,invasive growth, and bio lm formation ( Fidalgo et al., 2006 ) and it isregulated by at least three well-known signalling cascades, such as theRas-cAMP pathway, the Mitogen-activated protein kinase (MAPK)-dependent lamentous growth pathway, and the glucose repressionpathway ( Verstrepen and Klis, 2006 ).

A few studies dealt with the regulatory pathways employed by Z. rouxii to maintain and modulate the cell wall integrity in the faceof environmental challenges. In the two main model yeastsS. pombe and S. cerevisiae, the pathway mainly responsible for regu-lating cell wall changes is known as cell wall integrity (CWI) signal-ling pathway. Upon osmotic stress, this pathway transmits wallstress signals from the cell surface sensors to the Rho1 GTPase,which mobilizes a variety of effectors. Activation of CWI pathwayregulates the production of various cell wall carbohydrate polymersand their polarized delivery to the site of cell wall. Moreover, CWIserves different functions other than the osmotic stress response,such as the response against mechanical stress, cell shape maintain-ing, and scaffold for cell-surface proteins (as reviewed by Levin,2011 ). A fewproteinsinvolved in CWIpathwayhave been associatedto osmo and halotolerance phenotypes. In S. pombe, the MAPK Pmk1has been implicated in cell wall integrity, cytokinesis, and ion ho-meostasis ( Sengar et al., 1997 ). The MAPK kinase kinase (alsoknown as MEK kinase, MEKK or MAPKKK) Mkh1 and the MAPK ki-nase (also known as MEK or MAPKK) Pek1 act as the upstream sig-nalling components in the CWI pathway cascade and are essentialfor Pmk1 activation. The involvement of Mkh1, Pek1, and Pmk1 hasbeen demonstrated in salt stress response of S. pombe (Madridet al., 2006 ) and, recently, also of S. cerevisiae (Rodicio andHeinisch, 2010; Levin, 2011 ). In S. pombe, MKH1 gene-lacking cellsre-enter the cell cycle quite slowly after a prolonged arrest in stationaryphase and in the presence of NaCl or KCl they show a reduced growth(Sengaret al., 1997 ).In S. cerevisiae , Kcs1 kinase, whichis involvedin ino-sitol signalling, also ensures the cell wall integrity and consequentlycon-

fers adaptive responses to salt stress. The searchfororthologous genes innon-conventional yeast genomes has suggested that a similar pathwaycould also operate in Z. rouxii (Rodicio and Heinisch, 2010 ).

Inside thecell wall there is the plasma membrane, whichis involvedin a variety of cellular processes such as cell adhesion, ion conductivity,and signalling. Like prokaryotes, yeasts regulate the plasma membrane

uidity in osmostress adaptation ( Turk et al., 2011 ). The main factorsaffecting the membrane uidity are the length, branching and degreeof saturation of fatty acids, the amount of sterols, and the phospholipidcomposition ( Russell, 1989; Rodriguez-Vargas et al., 2007 ). The remod-ellingof these parameters strongly affects notonly themembrane uid-ity, but also the proper functioning of membrane-attached proteins,such as those involved in ion homeostasis, the glycerol transportsystems ( Marquez and Serrano, 1996; Kamauchi et al., 2002 ), and the

plasma membrane ATPase activity ( Coccetti et al., 1998 ).



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When theexternal salinity is shifted from lowto high concentrations,S. cerevisiae shortensthe fatty-acid chain length ( Turk et al., 2007 ) and in-creases their saturation level by synthesizing fatty acid desaturases thatintroduce double bonds into the fatty acids of membrane lipids ( Levin,2011 ). Whereas many eukaryotic organisms can synthesize dienoicfatty acids, S. cerevisiae can introduce only a single double bond at the9 position ( Tunblad-Johansson and Adler, 1987; Sharma et al., 1996;Rodriguez-Vargas et al., 2007 ). Under NaCl stress Z. rouxii increases the

amount of free ergosterol (decreasing the sterol-to-phospholipid ratio)and reduces both the lipid unsaturation index and the phospholipid-to-protein ratio ( Watanabe and Takakuwa, 1984, 1987; Hosono, 1992;Yoshikawaet al., 1995 ). The modi cations result in a reductionof mem-brane uidity. On the contrary, in the extreme halotolerant yeastD. hansenii high NaCl levels increase the sterol-to-phospholipid ratioand fatty acid unsaturation, without signi cantly affecting uidity(Turk et al., 2007 ). In the strongly halotolerant yeast-like fungusH. werneckii , high salinity conditions induce slight changes in the totalsterol content, but cause a signi cant increase both in the phospholipidcontent and the fatty acid unsaturation level ( Gunde-Cimerman andPlemenita , 2006). Therefore, H. werneckii tends to maintain thesterol-to-phospholipid ratio signi cantly lower thanother yeasts, mak-ingtheplasma membrane comparativelymore uidandofferinghigheracclimatization to salt stress conditions ( Turk et al., 2007 ).

Ionic and non-ionic solutes have differenteffects on theplasma mem-brane. When the sun ower ( Helianthus annuus ) oleate 12 desaturasesFAD2-1 and FAD2- 3 genes are expressed in S. cerevisiae, they increasethe content of dienoic fatty acids, especially 18:2 9,12 and the plasmamembrane unsaturation index ( Rodrguez-Vargas et al., 2007 ). Undersalt stress FAD-expressing cells display higher membrane uidity andsalt tolerance than the wild-type cells. In contrast, under high sorbitolconcentrations, the FAD-expressing cells do not differ in growth ratefrom the wild-type cells, suggesting that the dienoic fatty acid contentdoesn t affect the tolerance to non-ionic solutes. Although furtherresearches are required, these evidences collectively support thatthere are distinct mechanisms in modulating the cell wall integrity

and plasma membrane uidity in response to ionic and non-ionicstresses. Furthermore, it was demonstrated that certain osmolytes, es-pecially trehalose, stabilize phospholipid bilayers during osmostressconditions ( Hounsa et al., 1998; Gancedo and Flores, 2004 ). In prokary-otes, trehalose stabilizes lipid bilayers and prevents damages derivedfrom dehydration by inhibiting the fusion between vesicles and bymaintaining the membrane lipids in the uid phase (phase transition).Similarly, in S. cerevisiae trehalose also protects plasma membrane

against osmotic stress ( Iturriaga et al., 2009 and references herein).4.2. Cation homeostasis

High concentrations of Na + cations are toxic to most living cells andare frequent in food,whileK + cationsare lessabundant, but theyare es-sential for compensating negative charges and activating key metabolicprocesses, such as pyruvate synthesis and protein translation. Thus, themajority of yeasts maintain a high intracellular ratio of K + /Na+ , by se-lectively accumulating K + and actively extruding Na + . Intracellular ho-meostasis of these alkali metal cations affects physiological parametersand functioning, such as cell volume, plasma membrane potential, andintracellular pH ( Arino et al., 2010 ). When cation concentrations inthe external medium are higher than the physiological range, thedifference between the electrochemical potentials of the cations acrossthe membrane may be so high that the entrance cannot be annulled bysimply inhibiting the transporters mediating the uptake. To avoid aninternal toxic cation concentration, different types of ef ux systemshave been evolved to balance any excessive entrance.

