Evolutionary engineering and molecular characterization of cobalt-resistant Rhodobacter sphaeroides

With its versatile metabolism including aerobic and anaerobic respiration, photosynthesis, photo-fermentation and nitrogen fixation, Rhodobacter sphaeroides can adapt to diverse environmental and nutritional conditions, including the presence of various stressors such as heavy metals. Thus, it is an important microorganism to study the molecular mechanisms of bacterial stress response and resistance, and to be used as a microbial cell factory for biotechnological applications or bioremediation. In this study, a highly cobalt-resistant and genetically stable R. sphaeroides strain was obtained by evolutionary engineering, also known as adaptive laboratory evolution (ALE), a powerful strategy to improve and characterize genetically complex, desired microbial phenotypes, such as stress resistance. For this purpose, successive batch selection was performed in the presence of gradually increased cobalt stress levels between 0.1–15 mM CoCl2 for 64 passages and without any mutagenesis of the initial population prior to selection. The mutant individuals were randomly chosen from the last population and analyzed in detail. Among these, a highly cobalt-resistant and genetically stable evolved strain called G7 showed significant cross-resistance against various stressors such as iron, magnesium, nickel, aluminum, and NaCl. Growth profiles and flame atomic absorption spectrometry analysis results revealed that in the presence of 4 mM CoCl2 that significantly inhibited growth of the reference strain, the growth of the evolved strain was unaffected, and higher levels of cobalt ions were associated with G7 cells than the reference strain. This may imply that cobalt ions accumulated in or on G7 cells, indicating the potential of G7 for cobalt bioremediation. Whole genome sequencing of the evolved strain identified 23 single nucleotide polymorphisms in various genes that are associated with transcriptional regulators, NifB family-FeMo cofactor biosynthesis, putative virulence factors, TRAP-T family transporter, sodium/proton antiporter, and also in genes with unknown functions, which may have a potential role in the cobalt resistance of R. sphaeroides

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Evolutionary Engineering Of Phenylethanol-resistant Saccharomyces Cerevisiae

Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2013Saccharomyces cerevisiae, genetik ve moleküler biyoloji çalışmalarında çok sık kullanılan, özellikleri iyi bilinen model organizmalardan biridir. Bilimsel araştırmalardaki kullanım alanlarının yanı sıra, S. cerevisiae endüstriyel üretimde de önemli bir yere sahiptir. Özellikle etanol üretimi ve ekmek yapımında yaygın olarak kullanılmaktadır. S. cerevisiae, tek hücreli bir ökaryotik mikrorganizma olup, tomurcuklanma yolu ile hem eşeysiz, hem de mayoz bölünme gerçekleştirerek eşeyli olarak çoğalabilmektedir. S. cerevisiae’nin yüksek ökaryotların genomu ile gösterdiği yüksek homoloji de bir çok bilimsel çalışmada yarar sağlamaktadır. Özellikle insan genomu ile olan benzerliği sebebiyle, kanser, yaşlanma ve birçok hastalık mekanizmaları S. cerevisiae hücreleri kullanılarak araştırılmaktadır. Mikroorganizmalar, doğal ve endüstriyel ortamlarda sıkça stres koşullarına maruz kalmaktadır. Bunlar, yüksek yada düşük sıcaklık, ozmolarite, oksidatif stres, mekanik stres ve metal stresi gibi streslerdir. Araştırmacılar, mikrobiyel stres direnç mekanizmalarını araştırmakta ve aynı zamanda çeşitli streslere karşı direnç düzeylerini arttırmaya çalıştırmaktadırlar. Aynı zamanda, endüstriyel verimin arttırılması amacıyla, üreticiler de stres direnci yüksek mikroorganizmalar aramaktadırlar. Bu tez çalışmasında, feniletanole dirençli maya hücreleri elde edilerek feniletanole karşı geliştirilen direncin moleküler mekanizmalarının incelenmesi amaçlandı. Bunun için ilk olarak evrimsel mühendislik yöntemi ile feniletanole dirençli S. cerevisiae mutantları elde edildi. Ardından, feniletanole