Whole-Genome Duplication and Yeast's Fruitful Way of Life.

Studies on the fate of Saccharomyces cerevisiae paralogous gene pairs that arose through a whole-genome duplication event have shown diversification of retained duplicated genes. Paralogous functional specialization often results in improved function and/or novel function that could contribute to the adaptation of the organism to a new lifestyle. Here, we analyze and discuss particular case studies of paralogous functional diversification that could have played a role in the acquisition of yeast fermentative metabolism.

Synthetic negative genome screen of the GPN-loop GTPase NPA3 in Saccharomyces cerevisiae.

The GPN-loop GTPase Npa3 is encoded by an essential gene in the yeast Saccharomyces cerevisiae. Npa3 plays a critical role in the assembly and nuclear accumulation of RNA polymerase II (RNAPII), a function that may explain its essentiality. Genetic interactions describe the extent to which a mutation in a particular gene affects a specific phenotype when co-occurring with an alteration in a second gene. Discovering synthetic negative genetic interactions has long been used as a tool to delineate the functional relatedness between pairs of genes participating in common or compensatory biological pathways. Previously, our group showed that nuclear targeting and transcriptional activity of RNAPII were unaffected in cells expressing exclusively a C-terminal truncated mutant version of Npa3 (npa3∆C) lacking the last 106 residues naturally absent from the single GPN protein in Archaea, but universally conserved in all Npa3 orthologs of eukaryotes. To gain insight into novel cellular functions for Npa3, we performed here a genome-wide Synthetic Genetic Array (SGA) study coupled to bulk fluorescence monitoring to identify negative genetic interactions of NPA3 by crossing an npa3∆C strain with a 4,389 nonessential gene-deletion collection. This genetic screen revealed previously unknown synthetic negative interactions between NPA3 and 15 genes. Our results revealed that the Npa3 C-terminal tail extension regulates the participation of this essential GTPase in previously unknown biological processes related to mitochondrial homeostasis and ribosome biogenesis.

Two alpha isopropylmalate synthase isozymes with similar kinetic properties are extant in the yeast Lachancea kluyveri.

The first committed step in the leucine biosynthetic pathway is catalyzed by α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), which in the Saccaromycotina subphylum of Ascomycete yeasts is frequently encoded by duplicated genes. Following a gene duplication event, the two copies may be preserved presumably because the encoded proteins diverge in either functional properties and/or cellular localization. The genome of the petite-negative budding yeast Lachancea kluyveri includes two SAKL0E10472 (LkLEU4) and SAKL0F05170 g (LkLEU4BIS) paralogous genes, which are homologous to other yeast α-IPMS sequences. Here, we investigate whether these paralogous genes encode functional α-IPMS isozymes and whether their functions have diverged. Molecular phylogeny suggested that the LkLeu4 isozyme is located in the mitochondria and LkLeu4BIS in the cytosol. Comparison of growth rates, leucine intracellular pools and mRNA levels, indicate that the LkLeu4 isozyme is the predominant α-IPMS enzyme during growth on glucose as carbon source. Determination of the kinetic parameters indicates that the isozymes have similar affinities for the substrates and for the feedback inhibitor leucine. Thus, the diversification of the physiological roles of the genes LkLEU4 and LkLEU4BIS involves preferential transcription of the LkLEU4 gene during growth on glucose and different subcellular localization, although ligand interactions have not diverged.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Three, two, one yeast fatty acid desaturases: regulation and function.

Fatty acid composition of biological membranes functionally adapts to environmental conditions by changing its composition through the activity of lipid biosynthetic enzymes, including the fatty acid desaturases. Three major desaturases are present in yeasts, responsible for the generation of double bonds in position C9-C10, C12-C13 and C15-C16 of the carbon backbone. In this review, we will report data addressed to define the functional role of basidiomycete and ascomycete yeast desaturase enzymes in response to various external signals and the regulation of the expression of their corresponding genes. Many yeast species have the complete set of three desaturases; however, only the Δ9 desaturase seems to be necessary and sufficient to ensure yeast viability. The evolutionary issue of this observation will be discussed.

Gln3 is a main regulator of nitrogen assimilation in Candida glabrata.