4.2.1. Na+ inward and outward movementsFig. 1 illustrates ef ux and in ux systems recruited by Z. rouxii to

modulate the transport activity of the alkali metal cations across theplasma membrane. Like S. cerevisiae, Z. rouxii has been regarded as asodium excluder species, forwhichNa + is comparatively more cytotoxicat high concentrations compared to K + (Pribylova et al., 2008 ). Insodium excluder yeasts, two main mechanisms mediate the ef ux of

Fig. 1. Overview of the main plasma membrane systems mediating the alkali metal cation homeostasis and the glycerol uptake/retention in Zygosaccharomyces rouxii .



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excessive cations (Na + or K+ ) through the plasma membrane. The rstone is represented by the Na + (K+ ) P-type -ATPase, also known as so-dium pump, encoded by ENA1-4 genes in S. cerevisiae, and by ZrENA1in Z. rouxii. The yeast Na+ (K+ )-ATPases couple the ATP hydrolysis tothe cation transport against electrochemical gradients, and, unlikeNa+ speci c-ATPases from higher eukaryotes, they mediate both theNa+ and K+ ef ux. They are indispensable for the growth at high Na +

and K+ concentrations in alkaline environments ( Benito et al., 2002 ).

The second mechanism consists of Na+

/H+

antiporters encoded byNHA1 in S. cerevisiae , SpSOD2 and SpSOD22 in S. pombe, ZrNHA1and ZrSOD2-22 (with its variants ZrSOD2 and ZrSOD22) in Z. rouxii(Watanabe et al., 1995; Hahnenberg et al., 1996; Bauelos et al., 1998;Benito et al., 2002; Papouskova and Sychrov, 2007; Pribylova et al.,2008 ) (Table 2). Based on the substrate speci city, the Na + /H+ -antiporterscanbe furtherdividedinto twosubfamilies:those recognizingboth K+ and Na + (and their respective analogues Rb + and Li+ ), suchasS. cerevisiae Nha1 and SpSod22, are involved in K + homeostasis and arepresent in almost all hemiascomycetes, whereas those recognizing onlyNa+ (andits analogue Li + ) as substrate, such as SpSod2, Zr Sod2-22and Zr Sod2, determine salt detoxi cation in yeasts most distant fromS. cerevisiae (Kinclov et al., 2002 ). Furthermore, a third less studiedmechanism, which operates in acute response to salt stress, entailsthe sequestration of a surplus of toxic Na + cations in intracellularcompartments. A plethora of proton-coupled antiporters are includedin this process; in S. cerevisiae, the most studied ones are Nhx1, anendosomal Na + /H+ exchanger ( Nass et al., 1997; Nass and Rao,1998 ), and Vnx1, a vacuolar Na + and K+ /H+ exchanger ( Cagnac et al.,2007 ). An endosomal Na + /H+ exchanger homologous to S. cerevisiaeNhx1, has been also found in the halotolerant food yeast D. hansenii(Moniel and Ramos, 2007 ).

Yeast salt tolerance signi cantly depends on the genes encodingNa+ ATPases, plasma membrane and intracellular Na + /H+ -antiportersharboured in the yeast genome ( Table 2). The sodium pump Ena1 isthe most important salt tolerance determinant in S. cerevisiae (Prioret al., 1996; Bauelos et al., 1998 ). Deletion of ENA1 gene determinesthe salt-sensitivity in S. cerevisiae ( Haro et al., 1991; Marquez andSerrano, 1996 ). In the salt-sensitive S. pombe, the single ENA-relatedgene, called cta3 , only mediates the potassium ef ux ( Benito et al.,2002 ). The expression of S. cerevisiae ENA1 in S. pombe markedly

increases the tolerance to Na + (Bauelos et al., 1995 ). Other studiesreported that S. pombe Sod2antiporter complements theNa + sensitivityin S. cerevisiae ena1 mutants, suggesting that antiporters or sodiumpumps can both be used by S. cerevisiae to regulate the internal sodiumconcentration ( Hahnenberger et al., 1996 ). In D. hansenii two ENAparologs, namely DhENA1and DhENA2, havebeen found to complementthe salt sensitivity when heterologously expressed in S. cerevisiae ena1mutants ( Almago et al., 2001 ). Similarly, H. werneckii possesses two

highly salt responsive ENAgenes,namely HwENA1expressed in stressedcells exposed to high salt concentrations, and HwENA2 that is mainlyexpressed in stress-adapted cells ( Gunde-Cimerman and Plemenita ,2006 ). More recently, it was demonstrated that upon initial impositionof NaCl stress, S. cerevisiae extrudes intracellular Na + primarily byNha1, whereas the long term salt adaptation is mediated by the tran-scriptional up-regulation of ENA1 gene ( Proft and Struhl, 2004; Ruizet al., 2007; Ke et al., 2013 ).

In Z. rouxii, thesalttolerancehasbeenattributed to different variantsof Na+ /H+ antiporters and Na + -ATPase genes ( Hahnenberger et al.,1996; Pribylova et al., 2008 ). Like S. cerevisiae, Z. rouxii has a geneencoding Ena1-homologous protein named ZrENA1 (Watanabe et al.,1999; 2002 ). Transcriptional studies showed that, unlike S. cerevisiaeENA1, ZrENA1 has little relevance in Z. rouxii salt tolerance, as, underNaCl shock, the major Na + pumpout activity relies on the Na + /H+

antiporter Zr Sod22 (and their variants) ( Watanabe et al., 1995, 1999 ).Accordingly, under NaCl stress, Z. rouxii ena1 mutants and wide-typestrainsexhibit similargrowth rates ( Watanabe et al., 1999 ). Other stud-ies pointed out that Na + -ATPases and Na + antiporters almost servesimilar functions, but they are operational at different external pHlevels. In most sodium excluder species, Ena1 Na + -ATPase mediatesthe Na + export mainly at high external pH levels. When the externalpH is lower than the cytoplasmic pH, the function of Ena1 Na + -ATPase can be replaced by electroneutral Na + /H+ antiporters, whichdrive the Na + ef ux by the pH ( Bauelos et al., 1998 ). Similarly, in Z. rouxii Na+ -ATPase and Na + antiporters have different pH sensitivity.Since Z. rouxii is acidophilic yeast, Zr Ena1 could not be active at thelow pH usually encountered by Z. rouxii in food ( Watanabe et al.,1999 ). More recent evidences have demonstrated that the Na + extru-sion is mainly mediated by the Na + -speci c Na+ /H+ antiporter Zr Sod-22 and not by the substrate-unspeci c Na+ /H+ antiporter

Table 2Cation transport systems associated to halotolerance in different yeast species (adapted from Ramos et al., 2011 ). Number of parologs is reported in brackets.