dirençli S. cerevisiae mutantlarında, feniletanol direncinin moleküler mekanizmasını anlamak amacıyla fenotip analizleri, fizyolojik ve transkriptomik analizler gerçekleştirildi. Çalışma başlangıcında evrimsel mühendislik yaklaşımı yaban tip S. cerevisiae hücreleri üzerinde gerçekleştirildi. Bu amaçla ilk olarak başlangıç popülasyonunda genetik çeşitliliği arttırmak için kimyasal bir mutajen olan etil metan sülfonat (EMS) yaban tip maya hücrelerine uygulandı. Elde edilen mutajenize edilmiş maya kültürü, sonrasında seçilime maruz bırakılarak, kültür içinden istenen fenotipteki bireylerin seçilmesi planlandı. Seçilim süresince, ilk başta düşük konsantrasyonlarda (1.5 mL/L) feniletanol kültüre uygulandı ve inkübasyon gerçekleştirildi. Sonraki basamakta, hayatta kalan maya hücreleri, daha yüksek bir feniletanol konsantrasyonunda tekrar inkübe edildi. Her basamakta, OD600 değerleri ölçüldü ve hayatta kalma oranları kritik bir seviyeye düşene kadar bu seçilim işlemleri devam edildi. En son 3.6 mL/L feniletanol konsantrasyonuna kadar gelindi ve 56. nesilde seçilim işlemi durduruldu. Bu elde edilen son popülasyondan rastgele 10 birey seçildi ve direnç yeteneklerine göre kıyaslandı. On mutant birey, yaban tip ve son popülasyonun feniletanol dirençleri damlatma ve en muhtemel sayı (MPN) yöntemleri ile ölçüldü ve karşılaştırıldı. Elde edilen 10 birey arasından en yüksek direnci gösteren birey seçildi ve “C9” olarak adlandırıldı. C9 bireyinde genetik kararlılık testi uygulandı. Bu test ile maya mutantının feniletanol direncinin kalıcı olup olmadığı belirlendi. Damlatma ve MPN çalışmaları, bu bireyin feniletanol direncinin değişmediğini gösterdi. İlgili mutantta feniletanol direnci genetik olarak kararlı bulundu. Feniletanole dirençli maya mutantının çapraz direnç özellikleri de incelendi. Bunun için çapraz direnç testi uygulandı. Bu testte, seçilen mutant ve yaban tip, farklı konsantrasyonlarda feniletanol (2.5 mL/L ve 3 mL/L), etanol (8%, 10% ve 12% v/v), asetat (0.004% v/v), kobalt (1 mM ve 3 mM), bor (80 mM), bakır (0.5 mM), hidrojen peroksit (0.5 mM) ve nikel’e (0.2 mM) maruz bırakıldı, hayatta kalma oranları kıyaslandı. Tüm bu stres faktörleri içinde, feniletanol dirençli mutant, etanole karşı da direnç gösterdi. Etanol ve feniletanol’ün hücresel etki mekanizmalarının muhtemel benzerliklerinden dolayı bu iki stres faktörünün çapraz dirence neden olması beklenen bir durum olarak nitelendirilebilir. Feniletanol dirençli C9 mutantı, aynı zamanda kobalt’a karşı belirgin bir hassasiyet göstermektedir. Feniletanole dirençli mutantın direnç mekanizmasının moleküler düzeyde incelenmesi için transkriptomik analiz gerçekleştirilmiştir. Bu amaçla, DNA mikroarray yaklaşımı kullanılmış ve C9 ile yaban tip arasında, kontrol koşullarındaki transkripsiyon profilleri karşılaştırılmıştır. Analiz sonucunda, C9’un genel transkripsiyon profilinde ilgi çekici sonuçlara rastlanmıştır. Bu sonuçlardan biri, çok yüksek sayıda gende transkripsiyon artışı görülmesidir. S. cerevisiae genomunda bulunun yaklaşık 6000 gen içerisinde 1000 kadar genin anlatımı artarken 800’e yakın gende de anlatımda azalış olmuştur. Tüm bu genler, maya genomunun yaklaşık %30’una denk gelmektedir. Bu yüksek transkripsiyon profili, maya hücrelerinin stres anında gösterdiği kısa süreli cevaplar ile benzerlik göstermektedir. Normalde kısa süren ve çok sayıda kendini gösteren bu reaksiyonlar çevresel stres cevabı (Environmental stress response, ‘ESR’) olarak bilinmektedir. Feniletanole dirençli mutantta ESR’den sorumlu genler önemli düzeyde aktif durumdadır. Feniletanole dirençli mutanta ait transkripsiyon profilinde ilk göze çarpan anlatımı artan 1000 kadar gen arasında, karbonhidrat metabolizması ile ilgili genlerin önemli bir yer kaplamasıdır. Anlatımı artan 166 gen ile karbonhidrat metabolizmasından sorumlu genler, C9’un anlatımı artmış tüm genlerinin yaklaşık %20’sini oluşturmaktadır. Bunu 98 gen ile oksidatif stres cevabı izlemektedir. Aynı zamanda anlatımı artmış genler arasında 63 tanesi genel stres cevabından, 35 tanesi hücre duvarı