After Candida albicans, the yeast Candida glabrata ranks second as an aetiological agent of candidaemia and is the most frequently encountered non-Candida albicans species in patients with invasive candidiasis. Transcriptome analysis in C. albicans, C. glabrata and Cryptoccocus neoformans has revealed that, when engulfed by macrophages, these yeasts upregulate genes involved in nutrient acquisition, including nitrogen transporters such as the general amino acid permease Gap1, the dicarboxylic amino acid permease Dip5, the basic amino acid permease Can1 and the ammonium permeases Mep1 and Mep2. Nitrogen assimilation has been well studied in model species of fungi, such as Aspergillus nidulans, Neurospora crassa and Saccharomyces cerevisiae. However, little is known about nitrogen assimilation in C. glabrata. In the present study, we report a major role for Gln3 in the assimilation of glutamine, ammonium and proline. Ure2 also has a role in nitrogen assimilation, but it is only observable in ammonium and glutamine. In addition, Gat1 has a minor role, which is only observable in the absence of Ure2 and Gln3. Gln3 is absolutely necessary for full ammonium uptake from media. We have also shown that MEP2 gene expression in C. glabrata is completely dependent on Gln3, whereas GAP1 regulation is mainly exerted by Gln3, with the exception of proline where Gat1 has a minor role. In addition, in C. glabrata Ure2 appears to be a negative regulator of these NCR-sensitive genes, similarly to what has been described in S. cerevisiae. Our data place Gln3 as a key regulator of nitrogen assimilation.

Functional roles of a predicted branched chain aminotransferase encoded by the LkBAT1 gene of the yeast Lachancea kluyveri.

Branched chain amino acid aminotransferases (BCATs) catalyze the last step of the biosynthesis and the first step of the catabolism of branched chain amino acids. In Saccharomyces cerevisiae, BCATs are encoded by the ScBAT1 and ScBAT2 paralogous genes. Analysis of Lachancea kluyveri genome sequence, allowed the identification of the LkBAT1 locus, which could presumably encode a BCAT. A second unlinked locus (LkBAT1bis), exhibiting sequence similarity to LkBAT1 was also identified. To determine the function of these putative BCATs, L. kluyveri mutant strains lacking LkBAT1, LkBAT1bis or both genes were generated and tested for VIL metabolism. LkBat1 displayed branched chain aminotransferase activity and is required for VIL biosynthesis and catabolism. However, Lkbat1Δ mutant is a valine and isoleucine auxotroph and a leucine bradytroph indicating that L. kluyveri harbors an alternative enzyme(s) involved in leucine biosynthesis. Additionally, heterologous reciprocal gene complementation between S. cerevisiae and L. kluyveri orthologous LkBAT1, ScBAT1 and ScBAT2 genes, confirmed that the mitochondrial LkBat1 functions as BCAT in S. cerevisiae, restoring wild type phenotype to the ScBAT1 null mutant. Conversely, LkBAT1bis did not display a role in BCAAs metabolism. However, when ethanol was used as carbon source, deletion of LkBAT1bis in an Lkbat1Δ null strain resulted in an extended 'lag' growth phase, pointing to a potential function of LkBAT1 and LkBAT1bis in the aerobic metabolism of L. kluyveri. These results confirm the BCAT function of LkBAT1 in L. kluyveri, and further support the proposition that the BCAT function in ancestral-type yeasts has been distributed in the two paralogous genes present in S. cerevisiae.

Translation initiation factor eIF1A rescues hygromycin B sensitivity caused by deleting the carboxy-terminal tail in the GPN-loop GTPase Npa3.

The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.

Neo-functionalization in Saccharomyces cerevisiae: a novel Nrg1-Rtg3 chimeric transcriptional modulator is essential to maintain mitochondrial DNA integrity.