Category Gene Function Sc Sp Zr Dh Mf Ca Hw

Systems for K+ in ux TRK1 plasma membrane K + transporter belonging to HKT TRK family,with a main role in K + homeostasis

+ + + + + + +(8)

TRK2 plasma membrane K + transporter belonging to HKT TRK family,with a minor role in K + homeostasis

+ + + nd

HAK1 High Af nity K+ -H+ symporter belonging to the HAK KUP family + + + ACU1 K+ -Na+ P-type ATPase functionally similar to plant HKT transporters + ps

Systems of K+ ef ux TOK1 membrane depolarization activated K + channel + + + +(4)Systems for Na + and K+ ef ux NHA1 antiporter which uses a proton-motive force generated by the plasma

membrane H+

-ATPase to mediate the ef ux of Na+

, Li+

, K+

, and Rb+

through the plasma membrane

+ + + + +(2) + +(8)

SOD2 antiporter which uses a proton-motive force generated by the plasmamembrane H + -ATPase to mediate the ef ux of Na+ , and Li+ throughthe plasma membrane

+ + (v) nd +(2) nd

ENA1-4 P-type ATPase sodium pump; involved in Na + , K+ Li+ ef ux +(v) + + +(2) +(2) + +(4)Intracellular cations/H + transporters NHX1 endosomal Na + (K+ )/H+ antiporter which it regulates the acidi cation

of cytosol and vacuole lumen+ + + + + +(2) +(2)

KHA1 Putative K+ /H+ antiporter from Golgi with a probable role in intracellularcation homeostasis

+ + + + +(2) + +(2)

VNX1 Vacuolar Na+ (K+ )/H+ exchanger localized to the endoplasmicreticulum membrane

+ + + + +(2) +(2) +(2)

Nomenclature according to S. cerevisiae genome,with the exception of HAK1, ACU1, and SOD2 genes. When nofunctional data havebeen available, BLASTP and TBLASTN analyses werecarried out to identify homologues of K + , Na+ channel subunits, using the following queries: S. cerevisiae Trk1 (DAA08672.1); S. cerevisiae Trk2 (DAA09201.1); S. cerevisiae Tok1(DAA08707); D. hansenii Hak1 (ABI37006); M. farinosa Acu1 (CAF22247.1); S. cerevisiae Ena1 (DAA09449.1); S. cerevisiae Nha DAA11888.1); S. pombe Sod2 (CAB.69632.1), S. cerevisiaeNhx1 (DAA12290.1); S. cerevisiae Kha1 (DAA08706.1); S. cerevisiae Vnx1 (NP_014078.1).Abbreviations: Sc, S. cerevisiae ; Sp, S. pombe; Zr, Z. rouxii; Dh, D. hansenii ; Mf, M. farinosa (formerly P. sorbitophila );Ca, C. albicans ; Hw, H. werneckii ; nd,not determined; ps, pseudogene;v,

inter-strains variability.



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Zr Nha1 ( Prybilova et al., 2008 ). Synteny analysis demonstrated thatthese two genes arose from a duplication event. Furthermore, Z. rouxiigenome experienced a CNV in ZrSOD genes. The Z. rouxii type strainCBS 732T possesses one gene encoding the Na + /H+ antiporter, namely ZrSOD2-22, while the highly halotolerant allodiploid strains ATCC42981, CBS 4838 and CBS 4837 have two copies, namely ZrSOD2 and ZrSOD22 ( Watanabe et al., 1995; Iwaki et al., 1998; Kinclov et al.,2001; Solieri et al., 2013b ). In the related species Z. sapae, two genecop-

ies, ZrSOD2-22 and ZrSOD22, have been found ( Solieri et al., 2013a ).Generally, CNV can induce important adaptive phenotypes in yeast(Conrad et al., 2010 ). For instance, the expansion of gene repertoireencoding alkali metal cation transporters has been related to the ex-treme salt tolerance of H. werneckii (Lenassi et al., 2011 ). Although theCNV in Na+ /H+ antiporter-encoding genes has been supposed to con-tribute to the high halotolerance of Z. rouxii allodiploid strains(Gordon and Wolfe, 2008 ), no experimental evidences support this hy-pothesis. The ZrSOD2 gene encodes a functionally active antiporter in-volved in salt stress response ( Watanabe et al., 1995 ), whereas ZrSOD22 is a poorly transcribed gene and its disruption doesn t affectsalt-tolerant phenotype ( Iwaki et al., 1998 ).

In S. cerevisiae, the plasma membrane proton-pumping ATPasePma1 generates the electrochemical gradient required for nutrientuptake and ionic homeostasis ( Serrano et al., 1986 ). The PMA1 genetranscription is tightly regulated and the Pma1 activity is controlled bypH andglucose viaphosphorylationof itsC-terminal domain.Transcrip-tional studies demonstrated that, under salt stress, Z. rouxii drives theNa+ ef ux via Na+ /H+ antiporter through the H + gradient created by Zr Pma1 H+ -ATPase (Watanabe et al., 1995; Iwaki et al., 1998 ). The ZrATP2 gene, which encodes a mitochondrial F 1 ATPase subunit, isalso involved in the ATP production and salt tolerance ( Watanabeet al., 2003 ). The disruption of this gene is lethal in Z. rouxii but notin S. cerevisiae, suggesting that ZrATP2 is essential for maintaining Z. rouxii viability and functioning ( Watanabe et al., 2003 ). Based onthese evidences, it has been hypothesized that Z. rouxii is moreef cientin Na+ extrusion than S. cerevisiae due to the cooperative action of ef cient H + -ATPase systems and Na + /H+ antiporters with high Na + -speci city (Watanabe et al., 2005 ).

4.2.2. K + inward and outward movementsPotassium is an absolutely essential element for the livingorganisms

and, although theexternal K + concentrationcangreatly varydependingon the natural environment, it is in food much lower than whatmetabolically required inside the cells (200 300 mM). Therefore,besides the Na + ef ux, yeast cells also require ef cient systems for K +

uptake and, when necessary, extrusion ( Table 2 ).In S. cerevisiae , K+ cat-ions are continually taken up and extruded: the membrane potentialincreases when the potassium in ux is crippled and decreases in cellsdefective for K + ef ux (Kinclova-Zimmermannova et al., 2006 ). TheK+ uptake occurs mainly by facilitated diffusion through the high-af nity transporters Trk1 and Trk2, and it is driven by the electrochem-

icalH+

gradient across the plasma membrane generated by H+

-ATPasePma1 ( Michel et al., 2006 ). The homologous gene encoding a putativepotassium transporter, namely ZrTRK1, has been also characterized in Z. rouxii(Stbn andSychrov, 2011 ) ( Fig.1). Thefunctional expressionof the ZrTRK1 gene in S. cerevisiae trk1 trk2 mutants restores theability to grow at micromolar potassium concentrations, whereasthe Z. rouxii trk1 mutant grows more slowly than the wild-typestrain at low K + concentrations. Other non-conventional yeasts, suchas D. hansenii , possess the high af nity K+ transport Hak1, whichworks both as K + -H+ symporter and K + -Na+ symporter dependingupon the extracellular K + /Na+ concentrations ( Martnez et al., 2011 )(Table 2). The extremely halotolerant M. farinosa (formerlyP. sorbitophila ) possesses a peculiar p-type ATPase encoded by the ACU gene, which mediates the Na + and K+ uptake at high af nity

(Benito et al., 2004 ).

