Faculty of Biology Agnese Kokina Doctoral Thesis Impact of purine starvation on physiology of budding yeast Saccharomyces cerevisiae Purīna badināšanas ietekme uz maizes rauga Saccharomyces cerevisiae fizioloģiju Submitted for the degree of PhD of Natural sciences Subfield of Microbiology Supervisors: Dr. Biol., prof. Uldis Kalnenieks Dr. Biol. Jānis Liepiņš 2 “Pētīt.” Raiņa Kastaņolas perioda (1906.-1920.) dienasgrāmatās bieži lietots mudinājums 3 The work of this doctoral thesis was carried out at the Institute of Microbiology and Biotechnology, University of Latvia in 2011 to 2022. The thesis contains the introduction, 6 chapters, reference list. Form of the thesis: collection of research papers in biology, subfield – microbiology. Supervisors: Dr. biol., prof Uldis Kalnenieks, Dr. biol, Jānis Liepiņš Reviewers: Prof. Dr. Raffael Schaffrath, University of Kassel. Germany; Assist. Prof. Dr. Mo Motamedi, Division of Medical Sciences Harvard Medical School, U.S.A; Asoc. prof. Dr. Nils Rostoks, University of Latvia. The thesis will be defended at the public session of the Doctoral Committee of Natural sciences, University of Latvia, at 16:30 on April the 5th, 2023 in hybrid session in Jelgavas iela 1, Riga, Latvia and via Zoom. The thesis is available at the Library of the University of Latvia, Kalpaka blvd. 4. This thesis is accepted for the commencement of the degree of Doctor of Natural sciences on November the 1st, 2022) by the Doctoral Committee of Biology, University of Latvia. Chairman of the Doctoral Committee _______________/ Guntis Brumelis/ (paraksts) Secretary of the Doctoral Committee _______________/ Daina Eze/ (paraksts) © University of Latvia, 2023 © Agnese Kokina, 2023 4 ABSTRACT The budding yeast Saccharomyces cerevisiae is a commonly used eukaryotic model organism. It has evolved to live in environments with rapid influx of nutrients, which can be followed by nutrient scarcity. These conditions have created cellular mechanisms for nutrient uptake and sensing and allows metabolic adjustment for nutrient availability. In conditions where nitrogen, carbon or other macro nutrients are lacking, bakers yeast stops the metabolism and cell cycle, leaving the cells in a "fasting phenotype". Because budding yeast is often used in research due to its relatively simple maintenance, mutations in a number of amino acid or nucleotide biosynthetic pathways are commonly found in laboratory strains - auxotrophs, which serve as metabolic markers to facilitate genetic engineering in these strains. Several authors have shown that yeast cells are unable to "perceive" the lack of auxotrophic nutrients, so the lack of auxotrophic factors does not result in a fasting phenotype. This thesis deals with the physiology of budding yeast if it has a mutation in the pathway of adenine biosynthesis (ade8 or ade2). Adenine auxotrophy is a relatively common metabolic marker because mutants of the ade1 and ade2 genes accumulate red pigment in cells that can be used in cell selection. Our results show that adenine mutants in the absence of adenine are phenotypically more similar to cells with a "fasting phenotype." The cells successfully stop the cell cycle, their stress resistance increases, and a transcriptomic response is observed where adenine-starved cells are quite similar to cells in the stationary phase. The shift of cellular carbon metabolism from fermentation to the production of other metabolites - glycerol, acetate - is also observed. The thesis shows how several frequently used strains with different mutations in the adenine synthesis pathway respond to the inactivation of the adenine synthesis pathway and the consequent adenine deficiency. The results show a similar metabolic response, indicating that this is a general phenomenon. This is also indicated by the fact that several intracellular parasites have evolutionarily lost genes in the adenine biosynthetic pathway. One of these parasites (Leishmania) has been shown to have increased stress resistance in an adenine-free environment. This suggests that adenine deficiency in the cells are perceived in a wide range of living organisms and probably the mechanism behind this “sensing” is alike across many evolutionary distinct organisms. 5 KOPSAVILKUMS Maizes raugs Saccharomyces cerevisiae ir zinātnē bieži lietots eikariotu modeļorganisms. Tas evolucionāri ir attīstījies dzīvot vidēs ar fluktuējošu barības vielu pieejamību - straujam barības vielu pieaugumam var sekot barības vielu trūkums. Šie apstākļi radījuši raugā jutīgus mehānismus barības vielu daudzuma uztveršanai un vielmaiņas pielāgošanai barības vielu pieejamībai. Apstākļos, kuros trūkst slāpeklis, ogleklis vai citi makroelementi, maizes raugs aptur vielmaiņu un šūnas ciklu, šūnas nonāk “badošanās fenotipā”. Tā kā maizes raugu tā samērā vienkāršās uzturēšanas dēļ bieži izmanto arī zinātniskos pētījumos, laboratorijās izmantotajiem celmiem nereti ir sastopamas mutācijas vairākos aminoskābju vai nukleotīdu biosintēzes ceļos - auksotrofijas, kas kalpo kā metaboliskie marķieri, lai atvieglotu gēnu inženieriju šajos celmos. Vairāki autori ir pierādījuši, ka rauga šūnas nespēj “uztvert” auksotrofo barības vielu trūkumu, tādēļ pie auksotrofo faktoru trūkuma šūnas parasti nenonāk badošanās fenotipā. Šajā darbā aplūkota maizes rauga fizioloģija, ja tam ir mutācija adenīna biosintēzes ceļā (ade8 vai ade2). Adenīna auksotrofija ir samērā bieži izmantots metaboliskais marķieris, jo ade1 un ade2 gēnu mutanti uzkrāj šūnās sarkano pigmentu. Mūsu rezultāti norāda, ka adenīna mutanti adenīna trūkuma gadījumā fenotipiski drīzāk atbilst šūnām ar “badošanās fenotipu”. Šūnas sekmīgi aptur šūnas ciklu, tām pieaug stresa izturība, novērojama transkriptomiskā atbilde, kas tuvina adenīna badinātas šūnas šūnām stacionārajā fāzē. Novērojama arī šūnas oglekļa metabolisma pārvirzīšana no spirta uz citu metabolītu ražošanu – glicerīnu, acetātu. Darbā parādīts, kā uz adenīna sintēzes ceļa inaktivēšanu un tai sekojošu adenīna trūkumu reaģē vairāki pētniecībā bieži izmantoti celmi ar atšķirīgām adenīna sintēzes ceļa mutācijām. Rezultāti parāda līdzīgu metabolisko atbildi, norādot uz to, ka šis ir vispārīgs fenomens. Uz to norāda arī fakts, ka vairāki iekšsūnu parazīti ir evolucionāri zaudējuši adenīna biosintēzes ceļa gēnus. Vienam no šādiem parazītiem (Leishmania) novērota paaugstināta stresa izturība vidē bez adenīna. Tas ļauj spekulēt, ka adenīna trūkums šūnās tiek uztverts ar līdzīgiem mehānismiem plašā dzīvo organismu lokā. 6 Table of contents Abbreviations ....................................................................................................................... 8 Introduction ....................................................................................................................... 10 1. Literature review ........................................................................................................... 11 1.1. Basic biology of S. cerevisiae.................................................................................. 11 1.2. Starvation response ................................................................................................ 14 1.3. Natural and artificial starvations .......................................................................... 16 1.4. Purine biosynthesis in yeast ................................................................................... 18 1.5. Purine auxotrophy in nature ................................................................................. 21 2. Materials and methods.................................................................................................. 23 2.1. Strains and Cultivation Conditions .......................................................................... 23 2.2. Analysis of Extracellular Amino Acids and Purines ................................................ 25 2.3. Cell Morphology Measurements .............................................................................. 26 2.4. Flow Cytometry ....................................................................................................... 26 2.5. Fermentation and Metabolite Flux Measurements ................................................... 26 2.6. FTIR Analysis .......................................................................................................... 27 2.7. Cell Carbohydrate Extraction and Quantification .................................................... 27 2.8. Transcriptomics ........................................................................................................ 27 2.9. Sublethal Stresses ..................................................................................................... 28 3. Results ............................................................................................................................ 29 3.1. Adenine auxotrophy – be aware: some effects of adenine auxotrophy in Saccharomyces cerevisiae strain W303-1A .............................................................................. 29 3.2. Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast ............................................................................................................... 45 3.3. Adenine starvation is signalled through environmental stress response system in budding yeast Saccharomyces cerevisiae ................................................................................. 66 3.4. Purine auxotrophy: Possible applications beyond genetic marker .................... 73 4. Discussion ....................................................................................................................... 82 4.1. Care should be taken when using adenine auxotrophs in research .......................... 82 4.2. Internal purine resources are sufficient to finish the cell cycle ................................ 82 4.3. Are purine starved cells quiescent? .......................................................................... 83 4.4. Purine starvation is similar but not quite the same as nitrogen starvation ............... 85 4.5. How purine starvation is perceived in cells? We propose that mechanisms additional to the Gcn4p response play a role. Possibly TOR mediated Rim15p governed response. ........... 86 7 4.6. Transcription of purine starved cells does not agree with the observed metabolome ...................................................................................................................................................... 91 4.7. What is the place of purine starvation in evolutionary landscape ............................ 92 5. Conclusions and direction of following research ........................................................ 94 6.Theses for defence .......................................................................................................... 96 Approbation of the research............................................................................................. 97 Acknowledgments and funding ........................................................................................ 98 References .......................................................................................................................... 