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Impatto evolutivo del ciclo vitale e del sistema di mating sulla diversità genetica e funzionale dei lieviti appartenenti al complex Zygosaccharomyces rouxii
No full textStoricamente i lieviti della specie Zygosaccharomyces rouxii sono ceppi aploidi osmo e alotolleranti, che divergono da S. cerevisiae prima di una duplicazione del genoma (WGD), avvenuta nella famiglia Saccharomycetaceae 100 milioni di anni fa. La presente tesi si propone di studiare, mediante un approccio multifasico, la complessità genomica e la diversità fenotipica in un pool di ceppi di Zygosaccharomyces collettivamente descritti come complex Z. rouxii. Una nuova specie diploide correlata a Z. rouxii, Zygosaccharomyces sapae, è stata delineata. Lo studio comparativo polifasico di Z. sapae (ceppi ABT301T e ABT601) e ceppi depositati come Z. rouxii (CBS 732T, ATCC 42981, CBS 4837, CBS 4838 e OUT7136) evidenzia variabilità nel numero di cromosomi, dimensione del genoma e ploidia e divergenza in marcatori housekeeping e filogenetici. Z. rouxii complex si distingue in tre clusters, uno aploide di Z. rouxii (ceppo CBS 732T), il cluster diploide di Z. sapae e quello allodiploide/aneuploide (ATCC 42981, CBS 4837, CBS 4838 e OUT7136). Tutti i ceppi, con l’eccezione di CBS 732T, mostrano un’estesa eterogeneità intragenomica negli arrays rDNA, con indels/sostituzioni principalmente nelle regioni spaziatrici trascritte interne (ITS). Questi risultati indicano che i ceppi sono difettosi nei meccanismi di evoluzione concertata che omogeneizzano copie intra–ceppo di array rDNA, suggerendo un processo evolutivo “birth and death”. Quando un bacino più ampio di ceppi è screenato per l’eterogeneità ribosomale, 10 ceppi su 78 hanno mostrato mancanza di evoluzione concertata nei domini D1/D2 26S e nelle regioni ITS-5.8S. L’analisi quantitativa della risposta fenotipica a 15 perturbazioni ambientali è stato condotta con il software grofit. I ceppi ATCC 42981, CBS 4837 e CBS 4838 hanno mostrato un resistenza cationi metallici alcalini ed una capacità di crescita in glicerolo superiori a ABT301T e ABT601. L’indice logaritmico di crescita (basato su valori di μ) ha evidenziato come il ceppo ABT601 sia a lenta crescita ma poco sensibile alle condizioni di stress, mentre ABT301T ha una crescita più rapida in condizioni standard ma più sensibile alle condizioni sub-optimali. Il ciclo vitale e il sistema di mating sono forze evolutive trainanti la diversità genomica e funzionale dei lieviti. Sulla base della variabilità genetica e funzionale del complex Z. rouxii, si ipotizza che all’intero del gruppo il mating fra cellule aploidi eterotalliche divergenti possa risultare in progenie diploidi che danno origine a linee diploidi o allodiploidi/aneuploidi. Per confermare l’ipotesi, è stato studiato il sistema di determinazione del sesso nel ceppo ABT301T. Sono stati clonati tre loci MATα-idiomorfi divergenti (ZsMATa2a1_1, ZsMATa2a1_2, e ZsMATa2a1_3) e un singolo locus ZsMATA2A1 Z. rouxii-like. Inoltre, ABT301T possiede 2 geni HO divergenti codificanti per l’endonucelasi Ho (rispettivamente 92% e 100% di identità nelle sequenze aa rispetto a Z. rouxii). Per determinare la struttura a tre cassette (MAT, HML, HMR), abbiamo sequenziato le regioni fiancheggianti i loci idiomorfici MATa and MATA. Tutti i loci ZsMATa2a1 e ZsMATA2A1 hanno mostrato una organizzazione conservata DIC1-MAT-SLA2, identica a Z. rouxii. Inoltre, sono state individuate quattro putative cassette HML: una contiene la sequenza ZsMATa2a1_2 e le regioni fiancheggianti della cassetta Z. rouxii HML, le rimanenti tre (ZsMATa2a1_1, ZsMATa2a1_2, and ZsMATa2a1_3) divergono nella regione 3' da Z. rouxii. I dati supportano che ABT301T sia un ceppo Aaaa eterozigote che ha perso la cassetta HMR. La presente Tesi di Dottorato mostra come il