In S. cerevisiae, the extrusion of K + cation surplus is mediated bythe Ena Na + (K+ )-ATPase, the antiporter Nha1, and the voltage-gatechannel Tok1. While Nha1 and Ena ATPase are also involved in theNa+ detoxi cation, Tok1 represents the main system for the exclusiveK+ extrusion in S. cerevisiae (Ahmed et al., 1999 ). Tok1 is activated bythe plasma membrane depolarization and contributes to regeneratethe membrane potential by releasing intracellular K + outside the cell.Tok1 is also involved in the short term response to salt stress via the

HOG pathaway. Phosphorylated Hog1 (P-Hog1) has been recently pre-dicted to inhibit the K + extrusion mediatedby Tok1, leading to a plasmamembrane depolarization and a Na + in ux reduction ( Ke et al., 2013).The plasma membrane depolarization could thus be a short term adap-tation to osmotic (sorbitol) and ionic (Na + ) stress, because it reducestransporter activities and consequently the molecular import of thecell. Similarly, the phosphorylation of Nha1 by P-Hog1 increases theNa+ cationsef ux undersalt stress ( Proft andStruhl, 2004 ), andinhibitsthe K+ ef ux under sorbitol stress ( Kinclov-Zimmermannova andSychrov, 2006 ). In D. hansenii , the K+ ef ux is mediated by the Na + /H+ antiporter DhNha1 ( Velkova and Sychrova, 2006 ) and by the Na +

(K+ ) pumps DhEna1 and DhEna2, which seem able to protect the cellsfrom sodium or potassium stress at alkaline pH ( Almagro et al., 2001 ).In Z. rouxii, the response to potassium surplus has been poorly investi-gated. When Zr Nha1 and S. cerevisiae Nha1 have been expressedin S. cerevisiae lacking alkali metal cation ef ux systems ( ena1 4nha1 ), Zr Nha1 was less effective than S. cerevisiae Nha1 in restoringthetolerance to K + excess( Prybilovaet al., 2008 ). Therefore, differentlyfromorthologs in S. cerevisiae and D. hansenii , Zr Ena1 doesn tseemtobeinvolved in potassium homeostasis.

4.3. Sugar transporters

In yeasts hyperosmotic stimuli trigger a variety of regulatorymechanisms, which modulate the glucose uptake rate ( Horak, 2013 ).When glucose is available at high concentrations, S. cerevisiae uptakeshexoses via facilitated diffusion, and only when glucose is scarce, ituses the H + gradient and high-af nity symporters. In S. cerevisiae 17HXT genes encode facilitated diffusion carriers ( Boles and Hollenberg,I997),butonly4( HXT1-HXT4) are regulated in response to extracellularglucose concentrations ( Boles and Hollenberg, 1997; Ozcan and Johnston, 1999 ). The HXT2 and HXT4 genes encode high af nity and in-termediate af nity glucose transporters, respectively, which are up-regulated at low glucose concentrations and down-regulated underhyperosmotic stress ( Wendell and Bisson, 1994 ). On the contrary,HXT1expression increases during the exposure to 1.0 M salt, 1.5 M sor-bitol ( Hirayama et al., 1995 ) or high sugar (40% w/v) ( Erasmus et al.,2003 ). The osmotic stress-induced HXT1 transcription depends uponthe HOG pathway ( Rep et al., 2000 ), and it has been suggested to pro-vide additional glucose for the glycerol synthesis ( Hirayama et al.,1995 ). Furthermore, high glucose concentrations stabilize HXT1 mRNAtranscripts, indicating that both transcription and mRNA turnover are

regulated in yeast osmo-adaptation ( Greatrix and van Vuuren, 2005 ). Zygosaccharomyces rouxii is fructophilic yeast which prefers to

consume fructose overglucose.Therefore,hexose transporters mediatingglucose facilitate diffusion have been poorly investigated. The searchfor HXT orthologs in the Z. rouxii genome showed just one ORF(ZYRO0D13310g) similar to S. cerevisiae HXT10 (http://genolevures.org). In contrast, sugar facilitators for the fructose uptake, such as Ffzproteins (fructose facilitator of Zygosaccharomyces ) have been exten-sively characterized in Z. bailii (Pina et al., 2004 ) and Z. rouxii (Leandroet al., 2011 ). In particular, Z. rouxii has two low-af nity high-capacityfacilitators, Zr Ffz1and Zr Ffz2, which transport bothfructose andglucosewhen their external concentration is high ( Fig. 2). More recently,Leandro et al. (2013) characterized the high-af nity low-capacityfructose-H + symporter Zr Fsy1, which is up-regulated during the

growth of Z. rouxii at low extracellular sugar concentrations.

http://genolevures.org/http://genolevures.org/http://genolevures.org/http://genolevures.org/


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4.4. Production and accumulation of osmolytes

Compatible osmolytes are produced by yeasts to favour the celladaptation to osmotic stress. They signi cantly maintain the waterbalance, stabilize enzyme systems without interfering with the cellularmetabolism, and restore the original cell volume ( Brown, 1990 ). Toassure high intracellular osmolyte concentrations, yeast cells regulatethe cell-cycle progression and take action against the requirement of redox elements and energy demand.

Glycerol is the main osmolyte that is produced and accumulatedintracellularly in response to hyperosmotic stress, and also the moststudied one. The role of glycerol under varied osmotic conditions willbe discussed in detail. However, other less studied osmoprotectiveosmolytes, such as trehalose, arabitol, mannitol and erythritol, also getaccumulate under certain conditions and therefore will be mentioned.

4.4.1. Glycerol metabolic pathway

Yeasts possess two well-established pathways for glycerol biosyn-thesis, bothinitiating fromtheglycolyticintermediate dihydroxyacetonephosphate (DHAP) and ending up with the production of glycerol:(1) the Gcy-Dak pathway includes the dephosphorylation of DHAP todihydroxyacetone (DHA) by dihydroxyacetone phosphate kinase(Dak) followed by the production of glycerol from DHA in a reactioncatalyzed by glycerol dehydrogenase (Gcy); (2) the Gpd-Gpp pathwaycomprises the conversion of DHAP to glycerol 3-phosphate mediatedby glycerol 3-phosphate dehydrogenase (Gpd), and subsequently,the dephosphorylation of glycerol 3-phosphate by glycerol-3-phosphatase enzyme (Gpp), resulting in glycerol production ( Fig. 2).Even if the Gcy-Dak pathway seems to be active under a range of stressconditions,under osmotic stress glycerol is mainly synthesizedby Gpd-Gpp pathway ( Larsson et al., 1993; Albertyn et al., 1994; Eriksson et al.,

1995 ).