99 8 Abbreviations (p)ppGpp - Guanosine pentaphosphate and tetraphosphate, signalling molecules involved in stringent control in bacteria, leading to inhibition of RNA synthesis in the absence of amino acids ADE - ADEnine requiring, genes whose recessive alleles determine the requirement for adenine in the culture medium ADH2 - Alcohol DeHydrogenase - Glucose-repressible alcohol dehydrogenase II; catalyzes the conversion of ethanol to acetaldehyde ADP - adenosine diphosphate AICAR - 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate, a product of the Ade13p of the purine biosynthesis pathway AMP - adenosine monophosphate AMP - adenosine monophosphate, nucleotide ATP - adenosine triphosphate - the cell's main energy cofactor Bas1p - BASal transcription factor involved in regulation of basal and induced expression of genes of purine and histidine biosynthetic pathways; controls cellular ATP levels cAMP - cyclic adenosine monophosphate - secondary messenger in eukaryotic cells CFU - Colony Forming Unit - a cell of a microorganism that is capable of multiplying - forming a colony - on a solid medium CLS - Chronological Life-Span - measures the length of time nondividing cells survive. ESR- Environmental Stress Response - includes ∼900 genes whose expression is stereotypically altered when yeast cells are shifted to stressful environments. The coordinated expression changes of these genes is a common feature of the responses to many different environments, however the regulation of these expression changes is gene-specific and condition-specific. FCY2 - Purine-cytosine permease gene; mediates purine (adenine, guanine, and hypoxanthine) and cytosine accumulation FTIR - Fourier Transformation Infrared Spectroscopy is used to obtain infrared absorption or emission spectra of a solid, liquid or gas. G0 - G₀ phase describes the state of the cell outside the replicating cell cycle. G1 - Gap1 phase or growth phase 1 is the first of the four phases of the cell cycle that occur during eukaryotic cell division. GAAC - General Amino Acid Control - Gcn4p coordinated transcriptional response to amino acid depletion in the environment Gcn4p - General Control Nonderepressible, a transcriptional activator of amino acid biosynthetic genes; the activator responds to amino acid starvation. Gis1p - Histone demethylase and transcription factor; regulates genes during nutrient limitation. GLN3 - GLutamiNe metabolism- Transcriptional activator in nitrogen catabolite repression system; localization and activity regulated by quality of nitrogen source GMP - Guanosine monophosphate, nucleotide IMP - inosine monophosphate, nucleotide 9 Msn2p - Stress-responsive transcriptional activator; when activated, migrates to the nucleus in response to different stress conditions; binds to DNA to stress response elements of genes. Msn4p - Stress-responsive transcriptional activator; when activated migrates to the nucleus in response to various stress conditions; binds to the DNA to stress response elements of genes. mtDNA - mitochondrial DNA is the DNA located in mitochondria NGS - Next Generation Sequencing - a massively parallel sequencing technology that provides extremely high throughput, scalability and speed. NMR - Nuclear Magnetic Resonance - Spectroscopy is a technique that exploits the magnetic properties of certain atomic nuclei and can be used to determine the physical and chemical properties of the atoms or molecules in which they are located. OD - Optical Density, the absorption of light by a sample proportional to the density of cells in the sample Pho2p - PHOsphate metabolism transcription factor; regulatory targets include genes involved in phosphate metabolism. Pho85p - Cyclin-dependent kinase involved in regulating the cellular response to nutrient levels and environmental conditions and progression through the cell cycle PKA - Protein Kinase A, also known as cAMP-dependent protein kinase. PKA has several functions in the cell, including regulation of glycogen, sugar and lipid metabolism. Ras - GTPase encoded by the RAS1 gene, involved in G-protein signalling in adenylate cyclase activation; plays a role in the regulation of cell proliferation; localised to the plasma membrane. Rim15p - protein kinase encoded by the RIM15 gene, regulates cell proliferation by changing the location between the nucleus and cytoplasm rRNA- Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells SAICAR - 1-(phosphoribosyl)imidazole carboxamide, a product of the Ade1p of the purine biosynthesis pathway SAM - S-adenosylmethionine is a common cosubstrate involved in methyl group transfer, transsulfuration and aminopropylation. SD - Synthetic complete Dextrose, a type of synthetic medium with a known mineral content, glucose as a carbon source Snf1p - AMP-activated protein kinase; required for glucose-repressed gene transcription, heat shock, sporulation, and peroxisome biogenesis TOR - Target Of Rapamycin - a protein kinase involved in nutrient sensing and link to cell growth. tRNA - transport RNA - ensures amino acid delivery to ribosomes. YPD - Yeast Peptone Dextrose, a type of complete medium containing yeast extract, proteins hydrolysed by pepsin and glucose 10 Introduction Cells perceive available nutrients and tailor their metabolism accordingly. Starvation for basic nutrients elicits stress resistant phenotype. Purines are the basis of structure and functionality of every living cell, yet the response of the cells for lack of purines is scarcely described. This work aims to describe how Saccharomyces cerevisiae cells react to the depletion of purines and place purine starvation with the respect of other starvations. To achieve this aim following tasks were formulated: • to characterise ade auxotrophic strain growth in rich (YPD) media; • to describe metabolism, transcriptome and stress resistance of ade auxotrophic strains when starved for purines in defined media; • to describe phenotype of purine starved auxotrophs with truncated transcription factors for elucidation of purine starvation signalling in the cells; • to review the overall structure of the purine synthesis metabolic pathway and purine auxotrophy across all domains of life. 11 1. Literature review 1.1. Basic biology of S. cerevisiae Yeast Saccharomyces cerevisiae is a species of unicellular Ascomycota fungi that replicates by budding. The life cycle of this yeast consists of haploid and diploid phases. In the case if conditions are optimal - it rapidly proliferates mitotically (either as haploid or diploid), while in the case of poor nutrient supply it sporulates. Two haploid cells mate and form asci with 4 spores. Bakers yeast – S. cerevisiae as the name suggests is widely used in the food industry where its ability to quickly ferment simple sugars is employed in bread and alcohol production. The genome studies of S. cerevisiae point to the emergence of it as a species in the end of the Cretaceous age when sugar rich fruits appeared (Friis et al., 1996). Whole genome duplication event allowed S. cerevisiae to develop a “make-accumulate- consume” lifestyle, where available sugar is quickly converted into alcohol, prohibiting growth of other microorganisms. Alcohol is later consumed (Piškur et al, 2006). While S. cerevisiae employed by humans is selected for its quick fermentation capabilities and genetic analysis shows interspecific genome regions from other yeasts, wild yeasts isolated on various trees and primaeval forests still retain “make-accumulate-consume” lifestyle (Liti, 2015). To achieve this lifestyle S. cerevisiae exhibits Crabtree effect – if sugar concentration is high, most of energy is produced with fermentation even if oxygen is present (Verdyun et al., 1984), that is possible due to the glucose repressing mitochondrial enzyme transcription. Genome duplication gave rise to several enzyme isoforms, f. ex., ADH2 allowing the consumption of ethanol (Piškur et al, 2006; Thomson et al., 2005). It is worth noting that in both human generated or natural environments S. cerevisiae will have times of plenty when high quantities of nutrients are present and times of scarcity when nutrients are exhausted and cells must persist until the next nutrient influx (Lahue et al., 2020; Smets et al. 2010). In figure 1 is the scheme of central carbon metabolism adapted from Rintala 2010. 12 Figure 1. Carbon flow in the S. cerevisiae in presence of oxygen, in circles metabolites, in rectangles genes coding for respective enzymes (from Rintala, 2010). Note the amount of isoenzymes for many reactions. S. cerevisiae is a heterotroph organism, meaning cells produce energy and biomass from organic molecules that are acquired from the environment. The empirical estimated biomass equation for S. cerevisiae is C:H(1.613):O(0.557):N(0.158). The varying amounts and quality of nutrients present in the environment requires yeast cells to be able to sense and tailor metabolism according to it, sustaining cell composition and viability. Sugars are the main energy supply and also carbon source for yeast. Yeast cells prefer glucose or fructose to other mono-, di- or trisaccharides. Fermentable carbon sources are consumed prior to substrates that would yield energy with oxidative phosphorylation (Broach, 2012). 13 Glucose repression of mitochondrial function is the basis of the Crabtree effect. When cells are in a glucose rich environment various signalling systems activate genes required for fast growth such as ribosome biogenesis genes, at the same time stress response genes and alternate carbon source utilisation genes are repressed. Same as for the carbon sources also nitrogen sources are prioritised - ammonia ions that can be easily converted into central intermediates of nitrogen metabolism in cell - glutamate or glutamine, but S. cerevisiae can also use less preferred nitrogen sources such as proline. Use of nitrogen sources is also regulated by nitrogen source repression mechanism prioritising easily metabolised ones (Broach, 2012). When cells are using less preferable sources of C and N growth is slower and stress resistance genes are activated. Eventually with exhaustion of available nutrients the cell ceases to divide and becomes dormant (Smets et al., 2010). In full media S. cerevisiae has a relatively short doubling time – 1-2 h and is easily cultivated in laboratory conditions, but in difference to bacteria, yeast is a eukaryotic organism. This has led to usage of yeast as a “workhorse” of molecular biology. Several strains of yeast are developed that are widely used in research. W303 strain was constructed by Rodney Rothstein and is quite often seen in physiology related research such as ageing (Ralser et al., 2012). Another widely used strain is S288C or its derivatives. S288C was developed by Robert Mortimer for biochemical studies and was used to develop gene knock out collection and also serves as a reference genome (Mortimer, Jonston, 1986). W303 and S288C share more than 85% of their genome information (Ralser et al., 2012). System biology on other hand mostly uses strains derived from CEN.PK strain - yeast strain series developed by Michael Ciriacy and K.D. Entian (Entian, Kötter, 2007). Irrespective of field of studies quite often genetic manipulations are performed in these strains. To help in these manipulations strains harbour several mutations in biosynthesis genes of amino acids and nucleotides - W303 leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15, S288C derived BY4741 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (Branchman et al., 1998), and CEN.PK family strains usually carry ura3-52 his3-Δ1 leu2-3,112 trp1-289. By inserting a working copy of defective biosynthesis gene along with intended genetic modification it is easy to select modified strains. In laboratory conditions yeast cells are usually grown in a synthetic medium that is rich in carbon source (glucose) and good nitrogen source (ammonia) and all other nutrients in excess to support rapid growth/ proliferation of the yeast. 