In Saccharomyces cerevisiae, the transcriptional repressor Nrg1 (Negative Regulator of Glucose-repressed genes) and the ß-Zip transcription factor Rtg3 (ReTroGrade regulation) mediate glucose repression and signalling from the mitochondria to the nucleus, respectively. Here, we show a novel function of these two proteins, in which alanine promotes the formation of a chimeric Nrg1/Rtg3 regulator that represses the ALT2 gene (encoding an alanine transaminase paralog of unknown function). An NRG1/NRG2 paralogous pair, resulting from a post-wide genome small-scale duplication event, is present in the Saccharomyces genus. Neo-functionalization of only one paralog resulted in the ability of Nrg1 to interact with Rtg3. Both nrg1Δ and rtg3Δ single mutant strains were unable to use ethanol and showed a typical petite (small) phenotype on glucose. Neither of the wild-type genes complemented the petite phenotype, suggesting irreversible mitochondrial DNA damage in these mutants. Neither nrg1Δ nor rtg3Δ mutant strains expressed genes encoded by any of the five polycistronic units transcribed from mitochondrial DNA in S. cerevisiae. This, and the direct measurement of the mitochondrial DNA gene complement, confirmed that irreversible damage of the mitochondrial DNA occurred in both mutant strains, which is consistent with the essential role of the chimeric Nrg1/Rtg3 regulator in mitochondrial DNA maintenance.

Long-insert clone experimental evidence for assembly improvement and chimeric chromosomes detection in an allopentaploid beer yeast.

Lager beer is made with the hybrid Saccharomyces pastorianus. Many publicly available S. pastorianus genome assemblies are highly fragmented due to the difficulties of assembling hybrid genomes, such as the presence of homeologous chromosomes from both parental types, and translocations between them. To improve the assembly of a previously sequenced lager yeast hybrid Saccharomyces sp. 790 and elucidate its genome structure, we proposed the use of alternative experimental evidence. We determined the phylogenetic position of Saccharomyces sp. 790 and established it as S. pastorianus 790. Then, we obtained from this yeast a bacterial artificial chromosome (BAC) genomic library with its BAC-end sequences (BESs). To analyze these data, we developed a pipeline (applicable to other assemblies) that classifies BES pairs alignments according to their orientation. For the case of S. pastorianus 790, paired-end BESs alignments validated parts of the assembly and unpaired-end ones suggested contig joins or misassemblies. Importantly, the BACs library was preserved and used for verification experiments. Unpaired-end alignments were used to upgrade the previous assembly and provided an improved detection of translocations. With this, we proposed a genome structure of S. pastorianus 790, which was similar to that of other lager yeasts; however, when we estimated chromosome copy number and experimentally measured its genome size, we discovered that one key difference is the outstanding S. pastorianus 790 ploidy level (allopentaploid). Altogether, our results show the value of combining bioinformatic analyses with experimental data such as long-insert clone information to improve a short-read assembly of a hybrid genome.

Alanine Represses γ-Aminobutyric Acid Utilization and Induces Alanine Transaminase Required for Mitochondrial Function in Saccharomyces cerevisiae.

The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.

Transcriptome Analysis Unveils Gln3 Role in Amino Acids Assimilation and Fluconazole Resistance in Candida glabrata.

After Candida albicans, Candida glabrata is one of the most common fungal species associated with candidemia in nosocomial infections. Rapid acquisition of nutrients from the host is important for the survival of pathogens which possess the metabolic flexibility to assimilate different carbon and nitrogen compounds. In Saccharomyces cerevisiae, nitrogen assimilation is controlled through a mechanism known as Nitrogen Catabolite Repression (NCR). NCR is coordinated by the action of four GATA factors; two positive regulators, Gat1 and Gln3, and two negative regulators, Gzf3 and Dal80. A mechanism in C. glabrata similar to NCR in S. cerevisiae has not been broadly studied. We previously showed that in C. glabrata, Gln3, and not Gat1, has a major role in nitrogen assimilation as opposed to what has been observed in S. cerevisiae in which both factors regulate NCR-sensitive genes. Here, we expand the knowledge about the role of Gln3 from C. glabrata through the transcriptional analysis of BG14 and gln3Δ strains. Approximately, 53.5% of the detected genes were differentially expressed (DEG). From these DEG, amino acid metabolism and ABC transporters were two of the most enriched KEGG categories in our analysis (Up-DEG and Down-DEG, respectively). Furthermore, a positive role of Gln3 in AAA assimilation was described, as was its role in the transcriptional regulation of ARO8. Finally, an unexpected negative role of Gln3 in the gene regulation of ABC transporters CDR1 and CDR2 and its associated transcriptional regulator PDR1 was found. This observation was confirmed by a decreased susceptibility of the gln3Δ strain to fluconazole.