In S. cerevisiae, genesimplicatedin glycerolproductionare duplicatedin differentially regulated paralogs, namely GPD1 and GPD2 (Albertynet al., 1994; Eriksson et al., 1995 ), GPP1 and GPP2 (Norbeck et al., 1996;Phlman et al., 2001a ), and DAK1 and DAK2 (Norbeck and Blomberg,1997; Rep et al., 2000 ). These genes arose from either single geneduplication or whole genome duplication events, and show frequentlydivergence and functional differentiation ( Kondrashov et al., 2002;Conant and Wolfe, 2008 ). For example, while GPP2 is mainly involvedin osmoadaptation, GPP1has a role both in osmoadaptation and growthunder anaerobic conditions ( Phlman et al., 2001a,b ). Similarly, Gpd1and Gpd2 enzymes have similar kinetic characteristics, but differwith respect to cellular distribution and transcriptional regulation(Albertyn et al., 1994; Eriksson et al., 1995 ). They are located in thecytosol, but Gpd1 possesses a peroxisome-targeting sequence, whileGpd2 is partly translocated into the mitochondria of non-respiringcells (Valadi et al., 2004; Jung et al., 2010 ). The single deletion of GPD1resulted in strains sensitive to osmotic stress ( Albertyn et al., 1994 ),while the deletion of GPD2 reduced growth under anaerobiosis ( Rep

et al., 1999 ). Therefore,Gpd1 has a major role in osmoadaptation. How-ever, neither the deletion of GPD1nor thedeletion of GPD2 resultedin anoticeable change in glycerol yield. GPD1 and GPD2 genes could thushave roles which parltyoverlap to compensate compromised functions,as recently shown for GPD2, which is up-regulated in response to GPD1deletion ( DeLuna et al., 2010 ).

The Gpd-Gpp pathway is the main metabolic route leading fromDHAP to glycerol also in Z. rouxii (Fig. 2). Accordingly, the heterologousexpression of ZrGCY1, but not of ZrGPD1, restores the glycerol produc-tion and the salt tolerance in S. cerevisiae gpd1 gpd2 mutants unableto synthesize glycerol ( Watanabe et al., 2004 ). Furthermore, it wasfound that both the glycerol production and salt tolerance increasewhen ZrGPD1 is expressed along with ScGPP2 (Watanabe et al.,2004 ). Like in S. cerevisiae, in Z. rouxii allodiploid strain ATCC 42981

two isoforms of some glycerol synthesis genes have been found, such

Fig. 2. Interactions among redox balance, glycolysis, and glycerol production during the Zygosaccharomyces rouxii ethanol fermentation. Black, green and orange lines indicate theenzymatic steps in glycolysis, Gpd-Gpp and Gcy-Dak pathways, respectively. Blue line indicates the relationship among glycolysis, Gpd-Gpp and Gcy-Dak pathways. Red lines representtheremodelling inmetabolic uxesunder bisulphiteandosmostressconditions. All thedotted linesrepresent omittedsteps.The rolesof glycerolmetabolismin Pi recycling,redoxbalance,gluconeogenesis and fatty acid biosynthesis are reported in gray boxes.

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as ZrGPD1 and ZrGPD2, ZrGPP1 and ZrGPP2, ZrGCY1 and ZrGCY2 (Iwakiet al., 2001; Wang et al., 2002; Watanabe et al., 2004 ). On the basisof their deduced amino-acid sequence, these proteins have a closehomology with the S. cerevisiae orthologs. ZrGPD1/ ZrGPD2 and ZrGCY1/ ZrGCY2 are constitutively expressed in Z. rouxii cells, but their differen-tial roles have not yet been investigated ( Iwaki et al., 1999, 2001;Watanabe et al., 2004 ).

4.4.2. Glycerol biosynthesis in non-stressed cellsUnder anaerobic conditions, sugarsare reduced to glycerol for main-taining the cytosolic redox balance and consuming the excess of NADHproduced during the glycolytic pathway, amino acid biosynthesis andorganic acids anabolic routes ( van Dijken and Scheffers, 1986; Priorand Hohmann, 1997; Medina et al., 2010 ). The glycerol production isalso pivotal in lipid biosynthesis and recycling the inorganic phosphateconsumed during glycolysis via the dephosphorylation step catalyzedby glycerol phosphatase ( Nevoigt and Stahl, 1997 ) ( Fig. 2).

The glycerol production was rstly studied in some non-Saccharomyces yeasts that are naturally unable to produce glyceroland grow on glucose under anaerobic conditions. It was observed thattheir ability to produce glycerol can be restored by introducing oxygenor another external electron acceptor in the culture medium. This effectis called Custers effect (Nevoigt and Stahl, 1997 ). In subsequentstudies, it was found that a S. cerevisiae gpd2 mutant grows poorly inanaerobic conditions without acetoin as an external electron acceptor(Ansell et al., 1997; Bjrkqvist et al., 1997; Valadi et al., 2004 ). Whenacetoin is added in anaerobic conditions, it is converted to butanediolby NAD+ -dependent butanediol dehydrogenase ( Nevoigt and Stahl,1997; Bjrkqvist et al., 1997 ). The GPD2 and GPP1 expression is stimu-lated under anaerobic conditions, when the glycerol productionbecomes essential for the redox balance and ethanol production(Albertyn et al., 1994; Eriksson et al., 1995; Nevoigt and Stahl, 1997 ).Under aerobic conditions, bisulphite ions induce the GPD2 transcriptionandinhibit the nalreductivestep inethanolfermentation,avoiding theaccumulation of excessive NADH ( Ansell et al., 1997 ). Bisulphite ionsform a complex with acetaldehyde that limits the ethanol productionand promotes the reoxidation of glycolytically formed NADH by theglycerol synthesis ( Fig. 2). The effect can be reversed upon addition of acetaldehyde ( Ansell et al., 1997 ). All these evidences pointed out thatthe function of GPD2 is mainly linked to redox imbalance, and not toosmotolerance.

4.4.3. Glycerol biosynthesis in osmo-stressed cellsThe cellular redox balance is pivotal for several aspects of cellular

physiology and its perturbation is implicated in the cellular adaptationto sugar and salt-stress conditions. Under high external osmolarity, anequimolar increase in cytoplasmic NADH is required to enhance theglycerol production. This requirement seems to be partially ful lled bydecreasing the acetaldehyde reduction to ethanol and increasing theacetaldehyde oxidation to acetate ( Fig. 2).

In S. cerevisiae, high external osmolarity induces the glycerol

production both under aerobic and anaerobic conditions. Dependingon the strain, medium, and process parameters, 4 to 10% of the carbonsource may be converted to glycerol. Under stress conditions, theGPD1 and GPP2 genes are positively regulated by the HOG pathway toproduce glycerol ( Albertyn et al., 1994; Eriksson et al., 1995 ). BesidesGPD1 and GPP2 genes, S. cerevisiae positively regulates the expressionof DAK1 gene encoding the Dha1 kinase ( Rep et al., 2000). Accordingly,glycolytic pathway enzymes are slightly repressed in cells exposed tosaline medium ( Akhtaret al., 1997 ). Unlike S. cerevisiae , Z. rouxii doesn tincrease the expression of neither ZrGPD1 or ZrGPP2 in response to saltstress ( Iwaki et al., 2001 ) and the speci c activity of the correspondingenzymes remainsunaltered ( van Zyl et al., 1991 ). Although ZrGPD1and ZrGPP2have a main role in glycerol production ( Watanabe et al., 2004 ),these results suggest that these genes are constitutively expressed in

Z. rouxii and by-pass the HOG pathway control. In contrast, ZrGCY1

and ZrGCY2 are up-regulated by salt stress, indicating that these genesare target candidates of the HOG pathway ( Iwaki et al., 2001 ). More-over, differently from Zr Gpd1, glycerol dehydrogenase Zr Gcy1 and ki-nase Zr dak1 increase the activities under hyperosmotic conditions (a wb 0.96) ( van Zyl et al., 1991 ).