14 1.2. Starvation response When all nutrients are plentiful, yeast cells grow and proliferate until a certain nutrient is exhausted, afterwards growth halts until metabolism is reoriented to use any available alternative or stops completely if no substitute is available. If a microbial cell number in a fresh media would be recorded several population growth phases can be distinguished. In the beginning the population experiences a so-called lag phase where no or minimal growth is observed. After the lag phase and adapting to the environment cells proliferate exponentially giving the name of this phase - log phase. After a while nutrients are exhausted and a stationary phase where no population growth is observed will be reached. Due to the Crabtree effect S. cerevisiae populations grown aerobically on glucose exhibit so called diauxic growth, where the second log phase can be observed as cells switch from fermentation to the consumption of fermentation products (Figure 2). Figure 2. Typical growth curve of S. cerevisiae in aerated glucose rich environment. First growth phase is observed when glucose is used as the main carbon source, second growth observed on fermentation products. Lack of nutrients is common in nature. Cells in later population growth phases coincident with slow or no growth show increased stress resistance and longer lifespans if compared to log phase cells. This helps cells to survive until the next supply of fresh nutrients is available. Growth is regulated by cellular signalling pathways that perceive the state of nutrients in the environment. After receiving signal from the receptor usually with the help of a secondary messenger or G protein signal is passed via kaskades of signal 15 transduction pathway most commonly ending with activation of protein kinase that in turn phosphorylates enzymes or transcription factors causing changes in cellular metabolism and gene expression. Glucose being the preferred carbon source will activate several cell signalling systems. Most of the glucose effects on biosynthetic capacity and stress responses are mediated by the protein kinase A pathway, while repression of genes involved in use of alternative carbon sources are mediated predominantly by Snf1p. Protein kinase A (PKA) pathway is activated by G protein Ras that causes synthesis of secondary messenger cAMP, that in turn will activate PKA that directly and indirectly will affect enzyme activity and variety of transcription factors. TOR (target of rapamycin) is the second main cell growth speed regulating signalling system. TORp is a phosphatidylinositol kinase-related protein kinase that controls cell growth in response to nutrients. Rapamycin is an immunosuppressive and anticancer drug that acts by inhibiting TORp. The modes of action of TORp and rapamycin are remarkably conserved from S. cerevisiae to humans (Crespo, Hall, 2002). While the Ras/PKA pathway mainly relates to the carbon availability and TOR is described in connection with nitrogen sensing, both pathways are interconnected and share many downstream targets - transcription factors (Plank, 2022). Both pathways regulate ribosome production and repress autophagy and stress response, thus a suppression of either of the pathways results in slow down of the growth (Figure 3). Figure 3. General signalling pathways in yeast cells influencing cell growth/resistance phenotype. Snf1 is a protein kinase required for glucose-repressed gene transcription. Figure adapted from Busti et al., 2010. 16 Starvation for the nutrients is known to induce quiescence in the budding yeast. Quiescence is a state of the cell where no proliferation happens yet after returning in a nutrient rich environment proliferation resumes. As of yet no definite single marker for achieving quiescent state has been defined, but there is a cluster of characteristics that are typical for quiescent cells - smaller, denser cells with thicker cell wall, these cells show reduced metabolic activity with smaller amounts of RNA and ribosomes. Quiescent cells arrest cell cycle at G0 stage (Sun, Gresham, 2021) Yeast cells subjected to starvation for any nutrient exhibit a stereotypic pattern of gene expression changes, referred to as the environmental stress response (ESR). ESR is initiated also by a large number of environmental stressors, such as heat, oxidative stress, or high osmolarity (Gasch et al., 2000). The predominant components of the set of genes that are repressed in the ESR include those required for mass accumulation, primarily ribosome biogenesis genes. Stress resistance genes - such as catalase or heat shock protein expression is activated. The fact that other stresses also elicit a similar repression suggests that the individual stressors either engage nutrient signalling pathways, such as PKA and TOR, or interact with the same transcriptional regulatory apparatus that responds to nutrients (Broach, 2012). It has been observed that in some cases if cells are starved for some auxotrophic agents - uracil, leucine - they do not enter quiescent state and lose viability rapidly. This had led to distinguishing two types of starvations, dependent on the missing nutrient: ‘natural limitations’, which sets in when basic nutrients (carbon, phosphorous, sulphur and nitrogen) are scarce, and ‘artificial limitations’, which sets in when particular metabolites or metabolic intermediates are insufficient (Saldanha et al., 2004). 1.3. Natural and artificial starvations Cells starved for main nutrients carbon, nitrogen, phosphorus and sulphur share same characteristics - cell cycle arrest as unbudded cells, thickened cell walls, increased stress resistance and an accumulation of storage carbohydrates (Klosinska et al., 2011; Lillie & Pringle, 1980; Schulze et al., 1996). At the same time intracellular nutrients do change depending on the factor causing starvation and are not uniform across starvations. If cells experience a lack of nitrogen in the presence of carbon source, protein and RNA amount in cells drops, with almost no free amino acids in cytoplasm, but storage carbohydrates and 17 fats increase. Cells that are starved just for carbon, but have nitrogen present would have almost twice as much proteins and RNA and less storage molecules - carbohydrates (especially glycogen) and fats (Albers et al., 2007). Boer and colleagues published results of amounts on intracellular metabolites during various starvations. Cells starved for carbon were mainly limited in metabolites of main energy generating pathways - glycolysis and Krebs cycle, nitrogen starved cells experienced lack of amino acids, whereas in phosphate starved cells phosphorylated intermediates of pentose phosphate pathway and triphosphates were found to be main metabolites concentrations of which were significantly reduced (Boer et al., 2010). Interestingly, similar reduced amounts of metabolites were observed also in E. coli starved for carbon and nitrogen (Brauer et al., 2006). Also recent research on involvement of transcription factors during starvation shows various pathways how starvation is sensed and communicated via Rim15p in carbon or nitrogen starvation in budding yeast (Sun et at., 2020). Auxotrophy is a typical example of an artificial starvation that would not be experienced by prototrophic cells. Many common laboratory yeast strains (W303, S288C, CEN.PK and FY series) contain one or several auxotrophic markers. Histidine, leucine, uracil, adenine and tryptophan (his, leu, ura, ade and trp) are the most common auxotrophic markers of Saccharomyces cerevisiae strains used in physiology studies (Pronk, 2002; Da Silva, Srikrishnan, 2012). Insufficient concentration of an auxotrophic agent leads to artificial limitation that converts to starvation. When cells were starved for leucine and uracil it was observed that their viability rapidly decreased, which led to the concept of artificial starvations (Saldanha et al., 2004; Gresham et al., 2011). Before coinage of this concept it was known that fas1 cells starved for fatty acids die rapidly - cells lose viability by several folds of magnitude within 24h (Henry, 1973). Henry also observed survival of cells starved for lysine, tryptophan and adenine, where after 24h around 30% -50% were alive after starvation, that is considerably less as with fatty acids. Research with wine yeast shows that lack of vitamins in media also leads to the rapid loss of viability (Duc et al., 2017). Methionine has been shown to be an exception of the rule where methionine starved cultures resemble more of the natural starvations. It is argued that lack of methionine is perceived as sulphur starvation (Unger, Hartwell 1976; Petti et al., 2011) 18 It seems that in most of these “artificial” starvations the cell signalling system is not “aware” of intermediates lacking and does not arrest cell cycle, but tries to proceed with fast growth eventually “running out” of building blocks. If for some other reason growth speed affecting signalling systems are affected, leucine starved cells do not die as rapidly, that can be seen that if cells are starved for leucine in non fermentable carbon sources cell life span increases (Boer et al., 2008). It is further shown with quiescence and chronological life span screens where low survivability in artificial starvations is suppressed by mutations in TOR pathway or its targets (Boer, 2008; Gresham et al., 2011). Cell cycle arrest and quiescence do not occur for an artificial starvation with either leucine or uracil. Inability to complete cell division and to halt subsequent cell cycle leads to a decrease in viability in addition to an observable “glucose wasting” phenomenon, where auxotrophic starved cells converted higher quantities of glucose to ethanol compared to the phosphate starved ones (Boer et al., 2008). Both cell cycle arrest and mitochondrial respiration is governed by cell signalling systems further pointing to involvement of those in “sensing” nutrient scarcity. 1.4. Purine biosynthesis in yeast New purine bases in eukaryotic cells are obtained in two ways - salvage and de novo synthesis. In the salvage pathway, purine bases are taken up from the environment or recycled within the cell and attached back to the ribose 5‐phosphate, thus restoring nucleotides. Most eukaryotes have two purine salvage enzymes - one that can produce AMP (adenine phosphoribosyltransferase, EC 2.4.2.7) and hypoxanthine phosphoribosyl- transferase (EC 2.4.2.8) that can produce GMP or IMP, and sometimes xanthosine monophosphate. Purines are actively transported across cell membrane with the help of purine-cytosine permease Fcy2p (Kurtz et al., 1999) and stored in the vacuole (Nagy, 1979) presumably with the help of a Fun26p transporter that is a passive transporter (Boswell- Casteel, 2014). The purine synthesis pathway and its regulation are highly conserved in all eukaryotes from fungi to mammals (Agmon et al., 2020). Most probably, the last common ancestor had a pathway with the same structure that diversified into the now known three domains of life - Bacteria, Archaea and Eukarya (Armenta‐Medina et al., 2014; Vázquez‐Salazar et al., 2018). 19 In S. cerevisiae, the purine de novo synthesis pathway comprises sequential chain of reactions adding C and N atoms to ribose phosphate until inosine monophosphate (IMP) is formed, which is the common substrate for GMP and AMP synthesis. To synthesise IMP, the glycine backbone is fused with nitrogen provided by the amide groups of two glutamine molecules and one aspartate. Additional carbon atoms are provided by two formate and one CO2 molecule (Pedley, Benkovic, 2017). All de novo pathway gene expression is regulated by Bas1/2p transcription factors that respond to the concentrations of pathway intermediates phosphoribosylaminoimidazolecarboxamide (AICAR) and phosphoribosylaminoimidazole- succinocarboxamide (SAICAR), metabolites that are products of reactions catalysed by Ade13p and Ade1p, respectively (Denis et al., 1998). The total flux of metabolites through the purine de novo synthesis pathway is regulated by the first enzyme Ade4p that is sensitive to ATP and ADP concentrations in the cell (Pinson et al., 2009; Rébora, Desmoucelles, Borne, Pinson, & Daignan‐Fornier, 2001; Rébora, Laloo, & Daignan‐Fornier, 2005). See simplified purine salvage and de novo synthesis scheme in figure 4. Note that IMP provides both AMP and GMP, thus a mutation in IMP synthesis pathway in a media without nucleotides would result not only in adenine, but also guanine auxotrophy. Thus further on we will refer to this starvation as purine starvation. 