ciclo vitale e il sistema di mating influenzino la diversità funzionale e genetica del complex Z. rouxii, rendendolo un modello promettente per studiare evoluzione genomica in lieviti pre-WGD.Zygosaccharomyces rouxii traditionally includes osmotolerant and halotolerant haploid yeasts, which emerged from the S. cerevisiae lineage prior to a whole genome duplication (WGD) event occurred in Saccharomycetaceae family 100 million years ago. In this thesis, we applied multi-phasic approaches to elucidate the molecular basis of genomic complexity and functional diversity in a pool of selected Zygosaccharomyces strains, referred as Z. rouxii complex. Firstly, we delineated a novel diploid species related to Z. rouxii, namely Zygosaccharomyces sapae. Subsequently, a polyphasic comparative analysis of Z. sapae (strains ABT301T and ABT601) with strains deposited in culture collection as Z. rouxii (CBS 732T, ATCC 42981, CBS 4837, CBS 4838 and OUT7136) was performed, which revealed that the Z. rouxii complex strains show variability with respect to chromosome number, genome size, ploidy and sequences of housekeeping and phylogenetic markers. Our finding demonstrated that Z. rouxii complex could be split up into three lineage, namely haploid lineage Z. rouxii (CBS 732T), diploid Z. sapae, and allodiploid/aneuploid lineage comprising ATCC 42981, CBS 4837, CBS 4838 and OUT7136. Moreover, an extent pattern of rDNA heterogeneity was observed in intragenomic copies of rDNA array in all strains of Z. rouxii complex, with exception of CBS 732T, with indels/substitutions occurring mainly in internal transcribed spacer regions (ITS). These finding indicated that the strains are defective in mechanisms driving intra-strain homogenization by concerted evolution, suggesting a birth and death process of evolution. When a wider pool of Z. rouxii strains were screened on the basis of rDNA heterogeneity in D1/D2 26S and ITS-5.8S rDNA, 10 out of 78 strains showed lack of concerted evolution in rDNA array.
A quantitative profiling of Z. rouxii complex strains was carried out in response 15 environmental perturbations using statistical tool grofit. ATCC 42981, CBS 4837 and CBS 4838 displayed alkali metal cations resistance and glycerol respiration higher than ABT301T and AB601. μ-based logarithmic phenotypic index highlighted that ABT601 is a slow-growing organism insensitive to stress perturbation, whereas ABT301T grows fast on rich medium and is sensitive to sub-optimal conditions.
The life cycle and the mating system have been reported as the major evolutionary forces driving genetic and functional diversity in yeasts. In context to Z. rouxii complex strains, we hypothesized that mating between the highly divergent haploid, heterothallic parent cells could have resulted in diploid progenies with high genome complexity that subsequently lost the ability to undergo meiosis and given rise to diploid (Z. sapae) and allodiploid/aneuploid (ATCC 42981, CBS 4837, CBS 4838, OUT7136) strains. To test this hypothesis, we investigated sex-determination system in ABT301T. We cloned three divergent MATα-idiomorph loci (ZsMATa2a1_1, ZsMATa2a1_2, and ZsMATa2a1_3) and a single ZsMATA2A1. ABT301T possesses two divergent HO endonuclease genes (92% and 100% aa sequence identities compared to Z. rouxii). To establish the three-cassette structure (MAT, HML, HMR), we analyzed the flanking regions of MATa and MATA-idiomorph loci. ZsMATA2A1 and all ZsMATa2a1 loci exhibited a conserved DIC1-MAT-SLA2 organization, identical to Z. rouxii. Moreover, four putative HML cassettes were identified: one containing sequence ZsMATa2a1_2 and Z. rouxii flanking regions; the remaining three (ZsMATa2a1_1, ZsMATa2a1_2, and ZsMATa2a1_3) are divergent in 3’ regions from Z. rouxii. In conclusion, ABT301T is a highly heterozygous Aaaa strain which has lost HMR.