DNA-binding specificity determinants of replication proteins encoded by eukaryotic ssDNA viruses are adjacent to widely separated RCR conserved motifs.

Eukaryotic ssDNA viruses encode a rolling-circle replication (RCR) initiation protein, Rep, which binds to iterated DNA elements functioning as essential elements for virus-specific replication. By using the iterons of all known circoviruses, nanoviruses and nanovirus-like satellites as heuristic devices, we have identified certain amino acid residues that presumably determine the DNA-binding specificity of their Rep proteins. These putative "specificity determinants" (SPDs) cluster in two discrete protein regions, which are adjacent to distinct conserved motifs. A comparable distribution of SPDs was uncovered in the Rep protein of geminiviruses. Modeling of the tertiary structure of diverse Rep proteins showed that SPD regions interact to form a small beta-sheet element that has been proposed to be critical for high-affinity DNA-binding of Rep. Our findings indicate that eukaryotic circular ssDNA viruses have a common ancestor and suggest that SPDs present in replication initiators from a huge variety of viral and plasmid RCR systems are associated with the same conserved motifs.

In Kluyveromyces lactis a Pair of Paralogous Isozymes Catalyze the First Committed Step of Leucine Biosynthesis in Either the Mitochondria or the Cytosol.

Divergence of paralogous pairs, resulting from gene duplication, plays an important role in the evolution of specialized or novel gene functions. Analysis of selected duplicated pairs has elucidated some of the mechanisms underlying the functional diversification of Saccharomyces cerevisiae (S. cerevisiae) paralogous genes. Similar studies of the orthologous pairs extant in pre-whole genome duplication yeast species, such as Kluyveromyces lactis (K. lactis) remain to be addressed. The genome of K. lactis, an aerobic yeast, includes gene pairs generated by sporadic duplications. The genome of this organism comprises the KlLEU4 and KlLEU4BIS paralogous pair, annotated as putative α-isopropylmalate synthases (α-IPMSs), considered to be the orthologs of the S. cerevisiae ScLEU4/ScLEU9 paralogous genes. The enzymes encoded by the latter two genes are mitochondrially located, differing in their sensitivity to leucine allosteric inhibition resulting in ScLeu4-ScLeu4 and ScLeu4-ScLeu9 sensitive dimers and ScLeu9-ScLeu9 relatively resistant homodimers. Previous work has shown that, in a Scleu4Δ mutant, ScLEU9 expression is increased and assembly of ScLeu9-ScLeu9 leucine resistant homodimers results in loss of feedback regulation of leucine biosynthesis, leading to leucine accumulation and decreased growth rate. Here we report that: (i) K. lactis harbors a sporadic gene duplication, comprising the KlLEU4, syntenic with S. cerevisiae ScLEU4 and ScLEU9, and the non-syntenic KlLEU4BIS, arising from a pre-WGD event. (ii) That both, KlLEU4 and KlLEU4BIS encode leucine sensitive α-IPMSs isozymes, located in the mitochondria (KlLeu4) and the cytosol (KlLeu4BIS), respectively. (iii) That both, KlLEU4 or KlLEU4BIS complement the Scleu4Δ Scleu9Δ leucine auxotrophic phenotype and revert the enhanced ScLEU9 transcription observed in a Scleu4Δ ScLEU9 mutant. The Scleu4Δ ScLEU9 growth mutant phenotype is only fully complemented when transformed with the syntenic KlLEU4 mitochondrial isoform. KlLEU4 and KlLEU4BIS underwent a different diversification pathways than that leading to ScLEU4/ScLEU9. KlLEU4 could be considered as the functional ortholog of ScLEU4, since its encoded isozyme can complement both the Scleu4Δ Scleu9Δ leucine auxotrophy and the Scleu4Δ ScLEU9 complex phenotype.

Ancestry informative markers and admixture proportions in northeastern Mexico.