20 Figure 4. Simplified purine salvage and de novo synthesis scheme, all genes of de novo pathway and some intermediates shown. Genes shown in italics, genes in boxes - genes used in this thesis research. Adapted from Kowalski et al, 2008. Abbreviations: PRPP - 5-phospho-α-D-ribose 1-diphosphate, Gln - glutamine, Gly - glycine, Asp - aspartate, fum - fumarate, THF - tetrahydrofolate, Gua - guanine, Hypox - hypoxanthine, Ade- adenine, SAH - S-adenosylhomocysteine, SAM - S- adenosylmethionine, Met - methionine, HomoCys - homocysteine. As purine synthesis is connected to histidine synthesis via AICAR some biosynthesis mutants are not only adenine auxotroph but also require histidine if the mutation is below the ADE13 gene. AICAR and SAICAR both have regulatory roles. ADE13 mutant is not viable due to SAICAR accumulation and ADE16 ADE17 double mutant accumulates AICAR and shows slower growth (Tibetts, Appling, 2000). Histidine synthesis pathway reactions catalysed by HIS1, HIS4 and HIS7 are also affected by Bas1/2 transcription factors and adenine depletion in the cell (Denis et al., 1998). ADE3 gene is not directly involved in generation of purine rings but catalyses sequential reactions 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5), to supply forms of activated one-carbon units required for biosynthesis of purines, histidine, 21 methionine and pantothenic acid. Cells defective in the ADE3 gene are adenine and histidine auxotrophs. 1.5. Purine auxotrophy in nature Purine auxotrophy is a common phenomenon among monera, protozoans, and metazoans. For example, all parasitic protozoans and many intracellular bacterial pathogens are purine auxotrophs. Two of the well‐known examples are Toxoplasma gondii (causes toxoplasmosis) and Plasmodium falciparum (causes malaria) (Downie et al., 2008; Weiss, Kim, 2011). Parasitic worm genome analyses revealed that purine auxotrophy is common among parasitic platyhelminths and roundworms (nematodes). Parasitic worms lack some or all de novo purine pathway enzymes (International Helminth Consortium, 2019). These auxotrophic organisms save resources for expensive purine de novo synthesis as one nucleotide “costs” ~50 ATP molecules (cost calculated including energy spent for synthesis of all intermediate metabolites) (Lynch, Marinov, 2015). Many apicomplexan parasites lack all the genes encoding enzymes of de novo purine synthesis and rely only on purines harvested from their host. Using salvage pathways, parasites collect different species of purines - adenine, xanthine, hypoxanthine, adenosine, and inosine. Parasitic organisms absorb purine sources through specific nucleotide transporters (Chaudhary et al., 2004; Major et al., 2017). However, the inability to synthesise purine is not specific to parasites. For example, Tetrahymena is a genus of free‐living protozoans that requires exogenous purine and pyrimidine supply to sustain growth. It has not lost its entire purine synthesis pathway, but several steps are missing (Hill, 1972). As purine supply of parasitic organisms is dependent on host cells, purine starved cells will show specific phenotype. Leishmania cells will arrest cell cycle, increase stress resistance and reorient cell metabolism to deal with both purine deprivation and general stress. (Carter et al., 2010; Martin et al., 2014). Purine auxotrophy in yeast is not naturally occurring and is caused by genetic manipulations. Literature on effects of purine auxotrophy is scarce. It is known that purine depletion will stimulate Gcn4p - transcription regulator protein, responsible for increased transcription of more than 30 different amino acid biosynthetic genes in response to starvation for a single amino acid. (Rolfes, Hinnebusch, 1993). There are reports on Gcn4p 22 dependent purine biosynthesis gene activation (Mösh et al., 1991), as the ADE4 gene promoter contains three sequences ATGA (C/G)TCAT that bind Gcn4. This motive is shared with Bas1p binding site GAGTCA, which is proven to be involved in ADE4 transcription initiation (Som et al., 2005), which causes competition between these two transcription factors. Purine synthesis genes are also mentioned to be involved in determination of chronological life span (CLS), but reports are contradictory. Matecic describes the effect of purine de novo mutations on CLS that is comparable to glucose restriction. Mutations in GLN3, TOR1 and FCY2 also extend CLS but to a smaller extent than mutations in de novo synthesis pathway genes. CLS extension in ade de novo synthesis mutants is suppressed by adding extra adenine in media, but not in gln3, tor1 or fcy2 mutants (Matecic et al., 2010). Garay and colleagues on the other hand identify adenine de novo synthesis pathway gene mutations as CLS shortening ones. It is worth noting that in both cases strains harbouring several auxotrophies and synthetic media were used (Garay et al., 2014). Analysis of several quiescence studies stresses the fact that differences in strains used and media employed may explain vast discrepancies in genes affecting CLS (Smith et al., 2016). There are also reports on influence of adenine starvation cells on retrotransposon activity, where starvation induces transcription of retrotransposons (Todeschini et al., 2005; Servant et al., 2008). Stress induced transposon activity has been connected to the possibility of genome evolution (Fedoroff, 2012). 23 2. Materials and methods 2.1. Strains and Cultivation Conditions Two genetic backgrounds were used: W303 and CEN.PK. Strains used in research are summarised in table 1. All cultures were maintained on YPD agar and kept at 4◦ C. Fresh YPD agar plates were regularly reinoculated from stock cultures kept at − 80◦ C. Table 1. Strains used in research used for theses. 1 - Kokina et al., 2014, 2 - Kokina et al., 2022, 3 - Ozoliņa et al., 2017 Strain name used in text Genotype Source Used in research in W303 ade2 W303-1A MATa leu2-3,112 trp1-1 can1- 100 ura3-1 ade2-1 his3-11,15 Dr. Peter Richard 1 W303 ADE2 W303-1A ADE2 Dr. Arnold Kristjuhan 1 W303 prototroph 2832 – 1B MATa can1 Dr. Frederick R. Cross 1 CEN.PK prototroph CEN.PK 113-1A Dr. Peter Richard 1,2 CEN.PK ADE8 CEN.PK2 MATa leu2-3/112 ura3-52 trp1- 289 his3-1, MAL2-8c SUC2 Dr. Peter Richard 1,2 CEN.PK ade8 CEN.PK2 MATa leu2-3/112 ura3-52 trp1- 289 his3-1, ade8∆0, MAL2-8c SUC2 Our research 1,2,3 msn2 CEN.PK ade8 msn2::KanMX Our research 3 msn4 CEN.PK ade8 msn4::KanMX Our research 3 rim15 CEN.PK ade8 rim15::KanMX Our research 3 The ade8 knockout was induced by the ura3-URA3 5-FOA toxicity knockout technique, using ade8 knockout construct plasmid (Sadowski et al., 2008). Transcription factors were truncated by transforming yeast with linear PCR fragment containing flanking homologous sequences of the respective transcription factor and 6xHis 24 tag and G418 marker in between (Janke et al., 2004). After transformation strain identity was confirmed by colony PCR using gene specific test primers and internal primer (Nat_ctrl_Hb) from the insert (Lõoke et al., 2011). All the details regarding plasmids and primers are given in Table 2. All primers were synthesised by Sigma Aldrich. Table 2. Plasmid and primers used for generation of strains with truncated transcription factors used in Ozoliņa et al, 2017 Name Description or sequence Source pYM46 PCR template for C-terminal myc tag plus 7 His residues: marker pAgTEF-kanMX-tAgTEF, selectable phenotype: G418 resistance Janke et al. 2004, EUROSCARF MSN4_S2 CTTGTCTTGCTTTTATTTGCTTTTGACCTTATTTTTT TCAATCGATGAATTCGAGCTCG Our research MSN4_S3 GCATTCAGACGCAGTGAGCACTTGAAAAGGCATA TAAGATCGTACGCTGCAGGTCGA Our research MSN2_S3 GAAATTTAGTAGAAGCGATAATTTGTCGCAACACA TCAAGCGTACGCTGCAGGTCGA Our research MSN2_S2 TGAAGAAAGATCTATCGAATTAAAAAAATGGGGT CTATTAATCGATGAATTCGAGCT Our research RIM15_S2 CAGTTATTTTTTTTAATTATCTTTATCTTAAAATTT ATCAATCGATGAATTCGAGTCCG Our research RIM15_S3 CAGGAGGCGGCAACCAGTAGAGTCTTTGACGATG TTTTAGCGTACGTCGCAGGTCGA Our research MSN4_test_F01 AGAAGGCATTCAGACGCAGT Our research RIM15_test_F01 CCAATTGTGGCCATAACAAA Our research MSN2_test_F01 CCATTATCGCCTGCATCATCAT Our research NAT-HgB_ctrl ACGAGGCAAGCTAAACAGATCT Our research In Kokina et al., 2014 strains were cultivated in YPD - 10 g L-1 of yeast extract (Biolife), 20 g L-1 of peptone (Biolife), 20 g L-1 of dextrose (Sigma) or SD media. In Kokina et al. (2022) and Ozoliņa et al. (2017) cells were cultivated exclusively in Synthetic Defined (SD) media (Saldanha et al., 2004) with 80 mg tryptophan, 100 mg uracil, 480 mg leucine, 100 mg histidine, and 100 mg adenine added per litre, as suggested in (Pronk, 2002). For purine starvation exponentially grown cells were transferred to fresh SD media with adenine omitted but other additives same as previous. 25 To ensure that yeast cultures were in the exponential growth phase, we reinoculated overnight cultures (grown from a single colony) into fresh media, where at least 6 doublings occurred and OD600 0.5–1, corresponding to 1–2 · 107 cells mL− 1 , was reached. Cultures in the exponential growth phase (OD600 0.5–1) were washed with distilled water twice and resuspended at OD600 0.5 in full SD media (SD) or SD media with adenine omitted (SD ade−). All cultures used for further measurements were incubated on a rotary shaker at 30 ◦C and 180 rpm in flasks where broth volume does not exceed 20% of total volume. To demonstrate changes in optical density during starvation, 96-well Tecan Infinite M200 multimode reader was used with the following cultivation cycle: orbital (3.5 mm) shaking for 490 s, waiting for 60 s, optical density measurement at 600 nm. Alternatively, culture growth dynamics was measured with a Z2 Cell and Particle Counter (Beckman Coulter, Brea, CA, USA). 2.2. Analysis of Extracellular Amino Acids and Purines In Kokina et al. (2022) NMR spectroscopy was used. To obtain samples cell-free culture media was mixed with DSS (sodium 4,4-dimethyl-4-silapentane sulfonate) in D2O to obtain a final DSS internal standard concentration of 1.1 mM and transferred to a 5 mm NMR sample tube. NMR analysis was performed at 25 ◦C on a 600 MHz Bruker Avance Neo spectrometer equipped with a QCI quadruple resonance cryoprobe. The noesypr1d pulse sequence was used with water suppression during a recycle delay of 10 s. The spectral width was 11.9 ppm, and 128 scans were collected into 32K data points using an acquisition time of 2.3 s. The acquired 1H NMR spectra were zero-filled once, and no apodization functions were applied prior to Fourier transformation. Phase and baseline corrections were applied manually. Spectra were referenced to DSS (at 0.00 ppm). The identification and quantification of sample components were performed using Chenomx NMR Suite professional software (version 5.11; Chenomx Inc., Edmonton, AB, Canada). In Kokina et al., 2017 the concentration of adenine in media was determined enzymatically following a modified protocol from Zhang et al. (2003). More specifically, the concentration of adenine in media was quantified fluorometrically by hypoxanthine 26 oxidase (Sigma X4500)-coupled assay using horseraddish peroxidase (HRP; Sigma) and Amplex UltraRed dye (Molecular Probes, ex/em 530/590 nm). The reaction mix contained 20 μL of sample, 2 mL 0.1 M, pH 7.5 sodium phosphate buffer, 0.02 U xanthine oxidase, and 2 U HRP. Reaction mixtures were incubated at 30 °C for 30 min at which point the emission at 590 nm (ex 530) was measured with a FluoroMax-3 (Yvon Horiba) spectrofluorometer. 2.3. Cell Morphology Measurements Cell samples were fixed in formaldehyde 0.5% and examined with an optical microscope (Olympus BX51, Tokyo, Japan). Microphotographs (1360 × 1024 pixels) were obtained with a digital camera (Olympus DP71, Tokyo, Japan). Cell size and budding index were determined by microphotography analysis in the ImageJ program. Budding index was defined as the proportion between the number of cells with buds and the total cell number. Bud was defined as a cell with a cross-section area less than half the mother cell size. Cell size was determined as the cell cross-section area measured from the microphotographs using ImageJ. Cells were defined as ellipses, with area measured in pixels and recalculated to square micrometers (1 μm = 5.7 pixels). For each sample, at least 500 cells were measured. 2.4. Flow Cytometry Cell DNA content was determined by flow cytometry as described in (Sein et al., 2018). Briefly, 0.5 mL of yeast culture was fixed in 10 mL of ice-cold 70% ethanol for at least 15 min and washed once with 50 mM citric acid. RNA was degraded using RNase A (10 μg mL− 1 ) in 50 mM citric acid overnight at 37 ◦C. DNA was stained with 10 × SYBR Green (Invitrogen, Waltham, MA, USA) in 50 mM citric acid for 30 min. Cells were analysed with a FACSAria device (Becton Dickinson, Franklin Lakes, NJ, USA). Cell cycle distribution was analysed with Cyflogic software. 