Extreme halotolerant yeasts produceandaccumulate large amountsof osmo-protective metabolites. Although this feature has been widelyexploited in industrial bioprocesses ( Nevoigt and Stahl, 1997 ), its

molecular mechanism has been poorly investigated. In the blackyeast H. werneckii , a setof 95salt-responsive genes have been identi edand most of them have not previously been related to halotoleranceand HOG pathway in any other halotolerant yeasts ( Vaupoti andPlemenita , 2007). Furthermore, in H. werneckii the adaptation to highamounts of NaCl and sorbitol involves the differential expressionof mitochondria-related genes ( Vaupotic et al., 2008 ). Mitochondriapreferentially accumulate energy metabolism-related enzymes inhypersaline medium, and chaperones and heat shock proteins, such asKar2 and Hsp60, in medium supplemented with sorbitol. Live-cellimaging showed that the mitochondria condense differentally inresponse to different osmolytes. In hypersaline medium, the mitochon-drial condensation is accompanied by an increasing in ATP synthesisand oxidative damage protection, whereas in presence of non-ionicosmolytes it is accompanied by a decreasing both in ATP synthesis andlipid peroxidation level ( Vaupotic et al., 2008 ).

4.4.4. Glycerol retention and active transport Besides de novo biosynthesis, the glycerol retention is effective to

prevent the massive out ow of water from cells in response to anosmotic stress. Being a liposoluble molecule, glycerol has the tendencyto be leaked out through the plasma membrane, and its retention hasthus to be an active response by the cell. Under osmotic conditions,S. cerevisiae synthesizes a considerable glycerol amount most of whichleaks out of the plasma membrane ( Hohmann, 2002 ). In contrast, Z. rouxii synthesizes a smaller glycerol quantity than S. cerevisiae, andchanges membrane phospholipid and fatty acid compositions todecrease the membrane uidity and permeability. This strategy entails Z. rouxii to effectively retain polyols and maintain a very high gradientbetween the intra and extracellular environments ( van Zyl et al.,1990; Pribylova et al., 2007a ).

In S. cerevisiae, the glycerol enters into the cell by two differentmechanisms: a low af nity transport system (facilitated diffusion) anda high af nity proton symport system (active transport) ( Table 3).Fps1 is an aquaglyceroporin belonging to the MIP family that is mainlyinvolved in the glycerol transport by facilitated diffusion ( Sutherlandet al., 1997; Karlgren et al., 2005; Hohmann et al., 2007 ). FPS1 is consti-tutively expressed in a salt-independent manner and mutants lacking aregionin theFps1 N-terminal domain (amino acid residues from 150 to231) constitutively release glycerol ( Tams et al., 2003 ). A shift fromlow to high external osmolarity induces the Fps1 closure, whereas adecrease in osmolaritycauses thechannel opening, both withinsecondsafter thechange in external osmolarity( Luyten et al., 1995; Tams et al.,

1999 ). In the absence of osmotic stress, Fps1 is opened by the bindingof Rgc2 (regulator of the glycerol channel 2) to the Fps1 C-terminalcytoplasmic domain. In response to osmostress Fps1 is closed byP-Hog1, which binds the N-terminal cytoplasmic domain of Fps1 andphosphorilates the positive regulator Rgc2 ( Beese et al., 2009; Leeet al., 2013; Petelenz-Kurdziel et al., 2013 ). Furthermore, Fps1 affectsthe glycolipid and phospholipid composition of the plasma membrane(Sutherland et al., 1997; Toh et al., 2001 ).

Several evidences show that the Z. rouxii ortholog to Fps1, namely Zr Fps1, conserves structural features and regulatory mechanisms. Zr Fps1 is a 692 aa-long protein characterized by long hydrophilic N(228-LHQNPQTPTVLP-239) and C-terminal (537-HESPVNWPIATY-548)domains, both sharing high homology with their S. cerevisiae counter-parts ( Tang et al., 2005 ). Since Z. rouxii fps1 mutants retain the ability

to grow on glycerol as the sole carbonsource, ZrFPS1is not required for



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to two putative homologs to S. cerevisiae HOG1, namely ZrHOG1 and ZrHOG2 (Iwaki et al., 1999; Kinclov et al., 2001 ). A TGY motif similarto that of S. cerevisiae Hog1 has also been found in Zr Hogl and Zr Hog2,suggesting that a putative Zr Pbs2 MAPK kinase similar to S. cerevisiaePbs2 could exist also in Z. rouxii ( Iwaki et al., 1999 ). Although some Z. rouxii allodiploid strains possess two ZrHOG gene copies, namely ZrHOG1 and ZrHOG2, there is no relationship between this gene redun-dancy and their osmotolerance ( Kinclov et al., 2001 ). This could meanthat ZrHOG1 and ZrHOG2 are expressed differentially, or that one of these paralogs is transcriptionally silent, as it happens for ZrSOD22. ZrHOG1 and ZrHOG2 genes are functional as MAP kinase and areable to complement the salt-sensitivity in S. cerevisiae hogl null mu-

tants ( Iwaki et al., 1999 ). However, their overexpression in wild-typeS. cerevisiae doesn t improve the glycerol synthesis, indicating that theamountof Zr Hog1 and Zr Hog2is nota limitingfactor inthe glycerol pro-duction. These studies collectively supported that Z. rouxii possesses aHOG pathway functionally equivalent to that of S. cerevisiae.

STRE motifs are species-speci cally distributed in the yeast genome,so that each species has peculiar sets of osmo-responsive genes thatcontribute to functional diversities in response to osmotic cues. Underhigh osmolarity conditions, S. cerevisiae produces much more glycerolthan Z. rouxii, by up-regulating genes involved in glycerol synthesis viaHOG pathway ( Pribylova et al., 2007a ). Accordingly, the S. cerevisiaeGPD1 gene exhibits four STRE elements in its promoter region(Albertyn et al., 1994 ) and encodes a Gpd1 enzymewith higher activitythan Zr Gpd1 ( Akhtar et al., 1997; Norbeck and Blomberg, 1997 ). Like

S. cerevisiae P-Hog1, P- Zr Hogl also translocates into the nuclear

compartment and activates transcription factors homologous toMsn2p/Msn4p and Hot1, thus leading to the transcription of STRE-controlled genes. However, STREs were not found in the ZrGPD1-2 pro-moter regions, indicating that their expression is salt and HOGpathway-independent.The lack of STREs may account for the moderateglycerol amount produced by Z. rouxii to cope high external osmolarity.On the contrary, a putative STRE motif with core CCCCT sequence hasbeen identi ed in the upstream region of ZrFPS1, which is missing inS. cerevisiae FPS1 (Tang et al., 2005 ). Therefore, under high salt concen-trations, only the ZrFPS1 gene transcription is regulated via the HOGsignalling pathway, resulting in the higher ability of Z. rouxiito intracel-lularly retain glycerol compared to S. cerevisiae. In contrast, S. cerevisiae

P-Hog1 transiently induces the closure of Fps1channelduringthe shortterm salt response, but it cannot induce the FPS1 transcription duringthe long term salt adaptation.