To investigate the ancestral admixture in the Mestizo population in northeastern Mexico, we genotyped 74 ancestral informative markers (AIMs) and 15 Y-single-nucleotide polymorphisms (Y-SNPs) in 100 individuals. The Native American contribution is 56% (range: 27.4-81.2%), the European contribution is 38% (range: 16.7-70.5%) and the West African contribution is 6%. The results show a higher European contribution than was reported in other similar studies in the country, albeit with a predominant Native American ancestry. No remarkable differences in the ancestry proportions were observed using subgroups of 74, 54, 34 and 24 AIMs. The paternal lineage calculated by genotyping of 15 Y-SNPs, shows a major component of European and Eurasian ancestry markers ( approximately 78%), compared with Amerindian ( approximately 12%) and African markers (10%). This information will set a reference for future determinations of admixture proportions in the Mestizo population from Mexico and for population-based association studies of complex diseases.

High resistance to oxidative stress in the fungal pathogen Candida glabrata is mediated by a single catalase, Cta1p, and is controlled by the transcription factors Yap1p, Skn7p, Msn2p, and Msn4p.

We characterized the oxidative stress response of Candida glabrata to better understand the virulence of this fungal pathogen. C. glabrata could withstand higher concentrations of H(2)O(2) than Saccharomyces cerevisiae and even Candida albicans. Stationary-phase cells were extremely resistant to oxidative stress, and this resistance was dependent on the concerted roles of stress-related transcription factors Yap1p, Skn7p, and Msn4p. We showed that growing cells of C. glabrata were able to adapt to high levels of H(2)O(2) and that this adaptive response was dependent on Yap1p and Skn7p and partially on the general stress transcription factors Msn2p and Msn4p. C. glabrata has a single catalase gene, CTA1, which was absolutely required for resistance to H(2)O(2) in vitro. However, in a mouse model of systemic infection, a strain lacking CTA1 showed no effect on virulence.

A Lineage of Begomoviruses Encode Rep and AC4 Proteins of Enigmatic Ancestry: Hints on the Evolution of Geminiviruses in the New World.

The begomoviruses (BGVs) are plant pathogens that evolved in the Old World during the Cretaceous and arrived to the New World (NW) in the Cenozoic era. A subgroup of NW BGVs, the "Squash leaf curl virus (SLCV) lineage" (S-Lin), includes viruses with unique characteristics. To get clues on the evolutionary origin of this lineage, a search for divergent members was undertaken. Four novel BGVs were characterized, including one that is basal to the group. Comparative analyses led to discover a ~670 bp genome module that is nearly exclusive of this lineage, encompassing the replication origin, the AC4 gene, and 480 bp of the Rep gene. A similar DNA module was found in two curtoviruses, hence suggesting that the S-Lin ancestor acquired its distinctive genomic segment by recombination with a curtovirus. This hypothesis was definitely disproved by an in-depth sequence analysis. The search for homologs of S-Lin Rep uncover the common origin of Rep proteins encoded by diverse Geminiviridae genera and viral "fossils" integrated at plant genomes. In contrast, no homolog of S-Lin Rep was found in public databases. Consequently, it was concluded that the SLCV clade ancestor evolved by a recombination event between a primitive NW BGV and a virus from a hitherto unknown lineage.

Histone acetyltransferase TGF-1 regulates Trichoderma atroviride secondary metabolism and mycoparasitism.