2.5. Fermentation and Metabolite Flux Measurements Fermentation was done in a Sartorius Q-plus fermentation system with working volume of 0.3 L, gas flow 0.25 L · min − 1 , mixing rate 400 rpm, media pH set to pH 5.5. Biomass concentration was determined as absorbance in 590 nm (WPA Colorimeter Colourwave CO7500, Biochrom, Cambridge, UK). The following coefficient to convert 27 absorbance units to dry weight was used: 1 OD 590 = 0.278 g · L − 1 . Carbon dioxide evolution was recorded by an exhaust gas analyzer (Infors Gas Analyser, InforsHT, Basel, Switzerland) in parallel with harvesting metabolite samples. The contents of extracellular glucose, ethanol, acetate, and glycerol were measured simultaneously by an Agilent 1100 HPLC system with a Shodex Asahipak SH1011 column, and they were quantified with a refractive index detector (RI detector RID G1362A). The flow rate of the mobile phase (0.01 N H2SO4 ) was 0.6 mL min −1 and the sample injection volume was 5 μL. Biomass from fermentations was centrifuged and intracellular nucleotide pools were extracted via cold methanol extraction. ATP, ADP, and AMP were quantified by HPLC-MS-TOF analysis, as described in (Valgepea et al., 2010). 2.6. FTIR Analysis For cell macromolecular content analysis, Fourier-transform infrared (FTIR) spectroscopy was used as described in (Grube et al., 2002). For this analysis, 2 mL of cells (OD600 1–4) was harvested by centrifugation and washed 3 times with distilled water. Cell pellets were diluted with 50 μL of distilled water, and samples were spotted on 96-well spot- plates. Absorbance data were recorded by a Vertex 70 device with HTS-XT microplate extender, interval 4000–600 cm − 1 , resolution 4 cm − 1. For data collection and control, OPUS/LAB 6.5 software was used. 2.7. Cell Carbohydrate Extraction and Quantification Fractional cell polysaccharide purification for quantitative assays was done as described in (Stewart, 1975). Total carbohydrate content of each fraction was determined by anthrone assay, and results were expressed as glucose equivalent mg · gDW − 1 biomass (Dubois et al., 1956). 2.8. Transcriptomics Total yeast RNA after 4-h cultivation in synthetic dextrose (SD) or SD media with adenine omitted (SD ade−) was isolated with a RiboPureTM RNA Purification Kit for yeast (Thermo Scientific, Waltham, MA, USA). RNA samples for each condition were harvested in triplicate. Cell pellets from 50 mL suspensions were frozen in liquid N2 and stored at − 80 ◦C. RNA samples were prepared using 3.0 mRNA-Seq Library Prep Kit (Lexogen, 28 Vienna, Austria) according to the manufacturer’s protocol. Yeast transcriptome was analysed using MiSeq (Illumina, San Diego, CA, USA) NGS data analysis. Sequencing reads were quality filtered (Q = 30), Illumina adapters and poly-A tails were removed,and reads at least 100 nt in length were selected for further processing using cutadapt. S288C reference genome from yeastgenome.org was used to identify gene transcripts. Genes with lower than 1 count per million (CPM) in fewer than 2 samples were filtered out. The Benjamini and Hochberg method was used to calculate multiple comparison adjusted p-value as false discovery rate (FDR). FDR < 0.001 with logFC > 2 was set as a threshold for significance. Expression data set were submitted to the European Nucleotide Archive (ENA) database, under accession no. PRJEB40525. 2.9. Sublethal Stresses Cells were grown in SD media until the exponential phase, washed with distilled water twice, and inoculated in SD or SD ade − with cell density of 1 · 107 cells · mL − 1 . After 4 h incubation, cells were harvested by centrifugation, washed with distilled water once, and aliquoted in 1 mL, with OD 600 = 1 (corresponding to 2 · 10 7 cells · mL − 1 ). Three aliquots were exposed to each stress. For thermal stress, cells were kept at 53 ◦ C for 10 min. For oxidative stress, cells were incubated in 10 mM H2O2 for 50 min, then washed with distilled water. For desiccation, cells were sedimented by centrifugation, the supernatant was removed, and the pellet was air-dried in the desiccator at 30 ◦ C for 6 h. After drying, distilled water was added to resuspend cells. After all stress treatments, treated cells were serially diluted, and dilutions were spotted on YPD plates to assess CFU · mL − 1 . To check for cell loss during washing steps, the OD of the suspension was measured and CFU · mL − 1 corrected for OD value. Survival is expressed as % assuming that OD 600 = 1 corresponds to 2 · 10 7 cells · mL − 1 . To test weak acid stress resistance, cells were spotted on YPD plates supplemented with 0.1 M acetic acid, with pH of agar media set to 4.5 (Martynova et al., 2016). 29 3. Results 3.1. Adenine auxotrophy – be aware: some effects of adenine auxotrophy in Saccharomyces cerevisiae strain W303-1A DOI: 10.1111/1567-1364.12154 Key points: - When cultivated in YPD media strains with ade auxotrophy exhaust adenine before glucose. - After adenine exhaustion apparent increase in optical density of the culture is due to the cell swelling. - Long term survival places adenine starvation between carbon and leucine starvations. - Purine starved cells gain desiccation tolerance Graphical abstract 30 31 32 33 34 35 36 37 38 39 40 41 Supplementary figures from article 42 43 44 45 3.2. Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast DOI: 10.3390/jof8010029 Key points: - Purine starved yeast cells arrest their cell cycle at G1/G0 phase in the first two hours of purine starvation. - During the first four hours of purine starvation cells accumulate reserve carbohydrates and reduce glucose flow, reorienting more towards production of glycerol and acetate. - Transcription analysis shows that 4h purine starved cells downregulate transcription and translation processes. - Purine starved cells acquire stress resistance that depends on translation and is higher than rapamycin elicited resistance. - Pattern of gene regulation of purine starved cells highly overlaps with cells entering stationary phase. - Transcription analysis indicates involvement of environmental stress response programm, that is signalled through Msn2p and Msn4p. Graphical abstract 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Figure 6 from article in higher resolution 65 Figure 7 from article in higher resolution 66 3.3. Adenine starvation is signalled through environmental stress response system in budding yeast Saccharomyces cerevisiae DOI: 10.22364/eeb.15.29 Key points: - When transcription factors Msn2p, Msn4p, Rim15p are truncated ( reduced functionality) purine starvation dependent desiccation tolerance is lessened - Rim15p truncation shows a more prominent response as Msn2p, Msn4p indicating that coordinated response via signalling pathways controlling cell proliferation via nutrient is in play. 67 68 69 70 71 72 73 3.4. Purine auxotrophy: Possible applications beyond genetic marker DOI: 10.1002/yea.3434 Key points: - Purine synthesis is evolutionary conserved. - In S. cerevisiae ribosomal nucleotides may serve as nucleotide reserves in case of lack of deoxyribonucleotides. - Purine auxotrophy is a common phenomenon among parasitic monera, protozoans, and metazoans. - Parasites rely on scavenging of purines from their environment thus making that a potential therapeutic target. - Auxotrophic yeast cells are suitable as models for such parasites. 74 75 76 77 78 79 80 81 82 4. Discussion 4.1. Care should be taken when using adenine auxotrophs in research In research published in Kokina et al. 2014 we used strain W303-1A. This strain carries ade2-1 point mutation that introduces stop codon prematurely. Strains of ade2 and ade1 mutants have been used in yeast research quite extensively due to their red pigmentation that develops late phases of growth when adenine is depleted from media. The accumulation or lack of pigment has been the basis not only for selection of the ade1 and ade2 mutants, but also has been employed in the research of mitotic and meiotic recombination (Johnston, 1971), amyloid aggregation (Bharathi et al., 2016) and petite identification and mtDNA research (Shadel, 1999). The red pigment itself seems to be composed of polymerised ribosylaminoimidazole molecules varying in molecular weight and containing a number of amino acids. (Smirnov et al., 1967). Later research has shown that accumulated intermediate aminoimidazolribotyl (AIR) form cytosol are glutationated and delivered in vacuole via GRX4 transporter, where it is polymerised and modified with amino acids giving rise to the red pigment that is autofluorescent and does not leave vacuole. (Smirnov et al., 1967; Fisher, 1969; Sharma et al., 2003, Jainarayanan et al., 2020). While red pigmentation is fairly noticeable if cells are grown on solid media, in liquid cultures it is less distinguishable. Red pigment gives autofluorescence to cells during imaging and hinders cell visualisation (Weisman et al., 1987), so usually care is taken to use adenine-enriched media when growing cells for visualisation studies. On the other hand, stress physiology research is quite often performed on adenine auxotrophs after an exponential growth phase in rich media, without any additional supplementation (Carrasco et al., 2001, Petrezselyova et al., 2010). Our research points to drastic changes in cell physiology after adenine depletion such as changes in stress resistance, macromolecular composition and optical properties of the cells. Care should be taken when using full media, as this can lead to biased conclusions of causes of the effects observed if that happens after adenine depletion sets in. 4.2. Internal purine resources are sufficient to finish the cell cycle If adenine is present in the media it will be transported into the cells with the help of Fcy2p transporter at fairly consistent speed. Chemostat measurements for growth rate 0.015 - 0.14 h-1 show adenine uptake rate 30-40 mg adenine per g dry cell weight (vanDusen et al., 1997). If adenine is provided in excess in the media, only a fourth of uptaken adenine is incorporated in nucleic acids as purine bases. The rest is used for nucleotides and nucleosides needed for cell metabolism and the excess can be converted into hypoxanthine and stored in vacuole (Burrige et al, 1977, Reichert Winter, 1974, Sharma et al., 2003, Jainarayanan et al., 2020). Our measurements also show that growth of W303-1A in synthetic media happens with 83 the same rate if adenine is present and only after exhaustion of adenine cell growth curves start to differ (Kokina et al., 2014, Fig4), confirming that exponentially growing cells should be the same, irrespective of adenine content in the media. Yeast extract peptone (YEP) media is the most common media used in cultivation of yeast. Nucleotides and corresponding bases in this media are provided only by yeast extract that is obtained by autolysis of yeast cells. Most of the nucleotides are coming from RNA degradation as it is the most abundant nucleotide source in cells. Zhang and colleagues (2003) have shown that adenine content in yeast extract varies batch by batch due to variability of the autolysis process. Addition of extra adenine did increase biomass yield of adenine auxotrophic strain irrespective of adenine content in the YE batch, proving adenine to be a limiting nutrient in yeast extract. Our research (Kokina et al., 2014) agrees with VanDusen et al. (1997) observation that adenine auxotrophs stop their growth before exhaustion of glucose if grown in YEPD. We also show that additional 100 mg/L adenine is the amount of adenine when growth of adenine auxotrophs in SD is not limited by adenine. Our research published in 2021 shows that most adenine auxotrophs arrest cell cycle in G1 phase already after 1,5-2 h of adenine exclusion from media showing that internal supplies of nucleotides are sufficient for finishing the nucleic acid synthesis for cells that were in the middle of cell cycle when adenine was excluded. 