5.2. Calcineurin/Crz1 pathway

The calcineurin/Crz1 signal transduction pathway has an importantrole in cation homeostasis and salt stress adaptation, but it is relativelyless investigated compared to HOG pathway ( Matsumoto et al., 2002;Ke et al., 2013). Calcineurin, the Ca 2+ /calmodulin-regulated proteinphosphatase 2B, is a heterodimer containing a catalytic (A) subunitcomplexed with an essential regulatory (B) subunit and requires Ca 2+

and calmodulin for activity ( Cyert et al., 1991 ). Calcineurin controlsCrz1 activity by regulating its subcellular localization ( Stathopoulos-

Gerontides et al., 1999 ). When calcineurin-dependent signalling is

Fig. 3. Integrated overview of signalling transmission pathways involved in osmoadaptive gene regulation.Genes are referred to as Saccharomyces cerevisiae genome, with the exceptionof ZrFPS1 and ZrGCY1/2. Genes regulated by calcineurin pathway are reported in bold.

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low, Crz1 is phosphorylated and resides primarily in the cytosol. Upondephosphorylation by calcineurin, Crz1 enters into the nucleus andhere it regulates the expression of target genes with CalcineurinDependent Response Elements (CDREs) in the promoter region. Thesecis-elements consist of a common sequence motif, 5 -GAGGCTG-3,which Crz1 binds through a C2H2 zinc nger motif ( Stathopoulos andCyert, 1997; Matheos et al., 1997 ). Around 163 genes have been foundunder control of Calcineuirn/Crz1 pathway ( Yoshimoto et al., 2002 ).

Among them, the GSC2 gene encodes a subunit of beta-1, 3 glucansynthase, which is responsible for the synthesis of 1, 3-beta-D-glucan(Levin, 2005), and it is up-regulated after exposure to high sugarconcentrations ( Erasmus et al., 2003 ). CDRE motif was also foundin the anking region of ENA1(Kafadar and Cyert, 2004 ). High cytosolicCa2+ levels activate calcineurin in response to extracellular hyperionicstress and increase Na + ef ux by up-regulating the ENA gene(Matsumotoet al., 2002; Ruiz and Arino,2007 ). Ke et al. (2013) demon-strated that S. cerevisiae coordinates the HOG and the calcineurinpathways to achieve both immediate and longer-term adaptation toNaCl stress, respectively. In Torulaspora delbrueckii, a species close to Z. rouxii, crz1-null cells were insensitive to high Na + concentrations,indicating that TdCrz1 is notrequiredforthe salt-inducedtranscriptionalactivation of TdENA1 gene ( Hernandez-Lopez et al., 2006 ). This evi-dence suggests that yeast species could differ in regulatingsalt responsevia calcineurin/Crz1 pathway.

5.3. Ras-cAMP signalling pathway

The yeast cAMP-dependent protein kinase (PKA) is the effectorkinase of the Ras-cAMP signalling pathway. It is a conserved serine/threonine protein kinase, which, through the phosphorylation of differ-enttargets, has pleiotropiceffects onthecellgrowth, trehalose and glyco-gen metabolism, dimorphic shift, and stress adaptation ( Smith et al.,1998 ). Proteomic approaches were exploited to study the in uence of PKA on protein expression during the exponential growth of S. cerevisiae under osmotic stress ( Boy-Marcotte et al., 1998 ). Proteinsup-regulatedunderNaCl stress aregroupedinto three classes as regardsthe PKA activity: i) PKA-independent proteins (Gpd1, Gpp2 and Dak1);ii) fully PKA-dependent proteins (Tps1 and Gcy1); iii) partly PKA-dependent proteins (Eno1, Tdh1, Ald3, and Ctt1) ( Boy-Marcotte et al.,1998 ). Osmo-response seems to be mediated by PKA both at transcrip-tion level through the inhibition of STRE-dependent genes expression(Thevelein andde Winde, 1999 ), andat post-translationallevel throughthe regulationof trehalose synthesis ( Kobayashi andMcEntee,1993 ).Inparticular, Ras-cyclic AMP pathway negatively regulates Msn2/Msn4that are cytoplasmically accumulated, leading to the inhibition of osmostress response ( Grner et al., 1998 ) ( Fig. 3).

6. Non genetic regulation of osmostress tolerance

Understandingthe non-genetic regulationof cellularstress responseis an important biological question and it has received considerable

attention in recent years. In relation to yeast osmotic tolerance, twonon-genetic mechanisms have been implicated so far. The rst oneincludes epigenetic alterations in the chromatin structure that inducegenome-wide and local changes in gene transcription. The second oneis the non-genetic cell-to-cell phenotypic heterogeneity within anisogenic cell population under osmotic stress conditions.

6.1. Chromatin-mediated mechanisms

Post-translational modi cation of nucleosomal histone proteins andDNA methylation are two extensively characterized epigenetic mecha-nisms that regulate gene expression in plants grown under osmoticstress conditions ( Chinnusamy and Zhu, 2009; Grativol et al., 2012 ).On the contrary, few studies have dealt with this topic in yeasts. The

rst evidence about the epigenetic control of osmo-responsive genes

in yeasts was provided for the protein Sgd1, which is homologous inits N-terminal domain to Spt7, a subunit of the nucleosomal Spt-Ada-Gcn acetyltransferase (SAGA) histone acetylation complex ( Robertsand Winston, 1997 ). The SGD1 overexpression is able to partiallycomplement growth defects in S. cerevisiae hog1 and pbs2 mutantsand to increase their glycerol production ( Akhtar et al., 2000 ). Otherstudies suggested that changes in the chromatin structure contributeto the osmostress-stimulated expression of GPD1 gene. Under osmotic

stimuli, the transcriptional repressor activator protein Rap1 binds theGPD1 promoter and induced the GPD1 transcription. On the contrary,the speci c inactivation of all Rap1 binding sites completely abolishesthe osmostress-induced transcription of GPD1 (Eriksson et al., 2000;Morse, 2000 ).

A third evidence for the epigenetic regulation of cellular osmo-adaptiation involves the HOG pathway. Under osmotic shock, Hog1activates the transcription factors Hot1 and Msn2/4 which mediatethe recruitment of Rpd3-Sin3 histone deacetylase ( de Nadal et al.,2004 ). Active Rpd3-Sin3 complex binds to speci c promoters leadingto histone deacetylation, entry of RNA polymerase II, and transcriptioninitiation of osmoresponsive genes ( Alepuz et al., 2003; de Nadal et al.,2004 ). Upon unstressed conditions, the transcription factor Sko1,which is related to bZIP/ATF family of transcriptional regulators,represses the ENA1 transcription by binding CDRE ( Proft and Serrano,1999 ). Under hypertonic stress, Sko1 is phosphorylated by Hog1 andrecruits the SAGA histone deacetylase and the switch/sucrose non-fermenting (SWI/SNF) complex. The latters are transcription activatorswhich promote chromatin remodelling ( Pro t and Sturhl, 2002 ) andinduce the ENA1 expression in conjunction with Calcineurin/Crz1mediated pathway ( Pro t and Serrano, 1999 ). Yeast mutants lacking afunctional SWI/SNF complex are less tolerant to NaCl than wild-typecells (Pro t and Sturhl, 2002 ). Further studies are required to establishwhether similar epigenetic regulatory mechanisms also exist in Zygosaccharomyces yeasts.