Some filamentous fungi of the Trichoderma genus are used as biocontrol agents against airborne and soilborne phytopathogens. The proposed mechanism by which Trichoderma spp. antagonizes phytopathogens is through the release of lytic enzymes, antimicrobial compounds, mycoparasitism, and the induction of systemic disease-resistance in plants. Here we analyzed the role of TGF-1 (Trichoderma Gcn Five-1), a histone acetyltransferase of Trichoderma atroviride, in mycoparasitism and antibiosis against the phytopathogen Rhizoctonia solani. Trichostatin A (TSA), a histone deacetylase inhibitor that promotes histone acetylation, slightly affected T. atroviride and R. solani growth, but not the growth of the mycoparasite over R. solani. Application of TSA to the liquid medium induced synthesis of antimicrobial compounds. Expression analysis of the mycoparasitism-related genes ech-42 and prb-1, which encode an endochitinase and a proteinase, as well as the secondary metabolism-related genes pbs-1 and tps-1, which encode a peptaibol synthetase and a terpene synthase, respectively, showed that they were regulated by TSA. A T. atroviride strain harboring a deletion of tgf-1 gene showed slow growth, thinner and less branched hyphae than the wild-type strain, whereas its ability to coil around the R. solani hyphae was not affected. Δtgf-1 presented a diminished capacity to grow over R. solani, but the ability of its mycelium -free culture filtrates (MFCF) to inhibit the phytopathogen growth was enhanced. Intriguingly, addition of TSA to the culture medium reverted the enhanced inhibition growth of Δtgf-1 MFCF on R. solani at levels compared to the wild-type MFCF grown in medium amended with TSA. The presence of R. solani mycelium in the culture medium induced similar proteinase activity in a Δtgf-1 compared to the wild-type, whereas the chitinolytic activity was higher in a Δtgf-1 mutant in the absence of R. solani, compared to the parental strain. Expression of mycoparasitism- and secondary metabolism-related genes in Δtgf-1 was differentially regulated in the presence or absence of R. solani. These results indicate that histone acetylation may play important roles in the biocontrol mechanisms of T. atroviride.

Diversification of Transcriptional Regulation Determines Subfunctionalization of Paralogous Branched Chain Aminotransferases in the Yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae harbors BAT1 and BAT2 paralogous genes that encode branched chain aminotransferases and have opposed expression profiles and physiological roles . Accordingly, in primary nitrogen sources such as glutamine, BAT1 expression is induced, supporting Bat1-dependent valine-isoleucine-leucine (VIL) biosynthesis, while BAT2 expression is repressed. Conversely, in the presence of VIL as the sole nitrogen source, BAT1 expression is hindered while that of BAT2 is activated, resulting in Bat2-dependent VIL catabolism. The presented results confirm that BAT1 expression is determined by transcriptional activation through the action of the Leu3-α-isopropylmalate (α-IPM) active isoform, and uncovers the existence of a novel α-IPM biosynthetic pathway operating in a put3Δ mutant grown on VIL, through Bat2-Leu2-Leu1 consecutive action. The classic α-IPM biosynthetic route operates in glutamine through the action of the leucine-sensitive α-IPM synthases. The presented results also show that BAT2 repression in glutamine can be alleviated in a ure2Δ mutant or through Gcn4-dependent transcriptional activation. Thus, when S. cerevisiae is grown on glutamine, VIL biosynthesis is predominant and is preferentially achieved through BAT1; while on VIL as the sole nitrogen source, catabolism prevails and is mainly afforded by BAT2.

Diversification of the kinetic properties of yeast NADP-glutamate-dehydrogenase isozymes proceeds independently of their evolutionary origin.

In the yeast Saccharomyces cerevisiae, the ScGDH1 and ScGDH3 encoded glutamate dehydrogenases (NADP-GDHs) catalyze the synthesis of glutamate from ammonium and α-ketoglutarate (α-KG). Previous kinetic characterization showed that these enzymes displayed different allosteric properties and respectively high or low rate of α-KG utilization. Accordingly, the coordinated action of ScGdh1 and ScGdh3, regulated balanced α-KG utilization for glutamate biosynthesis under either fermentative or respiratory conditions, safeguarding energy provision. Here, we have addressed the question of whether there is a correlation between the regulation and kinetic properties of the NADP-GDH isozymes present in S. cerevisiae (ScGdh1 and ScGdh3), Kluyveromyces lactis (KlGdh1), and Lachancea kluyveri (LkGdh1) and their evolutionary history. Our results show that the kinetic properties of K. lactis and L. kluyveri single NADP-GDHs are respectively similar to either ScGDH3 or ScGDH1, which arose from the whole genome duplication event of the S. cerevisiae lineage, although, KlGDH1 and LkGDH1 originated from a GDH clade, through an ancient interspecies hybridization event that preceded the divergence between the Saccharomyces clade and the one containing the genera Kluyveromyces, Lachancea, and Eremothecium. Thus, the kinetic properties which determine the NADP-GDHs capacity to utilize α-KG and synthesize glutamate do not correlate with their evolutionary origin.