4.3. Are purine starved cells quiescent? In multicellular organisms cells are not dividing all the time. Also unicellular organisms have times when growth is limited. If such a non-dividing cell has lowered metabolic activity, increased stress resistance and is capable of resuming growth - these are called quiescent cells. Ability to stop the cell cycle is one of the prerequisites for the cell to become quiescent. When depleted of purines, purine auxotrophic cells arrest most of the cells within the first two hours of starvation as can be seen in Kokina et al. (2022) Fig 2a, allowing to speculate that adenine starved cells may be quiescent. When examining purine starved yeast cells many hallmarks of the quiescence are observed - not only cell cycle is stopped, but also we see accumulation of storage carbohydrates, decrease of cellular concentration of RNA and downregulation of ribosome biosynthesis. In figure 5 different aspects that had been reviewed in quiescent cells by Sun and Gresham 2020 are compared with our observations in adenine starved cells. 84 Figure 5. Comparison of fast growing cell and quiescence hallmarks from Sun and Gresham (2020) with properties of cells we observe in our research. NA - not assessed, data in a - Kokina et al., 2014, b - Kokina et al., 2022 Many quiescence hallmarks do correspond directly to our observations in purine starvation. Some others we can extrapolate from our data: for example glucose specific uptake rate and CO2 generation drops when cells are adenine starved, that would correspond to physiological parameter “slow down of metabolism”. While many hallmarks that we observe during 4 h starvation do correspond with quiescent cells, if we compare long term survival of purine starved cells with cells starved for leucine and carbon, then purine starvation would fall in the middle of natural and artificial starvations (Kokina et al., 2014, Fig6A), similar to the methionine starvation described by Petti et al. (2011). We see that adenine starved cells have lost half of their population by starvation day 1, which corresponds to the observations of Henry (1973). Chronological life span extension is characteristic for quiescent cells. Yeast strain used by us was not only adenine auxotroph but also histidine, leucine, uracil and tryptophan auxotroph, also strain used by Henry harboured several auxotrophies. It may be that these other auxotrophies play a role in long term starvation survival. To elucidate that pure adenine auxotroph long term survival without additional auxotrophies in genetic background would be needed. Most effects that we observed are pronounced already after 4h cultivation in adenine depleted media and are purely adenine starvation caused as other auxotrophic agents are still present (Kokina et al., 2022, Fig 1c). When starved for purines, yeast consumes less glucose and carbon is rerouted to acetate and glycerol (Kokina et al., 2022, Fig 3b). From biomass FTIR analysis we see an increase in lipid relative content (Kokina et al, 2022, Fig 4a). Glycerol in yeast can be used as osmoprotectant but is also important in lipid synthesis. Another purpose of glycerol synthesis is to act as a NADH sink if oxidation in the respiration chain is not available (Klein et al., 2017). Acetate production on other hand produces NADH thus the production of glycerol and acetate to balance redox cofactors is less probable. Acetate is used 85 in acetylCoA synthesis that is substrate for fatty acid synthesis. As we observe massive accumulation of trehalose and glycogen, it is possible that additionally carbon is stored in lipid form that causes an increase of glycerol and acetate synthesis. Lipid accumulation is also observed in nitrogen starvation if glucose is present (Albers et al., 2007). When comparing phenotype of purine starved cells with quiescent cells one observation clearly contradicts quiescent cell hallmarks. Our observations show an increase in the cell size of adenine starved cells. The increase in cell size was observed independent of genotype - W303 with ade2 (Kokina et al., 2014, Fig3 and S4) and CEN.PK background with ade8 mutations (Kokina et al., 2022, Fig2c). Cells continue to swell during starvation and will increase their cross section up to three times during 10 days of starvation (Kokina et al., 2014, S4). While we can not definitely say what is the reason for swelling, considering increase of storage carbohydrates in cells by approximately 20% of total dry weight during the first 4 h of starvation (Kokina et al., 2022, Fig 4b), one can speculate adenine starved cells accumulate storage molecules that leads to increase in cell size. Trehalose accumulation is observed during nitrogen starvation not only in S. cerevisiae (Klosinska et al, 2011) but also in fission yeast Schizosaccharomyces pombe (Sajiki et al., 2013). Trehalose and glycogen are storage carbohydrates, synthesis of which are regulated by several hierarchical systems namely TOR, PKA, Snf1p, Pho85p, and the energy sensor Pas kinase (François et al., 2012). It is known that depletion of sulphate, phosphate, or zinc is not accompanied by storage carbohydrate accumulation (François et al., 2012). As during the purine starvation researched by us there is abundant glucose in the media, we can assume that TOR signalling rather than PKA or Snf1 system is involved in regulation of observed increase of storage carbohydrates. 4.4. Purine starvation is similar but not quite the same as nitrogen starvation Starvation able to induce quiescence is considered a natural starvation. Some auxotrophic starvations fall in the same category as they mimic starvation for some macronutrient: methionine starvation mimics sulphur depletion (Petti et al., 2011). Our observations show that metabolically purine starved cells resemble nitrogen starved cells - accumulation of reserve carbohydrates, shifting carbon flow towards glycerol. We have also compared stress resistance of adenine auxotrophs on otherwise prototrophic background and purine starved cell stress resistance is very similar to the nitrogen starved cells (unpublished data). At the same time some differences to the nitrogen starvation can be observed - purine starved cells also produce considerable amounts of acetic acid, that is not observed in nitrogen starved cells (Albers et al., 2007). Nitrogen starved cells also arrest the cell cycle in G1 and finish the budding process for cells that were in other cell cycle phases, but newly formed daughter cells are smaller than mother cells (Johnston, 1977), purine starved cells do not show appearance of smaller cells (Kokina et al., 2014, Kokina et al., 2022). During nitrogen starvation a portion of the cytoplasm is non-selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, enzymatically cleaved until nucleosides and delivered into cytosol where they are 86 degraded further. After the start of the starvation nucleosides peak in the cytosol but they fall to initial or even below prestarvation levels within the first two hours of starvation. This is not observed in case of carbon or phosphate starvation or rapamycin induced growth arrest, where nucleosides stay in cytosol (Huang et al. 2015, Xu et al., 2013). Our results also show that purine starved purine auxotrophic cells will perform RNA degradation as the RNA amount in the cell decreases. We have not measured amounts of all nucleotides in cytosol, but the concentration of AMP, ADP and ATP has decreased. Interestingly decreasing amounts of ATP intracellular concentration is observed in phosphate starved cells, but not in nitrogen starved ones (Xu et al., 2013). Thus in the aspect of intracellular metabolite dynamics purine depletion resembles natural starvations - phosphate and nitrogen ones. 4.5. How purine starvation is perceived in cells? We propose that mechanisms additional to the Gcn4p response play a role. Possibly TOR mediated Rim15p governed response. Phenotypic similarity of the purine starvation to the natural starvations leads to the hypothesis that lack of purines is perceived and signalled in the cell. If cells are placed in adenine deficient media with cycloheximide - translation suppressor – present, stress resistance phenotype fails to develop. Also addition of cycloheximide after 2 h of starvation lessens resistance, proving that transcriptional activity after cell cycle arrest is crucial for stress resistance development (Kokina et al., 2022, Fig 5b). This leads to consideration of what kind of transcriptional programs are active in purine starved cells and what triggers these programs in action. While it is not known if the low levels of nucleotides would be signalled in the yeast cell per se, research on Arabidopsis shows that Rnr2 mutants with defective vacuolar rRNase undergo constitutive apoptosis, that can be rescued additional purines, but not pyrimidines in the cytoplasm. Other authors show that autophagy is induced via TOR signalling system (Kazibwe et al., 2020) This would indirectly suggest that cellular response to lowered cytoplasmic purine content is mediated via TOR. Similar research has not been performed on yeast cells, but mutant with overexpressed vacuolar RNase of the T(2) family RNY1, shows decreased oxidative stress resistance and chronological lifespan in stationary phase (MacIntosh et al, 2013), thus proposing that nucleotide balance plays a role in stress resistance also in yeast cells. PHO2, also known as BAS2 or GRF10, encodes a homeodomain transcriptional activator. Pho2p is required to express genes in several different pathways such as purine nucleotide biosynthesis, histidine biosynthesis, and phosphate utilisation. Genes that are known to be regulated by Pho2p include PHO5, PHO81, HIS4, CYC1, TRP4, HO, ADE1, ADE2, ADE5,7 and ADE8 (Liu et al., 2000, Daignan-Fornier, Fink, 1992). Pho2p activates transcription along with one of at least three distinct partner proteins: Swi5p, Pho4p, and Bas1p. Pho2p and Pho4p cooperatively bind to the promoter site of PHO5 (which encodes for a secreted acid phosphatase) and are required for PHO5 expression when cells are starved of phosphate (Bhoite et al., 2002). Pho2p and Swi5p together activate HO (Brazas, Stillman, 87 1993) while Pho2p and Bas1p activate genes in the purine and histidine biosynthesis pathways. Presence of AICAR promotes Pho2p and Pho4p interaction, whereas SAICAR - Pho2p and Bas1p, to upregulate ade de novo biosynthesis gene transcription (Pinson et al., 2009). While this shows interconnectedness of purine pathway regulation with phosphate pathway, in our case (ade2 and ade8 mutations) synthesis pathway is interrupted above synthesis of either - AICAR and SAICAR, thus, none of these interactions should be promoted and while there is some overlap in metabolomics with phosphate starvation it is not signalled on Pho2p level. Ability to stop proliferation and acquire stress resistance phenotype is connected to the chronological lifespan of the cell. Mechanisms influencing chronological lifespan frequently overlap with quiescence ensuring ones. Most interventions that extend lifespan are, or induce, limited amounts of stress that have a beneficial effect via the phenomenon of hormesis, because these stresses would be toxic or lethal at higher doses. These lifespan-extending hormetic stresses induce a protective cellular stress response. This conserved stress response in yeast was termed the General Amino Acid Control (GAAC) because it was initially identified as a response to amino acid depletion that upregulates the genes required for amino acid synthesis (Hinnebusch, Fink, 1983). The scheme of the GAAC can be seen in figure 6. This pathway is also induced by a variety of conditions including starvation for purines (Rolfes, Hinnebusch, 1993) and is now frequently called Integrated Stress Response especially in mammalian cell research. When analysing transcriptome and phenotype of purine starved cells we also see that starved cells become stress resistant for variety of stressors (Kokina et al., 2002, Fig 5a) and Gcn4p responsive genes are upregulated - for example, autophagy connected (ATG33, ATG34, ATG8 and others) for full list see Kokina et al., 2022, S2 and S3. In total around 70% of significantly upregulated or downregulated genes in our data set are connected to regulation by Gcn4p. 88 Figure 6. Model of Gcn4 expression regulation adapted from Postnikoff et al., 2017. In rich media conditions TOR suppresses Gcn2p and translation initiation factor eIF2 is able to mediate aminoacylation of 40S ribosome subunit. Several small inhibitory microORFs are located before the Gcn4 gene, thus the ribosome is dissociated before Gcn4 gene translation. In amino acid deplete conditions unloaded tRNA accumulation stimulates Gnc2 preventing functioning of eIF2. Due to leaky scanning of mRNA, Gcn4 protein is translated and induces transcription of a variety of genes. Research by Rolfes and Hinnebusch (1993) shows that Gcn1p, Gcn2p, Gcn3p (alpha subunit of eIF2b) and Gcn4p are required for response to purine starvation, proposing that purine starvation is sensed with the same mechanisms as amino acid starvation. In severe purine starvation Gcn4 system is responding not only on translational, but also transcriptional level. Researchers note that purine starvation is sensed also in presence of all amino acids. Amino acid starvation is sensed by uncharged tRNAs. When examining gcn2 mutants with point mutations researchers identified that response to the purines involve the same domains of Gcn2p as amino acid starvation. As for the aminoacylation of tRNAs ATP is needed and we know that during purine starvation absolute ATP concentration decreases, it may be that this causes accumulation of uncharged tRNAs that are sensed by Gcn2p. To elucidate this, additional research on the amount of tRNA and their charge during purine starvation would be needed. tRNAs have been shown to have additional roles except delivery of amino acids to the ribosomes. TOR complex is directly regulated by presence of tRNA (Kamada, 2017). Nutrient starvation induces transport of tRNA to the nucleus (Whitney et al., 2007), thus there is possible overlay of tRNA control over cell signalling systems besides Gcn2p mediated GAAC that may be involved also in purine starvation perception. 