6.2. Phenotypic heterogeneity

Phenotypicheterogeneity is a super-organism feature of prokaryotesandyeasts that provides a dynamic sourceof diversity andadaptivephe-notypes and increases the microbial tness in stressful environments(Avery, 2006 ). Within an isogenic (genetically uniform) microbial pop-ulation in a homogenous environment, individual cells can still exhibitdifferences in phenotype. The precise mechanisms of such cell-to-cellheterogeneity are elusive and few studies have linked variations inyeast morphology to molecular effectors. Hsieh et al. (2013) havehighlighted that reduction in the intracellular amount of chaperonprotein Hsp90 triggers morphological heterogeneity in Z. rouxii clonalpopulations. Under standard conditions high Hsp90 levels assure thestability of Cla4, a key regulator of spectrin formation that inhibitsmorphological switching of the cell from budding to lamentousgrowth. Under salt stress, low Hsp90 levels reduce the Cla4 stabilityand stimulate the cellular switching towards a lamentous form.

Additionally, the stress-induced Hsp90 inhibition is known to favourchromosomal instability and aneuploidy, which in turn potentiate thecellularadaption to stressful environments ( Chen et al., 2012 ). Interest-ingly, aneuploidy has been also frequently found in highly salt-tolerant Z. rouxii strains ( Solieri et al., 2013b, 2014 ).

7. Food exploitation and biotechnological perspective

The knowledge on the genetic and regulatory networks underlyingimportant phenotypic outcomes is a prerequisite for the successfulexploitation and control of microorganisms in food ( Giudici et al.,2005 ). In the last decade, researches in food science and microbiologymoved from classical methodologies to more advanced strategies,and usually borrowed well-established methods in medical, phar-

macological, and/or biotechnology research. As a result,

omics



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approaches and bioinformatics have been recently applied to identify:1) candidate genes to use in genetic improvement of industrial yeaststrains; 2) molecular targets for new food preservation technology;3) biomarkers for the early prediction of food process outcomes.

In particular, new perspectives have been opened by the identi ca-tion of genetic mechanisms that shape the osmostress response in Z. rouxii and other osmotolerant and halotolerant food spoilage yeasts.To prevent their growth in food, it is crucial to understand what causes

yeast stress tolerance. This knowledge could be exploited to set upquantitative prediction methods for the growth of food spoilage yeastsand, consequently, to reduce the economical loss derived from foodspoilage ( Loureiro and Malfeito-Ferreira, 2003; Plemenita et al.,2008 ). On the other hand, genetic loci that determine yeast sugar andsalt-tolerance could be cloned and expressed in industrial strains to im-prove their growth and fermentation performance in stressful condi-tions. For example, the ZrSod22 expression in S. cerevisiae increasesthe expulsion of toxic Na + and Li+ cations from cells at external acidicpH values and enhances the salt tolerance ( Iwaki et al., 1998;Watanabe et al., 2005 ). Similarly, salt stress-responsive genes fromhalotolerant fungi and bacteria can be expressed in plants to alleviateproblems caused by soil salinization in agriculture ( Gostin ar et al.,2012 ). A similar strategy was successful to reduce stuck fermentationin the bioethanol production ( Zhao and Bai, 2009 ).

The ability to grow at very low a w makes the Zygosaccharomycesstrains very attractive as microbial cell factories both for the expressionof heterologous proteins and metabolite production ( Branduardi et al.,2004, 2006; Vigentini et al., 2005 ). Zygosaccharomyces bailii has beenused as host for the production of interleukin-1 due to its ability towithstand hyper-osmotic, hyper-thermic, and acidic environments(Vigentini et al., 2005 ). Zygosacchaomyces rouxii strains have beenexploited for producing glutaminase ( Kashyap et al., 2002; Iyer andSinghal, 2008 ), chiralcompounds( Haucket al.,2003 ), andheterologousproteins ( Ogawa et al., 1990 ). Taking advantage of their ability to pro-duce polyols, osmotolerant and halotolerant yeasts, such as M. farinosa(formerly P. sorbitophila ) and C. tropicalis, have been employed on alarge-scale fermentation processes for the glycerol production ( Liuet al., 2006 ). Owing to the problems associated with the chemical pro-duction, halotolerant and osmotolerant yeasts became valuable for thefermentation-based production of sweet-tasting bodying and texturingagents, such as mannitol, D-arbitol, and xylitol ( Saha et al., 2007; Sahaand Racine, 2011 ).

Zygosaccharomyces species have been exploited as biocatalysts infood and beverage fermentation. Zygosacchaomyces rouxii plays acentral role in the avour formation of soy sauce ( Sluis et al., 2001;Cao et al., 2010; Wei et al., 2013 ). Flavour components, such as4-hydroxyfuranone derivatives, 4-hydroxy-2 (or 5) -ethyl-5 (or 2) -methyl-3 (2H) -furanone (HEMF), are produced from D-fructose-1,6-bisphosphate by Z. rouxii and positively affect soy sauce avour andquality ( Hauck et al., 2003 ). A highly salt-tolerant Z. rouxii mutant con-structed by whole genome shuf ing grows better in soy sauce than theparental strain and produces higher amounts of aroma compounds,

such as ethyl acetate, HEMF, and 4-ethylguaiacol ( Cao et al., 2010).The novel species Z. sapae and Zygosaccharomyces gambellarensis wereisolated from Italian traditional balsamic vinegar ( Solieri et al., 2007 ;Solieri et al., 2013a ) and highly sugary wine ( Torriani et al., 2011 )respectively, where they have a role in the fermentation performanceand in tailoring the sensory characteristics of these products ( Solieriand Giudici, 2008 ).

8. Concluding remarks

A critical reviewon the mechanisms underlyingsugarandsalt stresstolerance in non-conventional yeasts unveils the complex nature of thecellular adaptationto lowa w environments. In thepast, mostresearchesabout this topic have been carried out using S. cerevisiae as model

organism. Recently, the availability of transformation tools and the

genomesequences from osmoand halotolerant food species highlightedhow these yeasts combine conserved and species-speci c strategies tocounteract better sugar and salt stress than S. cerevisiae. These differ-ences are operational at genetic, metabolic, and regulatory levels. Inparticular, new insights on Z. rouxii functional biology and genomicspoint out how sugar and salt tolerance arise from distinct adaptivemechanisms, which integrate solute-unspeci c and solute-speci croutes. Future investigations will bene t by the application of systems

biology tools. They will contribute to complete the detailed map of osmo and halotolerance determinants in Z. rouxii. All these efforts holdpromise for i) rational strain engineering for biotechnological and foodexploitation; ii) prevention of yeast food spoilage; iii) understandingthe yeast adaptation to adverse environments.

Acknowledgements

Thisworkhas beensupportedbya grant of Regione Toscana (Misura124 Acetoscana: PSF 2007/2013).

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