The results presented in this thesis globally provide evidences of evolutionary impact of life cycle and mating systems on genetic and functional diversity in Z. rouxii complex strains, which represents a promising model for studying genome evolution in pre-WGD yeasts species
Getting insights from genomic complexities in Zygosaccharomyces rouxii complex
No full textGenus Zygosaccharomyces includes osmo/halotolerant yeasts that diverged from the S. cerevisiae lineage prior to its genome duplication (WGD). Recently, we delineated a novel species, Zygosaccharomyces sapae. Z. sapae has phylogenetic importance as its conspecific strain NCYC3042 has been implicated in allodiploid nature of hybrid ATCC42981. We carry out an extensive genetic survey on some Zygosaccharomyces strains (called Z. rouxii complex) to unravel intra and inter-strain DNA sequences heterogeneity; and diversity in chromosome patterns. Intragenomic copies of rDNA genes are not homogenized by concerted evolution in them, with indels and substitutions occurring mainly in internal transcribed spacer regions. This suggests a birth and death process of evolution. Variability in protein-coding sequences and ploidy level showed a marked intergenomic diversity in diploid and aneuploid cells. Functional analysis revealed a broad range tolerance to alkali metal cations, congruent with clusters obtained through genetic survey. Life cycle and breeding system are suggested to shape these diversities in this complex, making it an interesting pre-WGD model for studying evolutionary forces driving genome plasticity in low-aw environments
Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii
No full textThe osmotolerant and halotolerant food yeast Zygosaccharomyces rouxii is known for its ability to grow and survive in the face of stress caused by high concentrations of non-ionic (sugars and polyols) and ionic (mainly Na(+) cations) solutes. This ability determines the success of fermentation on high osmolarity food matrices and leads to spoilage of high sugar and high salt foods. The knowledge about the genes, the metabolic pathways, and the regulatory circuits shaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation control, optimization of food starter culture, and prevention of food spoilage. This review summarizes recent insights on the mechanisms used by Z. rouxii and other osmo and halotolerant food yeasts to endure salts and sugars stresses. Using the information gathered from S. cerevisiae as guide, we highlight how these non-conventional yeasts integrate general and osmoticum-specific adaptive responses under sugar and salts stresses, including regulation of Na(+) and K(+)-fluxes across the plasma membrane, modulation of cell wall properties, compatible osmolyte production and accumulation, and stress signalling pathways. We suggest how an integrated and system-based knowledge on these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control in food, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms
Sex-determination system in the diploid yeast Zygosaccharomyces sapae
No full textSexual reproduction and breeding systems are driving forces for genetic diversity. The mating-type (MAT) locus represents a mutation and chromosome rearrangement hotspot in yeasts. Zygosaccharomyces rouxii complex yeasts are naturally faced with hostile low water activity (aw) environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy. Here, we investigated sex-determination system in Zygosaccharomyces sapae diploid strain ABT301(T), a member of the Z. rouxii complex. We cloned three divergent mating type-like (MTL) α-idiomorph sequences and designated them as ZsMTLα copies 1, 2, and 3. They encode homologs of Z. rouxii CBS 732(T) MATα2 (amino acid sequence identities spanning from 67.0 to 99.5%) and MATα1 (identity range 81.5-99.5%). ABT301(T) possesses two divergent HO genes encoding distinct endonucleases 100% and 92.3% identical to Z. rouxii HO. Cloning of MATA: -idiomorph resulted in a single ZsMTLA: locus encoding two Z. rouxii-like proteins MATA: 1 and MATA: 2. To assign the cloned ZsMTLα and ZsMTLA: idiomorphs as MAT, HML, and HMR cassettes, we analyzed their flanking regions. Three ZsMTLα loci exhibited the DIC1-MAT-SLA2 gene order canonical for MAT expression loci. Furthermore, four putative HML cassettes were identified, two containing the ZsMTLα copy 1 and the remaining harboring ZsMTLα copies 2 and 3. Finally, the ZsMTLA: locus was 3'-flanked by SLA2, suggesting the status of MAT expression locus. In conclusion, Z. sapae ABT301(T) displays an aααα genotype missing of the HMR silent cassette. Our results demonstrated that mating-type switching is a hypermutagenic process in Z. rouxii complex that generates genetic diversity de novo. This error-prone mechanism could be suitable to generate progenies more rapidly adaptable to hostile environments
Complex nature of Zygosaccharomyces wild strains: a genotypic and functional characterization
No full textGenotypic and functional characterization of Zygosaccharomyces wild strain
Differential hypersaline stress response in Zygosaccharomyces rouxii complex yeasts: A physiological and transcriptional study
Full text linkThe Zygosaccharomyces rouxii complex comprises three distinct lineages of halotolerant yeasts relevant in food processing and spoilage, such as Z. sapae, Z. rouxii and a mosaic group of allodiploid strains. They manifest plastic genome architecture (variation in karyotype, ploidy level and Na+/H+ antiporter-encoding gene copy number), and exhibit diverse tolerances to salt concentrations. Here, we investigated accumulation of compatible osmolytes and transcriptional regulation of Na+/H+ antiporter-encoding ZrSOD genes during salt exposure in strains representative for the lineages, namely Z. sapae ABT301T (low salt tolerant), Z. rouxii CBS 732T (middle salt tolerant) and allodiploid strain ATCC 42981 (high salt tolerant). Growth curve modelling in 2 M NaCl-containing media supplemented with or without yeast extract as nitrogen source indicates that moderate salt tolerance of CBS 732T mainly depends on nitrogen availability rather than intrinsic inhibitory effects of salt. All the strains produce glycerol and not mannitol under salt stress and use two different glycerol balance strategies. ATCC 42981 produces comparatively more glycerol than Z. sapae and Z. rouxii under standard growth conditions and better retains it intracellularly under salt injuries. Conversely, Z. sapae and Z. rouxii enhance glycerol production under salt stress and intracellularly retain glycerol less efficiently than ATCC 42981. Expression analysis shows that, in diploid Z. sapae and allodiploid ATCC 42981, transcription of gene variants ZrSOD2-22/ZrSOD2 and ZrSOD22 is constitutive and salt unresponsive
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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