89 Rolfes and Hinnebusch (1993) also note that expression of purine de novo synthesis genes is regulated by additional mechanisms, as Gcn4p mediated response does not fully explain transcriptional answer during purine starvation. Our data (Ozoliņa et al., 2017) shows that other transcription factors, especially Rim15p plays a role in purine starvation phenotype development. Rim15p also have been shown to be the hub connecting various starvations to the quiescence phenomenon (Sun et at., 2020), as purine starved cells do show several quiescent cell hallmarks, Rim15p mediated response could be responsible for at least a part of purine starved cell phenotype. Transcription analysis of our strain when starved for purines shows a high number of genes that are up or downregulated compared to the fast growing cells (Kokina et al., 2022). When performing analysis with these genes in YEASTRACT to identify possible transcription factors that would cause this expression pattern Gcn4p does correspond to roughly 70% of our upregulated and downregulated genes, but is deemed as statistically insignificant (Kokina et al., 2022, S2) as opposed to Msn2p and Msn4p, that does explain roughly the same amount of genes, but are shown as statistically significant (p<0.05) possible regulators of the dataset. This also strengthens our hypothesis that Gcn4p driven response is not the only factor responsible for purine starved phenotype development. When analysing possible transcription factors governing transcriptional response during purine starvation, a variety of transcriptional factors are proposed as statistically significant. Highest coverage of all significantly regulated genes is shown by transcription factors Msn2p and Msn4p that are shown to upregulate 75% and 68% of upregulated genes in our dataset. Msn2p and Msn4p mediate the so-called environmental stress response (ESR) (Gasch, Verner-Washburne, 2002). ESR is also considered a hormetic response. When we compare genes regulated by these transcription factors, we can see that while there is an overlap each of these transcription factors do regulate different gene sets (Figure 7). For example, Msn2p and Msn4p regulates trehalose accumulation process, that is not regulated by Gcn4p 90 Figure 7. Comparison of purine starvation upregulated genes (logFC>2) proposed to be regulated by Msn2p, Msn4p and Gcn4p by YEASTRACT. Both Msn2p and Msn4p are regulated by Rim15p (Orzechowski Westholm et al., 2012), that corresponds to our observations of significance of Rim15p in purine starvation phenotype (Ozoliņa, 2017), Rim15p also induces Gis1p, that would explain 40% of our upregulated genes. Suppression of Gis1p has been shown to produce higher glycerol and acetate yields (Orzechowski Westholm et al., 2012). Rim15p transport in the nucleus depends on PKA and TOR signalling systems. Partial TOR involvement in purine starvation phenotype is also confirmed by rapamycin treatment. If fast growing cells are treated with rapamycin, stress resistance grows, but does not reach purine starvation levels. On the other hand, purine starved cells do not increase stress resistance after rapamycin treatment (Kokina et al., 2022). This would confirm that the TOR system is involved, but not the only one, governing purine starvation phenotype. A link between TOR and GAAC has been demonstrated in S. cerevisiae. TOR prevents dephosphorylation of Gcn2p by inhibiting one or more phosphatases. Phosphorylated Gcn2p will have lower ability to bind uncharged tRNA, thus a suppression of TOR system is needed for pronounced GAAC response (Cherkasova, Hinnebusch, 2003; Kubota et al., 2003). Our experiments show that stress resistance when cells are incubated in ade- environment with rapamycin added in the very beginning of starvation is lower than cells that have spent 2 h in environment without purines and then incubated for two more hour in presence of rapamycin (Kokina et al., 2022, Fig 5b) thus the interplay between TOR and GAAC in purine starvation is more complex than TOR repression allowing GAAC to take place. At the same time several other transcription factors are highlighted by YEASTRACT - Fhl1p, Rme1p, Cat8p, that explain smaller amounts of upregulated genes (56%, 18%, 7%) are involved in stress response that is connected to the DNA replication stress. There is evidence that purine starvation slows down replication forks in purine auxotrophic chinese hamster cell line (Zannis-Hadjopoulos, 1979). We know that purine starved cells finish their cell cycle even if purine deprivation happens in the middle of cell cycle. While we can assume that during 91 purine starvation there will be purine shortage for DNA building process that may cause replication stress, it is not the main reason for purine starvation phenotype judging from the gene expression data. 4.6. Transcription of purine starved cells does not agree with the observed metabolome While we see strong phenotypic response to the purine starvation on transcriptional level there are several conflicting pathways with observed changes - upregulation of glycolysis and citric acid cycle enzymes while glucose flow is diminished. Upregulation of trehalose and glycogen cleaving enzymes while we see accumulation of trehalose and glycogen. Upregulation of some glycolysis and gluconeogenesis, fatty acid synthesis and degradation enzymes at the same time. We have also seen that translation is crucial for stress resistance phenotype development in purine starvation (Kokina et al., 2022). This leads to the thought that while there may be active transcriptional response to the purine starvation, endeffect of the purine starvation phenotype on the metabolic level is governed mostly by posttranscriptional regulation. Flux through the metabolic pathways is governed not only by the amount of enzymes but also availability of the substrate and activity of the enzymes. Activity of the enzymes can be influenced by posttranslational modifications, of which phosphorylation is the most common one (Oliveira, Sauer, 2012). For example key enzymes governing equilibrium between glycogen and trehalose accumulation or cleavage are regulated by phosphorylation. Glycogen synthase (Gsy2p) is more active in a non phosphorylated state, while glycogen phosphorylase is active in its phosphorylated state. Phosphorylation of Gsy2p is governed by cyclin dependent regulators, most notably Pho85. Similarly, the trehalose cleavage enzyme neutral trehalase Nth1p is active when phosphorylated by the PKA system (François et al., 2012). While ATP concentration in purine starved cells falls to 0.3 of initial amount (Kokina et al., 2022) it is unlikely that lack of ATP per se is the cause of changes in protein phosphorylation levels, as the common cofactors such as NAD, ATP are usually found in concentrations that are one or two fold higher that Km of the enzymes (Nelson, Cox, 2017). Pinson et al. (2019) shows that the amount of NAD+ in the cell depends on the amounts of ATP, thus, we may assume that in purine starved cells NAD+ may be in lower concentration. Database (http://growthrate.princeton.edu/metabolome), that was created from chemostat data produced by (Boer et al., 2010) allows to assess cellular concentrations of various intracellular metabolites during growth limited by C, N, P or uracil or leucine. Of the available metabolites I selected all containing adenine - AcetylCoA, adenosine, ADP, ATP, SAM, NADP+, NAD+, FAD, cAMP - ATP, ADP and NAD+ are deemed as growth limiting in phosphate limitations, whereas adenosine shows overflow metabolism in phosphate limitation and NAD+ overflows in C limitation. Rest of metabolites are not shown as growth limiting or showing overflow metabolism in any of the mentioned limitations. It must be kept in mind that in chemostats cells are still growing albeit at different speeds - a situation that is different from starvation, where no growth would be observed. 92 Methionine starvation is shown to influence mTOR via the concentration of SAM in mammalian cells (Gu et al., 2017). In yeast, methionine levels are monitored and specific Met4p acts as a transcription factor that is activated during lack of methionine. But also in the yeast cells methionine influences TOR activity via SAM concentration and Ppa2p methylation. In low SAM concentrations TORC1 is inactive, tRNAs are not thiolated, growth stops and autophagy is promoted (Lauinger, Keiser, 2021). As for synthesis of SAM adenine is needed and we see similarities in purine starvation and methionine starvation, intracellular SAM concentration measurements during purine starvation would be a valuable tool to assess the impact of this TOR system branch on yeast cells. Data from chemostats indicate that SAM concentrations rise in cells as growth rate increases in C, N and P limitations and overall SAM concentration is higher in P limited cells, but SAM concentration and growth speed shows negative correlation in leucine and uracil limitations. It must be also considered that we see downregulation of SAM synthesis (MET6, SAM1) and THF metabolism connected (MIS1, MET13, ADE3) genes in purine starved cells (Kokina et al., 2022, Sup3). If methylation processes are affected, we could also see a regulation of posttranslational modifications on this level. 4.7. What is the place of purine starvation in evolutionary landscape The most widely accepted model of the origin of life states that organic molecules arose via simple chemical reactions. The Miller-Urey experiment is one of the most famous examples of such a process. It has been shown that purine nucleotides also could form under conditions likely present on primitive Earth (Oro, 1961, Nam et al., 2018). ATP - the main energy carrier in the cell is also shown to be able to form in prebiotic conditions, and accept phosphoryl group more readily as other nucleotide diphosphates (Pinna et al., 2022). As purines form DNA and RNA required for maintenance and expression of genetic information and also are included in a variety of cofactors purine synthesis are under strong positive selective pressure. Purine synthesis pathway is conserved across all domains of life. While enzymes and their cofactors may differ in eukaryotes, prokaryotes and archaea, the intermediates of the pathway will always be the same with the exception of N5-CAIR (N5-carboxyaminoimidazole ribonucleotide), which is bypassed in eukaryotes (Chua, Fraser, 2020). Notable exceptions are intracellular parasites, especially parasitic protozoans, who have lost their de novo synthesis pathway completely. Parasitic protozoa are an evolutionarily divergent group of unicellular eukaryotes that are responsible for a wide range of human and veterinary diseases. The most clinically relevant protozoan parasites are the apicomplexans Plasmodium spp. and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively) and the trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., which cause African sleeping sickness, Chagas’ disease, and leishmaniasis, respectively (Gazanion, Verges, 2018). These parasites are usually intracellular parasites and during the evolution have obtained various auxotrophies thus they must rely on a host for these metabolites. Although many of these parasites have complicated life cycles with two or more hosts, loss of the purine de novo pathway has been evolutionarily advantageous for them. Loss of biochemical synthesis 93 pathways (asparagine, phenylalanine, biotine and others) in the nutrient rich environment has been shown with E. coli (D’Souza et al., 2016, D’Souza, Kost, 2014). This loss leads to the fitness increase in the new evolved strains. Overall, in long term evolutionary laboratory experiments with fixed media, an increase of fitness with the cost of adaptability has been observed (Couce, Tenaillon, 2015). Interestingly, genetic analysis points to the auxotrophic lifestyle may have evolved before these protozoans became parasites. For example Bodo saltans - free living relative of Trypanosoma and Leishmania is already a purine auxotroph (Jackson et al., 2016, Januskovec, Keeling, 2016). Most of parasitic protozoans have lost purine de novo biosynthesis genes and rely on productive salvage reactions for their intracellular purine pools, interestingly, pyrimidine auxotrophs are less common (Gazanion, Verges, 2018). Purine auxotrophy was also more prominently found in the genetic screen for auxotrophies in Gram negative bacterias (Seif et al., 2020). Frequently loss of function is driven by higher fitness of the resulting strain, while it is known that amino acid auxotrophies identified by the Seif et al., are connected with host– pathogen interactions, suggesting that these auxotrophies may give selective advantage during host–pathogen interactions, the reason why purine auxotrophies are common is not yet well established. Our data and data from Leishmania research (Carter et al., 2010) point to gain of stress resistance phenotype in the absence of purines. Gcn4p is proven to react to purine limitation in yeast (Rolfes, Hinnebusch, 1993) and plants (Lageix, et al., 2008). In bacteria nutrient limitation or starvation induces the stringent response. Stringent response depends on a transient increase in the level of (p)ppGpp that causes reduced accumulation of stable RNAs (rRNA and tRNA), transcriptional downregulation of genes linked to growth (e.g., ribosome biogenesis) and upregulation of genes required for survival (e.g., nutrient acquisition and stress responses), and it also directly binds a number of proteins to regulate their activity. While specific binding targets may differ between Gram-negative and Gram-positive species, (p)ppGpp has been shown to regulate replication, transcription, translation and GTP biosynthesis by binding to proteins that participate in these processes, including DNA primase, RNA polymerase, small GTPases, enzymes involved in purine biosynthesis, and transcriptional regulators. Considerable differences exist between bacterial species with regard to both the mode of action and metabolism of (p)ppGpp. In Gram-negative species such as E. coli, ribosomes sense the uncharged tRNAs at the ribosomal A site during amino acid starvation, causing protein synthesis to stall. This leads to synthesis of (p)ppGpp, which acts as an allosteric regulator of RNA polymerase (Sivapragasam, Grove, 2019). Similarities in stringent response and Gcn4 mediated GAAC point to a widespread purine sensing mechanism that is observed in various lineages of life. 94 5. Conclusions and direction of following research - Yeast extract may be insufficient purine source for adenine auxotrophic strains and adenine is exhausted before glucose. - After adenine exhaustion apparent increase in optical density of the culture is due to the cell swelling. - Long term survival places adenine starvation between carbon and leucine starvations. - Purine starved yeast cells arrest their cell cycle at G1/G0 phase in the first two hours of purine starvation. - Purine starved yeast cells increase thermal, oxidative, weak acid and desiccation stress resistance by several orders of magnitude compared to fast growing cells. - During the first four hours of purine starvation cells accumulate reserve carbohydrates and reduce glucose flow, reorienting part of carbon flux towards production of glycerol and acetate. - Transcription analysis shows that 4 h purine starved cells downregulate transcription and translation processes similarly to other environmental stresses. - Purine starved cells acquire stress resistance that depends on translation and is higher than rapamycin elicited resistance indicating additional cell signalling sistem activity. - Pattern of gene regulation of purine starved cells highly overlaps with cells entering stationary phase. - Transcription analysis indicates involvement of environmental stress response programm, that is signalled through Msn2p and Msn4p via Rim15p. - Purine auxotrophic yeast cells are suitable as models for intracellular parasites as purine auxotrophy is a common phenomenon among parasitic monera, protozoans and metazoans. 95 After agglomeration of the results and the knowledge from literature following model of development of purine starvation phenotype can be made Fig 8 Figure 8. Summary of main characteristics of observed purine depletion phenotype and possible causes and signalling events leading to the phenotype discussed in the thesis. Achieved results allows us to describe purine starved cell yeast cell phenotype that has not yet been described before. Results show that care should be taken if working with adenine auxotrophs to avoid purine depletion as that results in a pronounced stress resistance phenotype. While we have described the phenotype and some hypothesis on the signal for this phenotype are being made, to elucidate mechanism of cell signalling of purine starvation additional experiments are needed. Firstly a long term starvation without additional auxotrophies in strain background would allow us to better describe chronological life span of adenine synthesis mutants during purine starvation. To better elucidate changes happening in cells during purine starvation tRNA dynamics and loading should be assessed during purine starvation. This would better describe the involvement of GAAC. For TOR system involvement, mutants defective in different parts of the TOR signalling system may be used to understand which parts are involved in purine depletion response. To help with understanding if some intracellular adenosine metabolite such as SAM, cAMP is influencing phenotype development, the amount of all adenine containing metabolites could be measured. If some of these metabolites are indeed in significantly smaller amounts, it would be worthwhile to see if the changes in the metabolites are the cause or consequence of the purine starvation phenotype. 96 6.Theses for defence Traditional compositions of rich media may be purine limiting for adenine auxotroph cells. Internal purine reserves are sufficient for yeast cells to finish DNA synthesis after onset of purine starvation. Yeast cells effectively react to the purine depletion by reorienting metabolism and arrest cell cycle via transcriptional response that is at least partially coordinated via Rim15p. Purine starved cells exhibit quiescence like phenotype. Purine auxotrophic starvation might be beneficial to parasitic organisms as preconditioning for other environmental stressors. 97 Approbation of the research Conferences: Poster presentation Ozoliņa Z., Zīle A., Kokina A., Pleiko K., Kristjuhan A., Liepiņš J. Mutation location in the eukaryotic purine synthesis pathway determines response to nitrogen or purine starvation. "Levures, Modèles et Outils" meeting (LMO14), Strasbourg, France, 27. - 29.10.2021. Poster presentation Ozoliņa Z., Zīle A., Kokina A., Pleiko K., Liepiņš J. Purine deprivation resembles nitrogen starvation in budding yeast. ICY15 meets ICYGMB30 congress, Vienna, virtual, 23.-27.08.2021 Oral presentation Agnese Kokina, Kārlis Pleiko, Zane Ozoliņa, Jānis Liepiņš Purīna trūkuma izraisītā globālā transkriptomikas atbilde maizes raugā. Global transcriptomic response to purine starvation in budding yeast. LU konference, Latvija, 19.02.2021 Poster presentation Kokina A., Pleiko K., Ozoliņa Z. and Liepiņš J. Role of mutations in purine de novo synthesis pathway in cell cycle arrest and stress resistance phenotype during purine starvation. ICYGMB 2019, Gothenburg, Sweden,18.08.22.08.2019. Poster presentation Ozoliņa Z., Kokina A., Martynova J., Liepins J. 2017. How purine starvation is communicated inside the baker’s yeast cell. 28th International Conference on Yeast Genetics and Molecular Biology (ICYGMB 2017), Prague, Czech Republic, 27.08. - 1.09.2017. Oral presentation A.Kokina; I. Vamža, D. Lubenets, J. Liepins. General stress resistance phenotype is no cell cycle dependent in budding yeast Saccharomyces cerevisiae, 3rd Congress of Baltic Microbiologists 18-21.10. 2016 Vilnius, Lithuania Oral presentation Liepins J, Kokina A, Nutritional effects on baker’s yeast desiccation tolerance, 2nd Congress of Baltic Microbiologists 16.-18.10. 2014, Tartu, Estonia Poster presentation A. Kokina, J. Kibilds, J. Liepins “Adenine auxotrophy effects on general and stress physiology in bakers yeast S. cerevisiae”, Yeast 2013 26th International Conference on Yeast Genetics and Molecular Biology, Frankfurt am Main, German, September, 2013. Oral presentation A. Kokina; J. Liepins “Trehalose metabolism in yeast S. cerevisiae - effects of common auxotrophies” 1st Congress of Baltic Microbiologists, Riga, Latvia, November 2012. Poster presentation A. Kokina; J. Liepins “Role of ade8 mutation in stress resistance of yeast Saccharomyces cerevisiae CEN.PK strain background” 22nd IUBMB and 37th FEBS congress, Spain, Sevilla, September, 2012 98 Acknowledgments and funding The results presented in this thesis have been obtained in four research projects: ESF funding ESF 2009/0207/1DP/1.1.1.2.0/09/APIA/VIAA/128 Establishment of a Latvian InterUniversity Research Group in Systems Biology, ESF 8.2.2.0/20/I/006 Strengthening the PhD Capacity of the Faculty of Science in the Framework of the New PhD Model Funding from the Latvian Research Council LZP-2018/2-0213 - Purine auxotrophy - a new superpower?, LZP-2021/1-0522 - From purine starvation to a stress-tolerant phenotype - uncovering the mechanisms. I would also like to thank my supervisors Dr. biol. prof. Uldis Kalnenieks and Dr. biol Jānis Liepiņš for their support and valuable discussions, which contributed to new perspectives on the results. The work would not have been possible without the help of my colleagues Kārlis Pleiko, Kārlis Švirksts, Rita Ščerbaka, Jekaterina Martinova, Kristel Tanilas, Nina Gaļiņina and Kristaps Jaundzems as they helped with learning the methods. I am grateful for the involvement of students Zane Ozoliņa, Jura Ķibilds, Ilze Vamža, Katrīna Daila Neiburga, Reinis Vangravs, Aleksandrs Fjodorovs that asked questions and were generally inquisitive as good students are supposed to be. 99 References Agmon, N., Temple, J., Tang, Z., Schraink, T., Baron, M., Chen, J., Mita P., Martin J.A., Tu B.P., Yanai I., Fenyö D., & Boeke, J. D. (2020). Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast. Nucleic acids research, 48(1), 486-49. https://doi.org/10.1093/nar/gkz1098 Albers, E., Larsson, C., Andlid, T., Walsh, M. C., & Gustafsson, L. (2007). Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae. 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