organizasyonundan, 21 gen ise otofaji ve mitokondri yıkımından sorumludur. Belirtilen %20’lik katkı karbonhidrat metabolizmasının, C9 mutantında önemli bir şekilde tetiklenmiş olduğunu göstermektedir. Benzer durum, daha önce tanımlanan ESR koşullarında da görülmüştür. Hücreler, stres altında kısa süreliğine glikoz metabolizmasını hızlandırmaktadır. Ancak, C9 mutantında bu genlerin anlatımları ortamda stres koşulları bulunmasa da aralıksız olarak gerçekleşmiştir. Benzer durum, anlatımı azalan genlerde de görülmüştür. Analiz sonuçlarına göre, C9 bireyinde özellikle nükleik asit metabolizması ve protein, ribozom sentezinde görev alan çoğu genin anlatımı ciddi oranda azalmıştır. Anlatımı azalan 821 genin %81’i rRNA ve tRNA’ların sentezi ve bağlanmasında, translasyonun başlamasında, RNA-DNA bağlanmasında, helikaz aktivitesinde görev almaktadır. C9’da protein sentezini azaltacak yönde görülen bu değişiklikler aynı zamanda genel ESR koşullarında da görülmektedir. Bu sonuçlar da feniletanol dirençli C9 bireylerinin sürekli bir ESR durumunda olduğu görüşünü desteklemektedir. C9’un aynı zamanda, özelleşmiş stres cevapları da verdiği görülmüştür. Yaban tipe kıyasla, aldehit dehidrogenaz 3 adlı genin 234 kat daha fazla anlatımı gerçekleşmiştir. Alkolün yıkılması sırasında ortaya çıkan bir toksik madde olan aldehidin yıkılmasından sorumlu bu genin yüksek şekilde anlatılması, C9’un sahip olduğu feniletanol direnci için önemli olabilir. Bu tez çalışmasında, feniletanol dirençli S. cerevisiae hücreleri evrimsel mühendislik yöntemleri ile elde edilmiş ve transkripsiyon seviyesinde karakterizasyonu gerçekleştirilmiştir. Dirençli maya mutantının yüksek seviyede gösterdiği gen anlatımı, yaban tip hücrelerin stres anında verdiği anlık tepkilerle benzerlik göstermektedir. Anlatımın yüksek ve karmaşık olması, feniletanol direncinin tek bir gen veya gen grubu ile ilişkilendirilmesini zorlaştırmaktadır. Bu sebeple, çevresel stres cevaplarının daha iyi anlaşılması ve feniletanol direncinin temel kökeninin bulunması için anlatımı önemli ölçüde artmış ya da azalmış genlerin delesyonu ya da aşırı anlatımı gibi ilave moleküler araştırmaların yapılması önerilebilir.Saccharomyces cerevisiae is one the most widely used model organisms in genetics, molecular biology and metabolic studies. In addition to its use in scientific research, it is one of the oldest microorganisms used for ages for industrial applications. S. cerevisiae is a unicellular eukaryotic organism, which can be found in haploid and diploid form, and can induce meiosis to generate new progeny of haploid from diploids (so called sporulation event) or reproduce asexually by budding. It shares high degree of homologies with higher eukaryotes like human. Due to these functional similarities, S. cerevisiae can be used in research related to cancer, aging and other human diseases. In natural environment and in industrial applications, S. cerevisiae cells are often under stress resistance that results them environmental changes. These changes can be named as osmotic, high or low temperature, dehydration, starvation, metal ion stresses etc. Researchers are interested in the microbial resistance mechanisms to these different types of stresses. Additionally, they are searching for strategies to increase stress tolerance. Producers are also interested in increasing yield and for this reason; they are searching for stress-resistant microorganisms. The aim of the present study was to obtain phenylethanol (PEA) resistant yeast strains via evolutionary engineering approach and perform transcriptomic and metabolic characterization to identify responsible pathways ans molecular factors in this resistance. In this thesis study, firstly, phenylethanol-resistant S. cerevisiae mutants were obtained by evolutionary engineering approach. Phenotypic and genetic characterization was then carried out to identify the molecular principles of phenylethanol resistance in S. cerevisiae. To apply evolutionary engineering to wild type S. cerevisiae cells, these cells were treated with a chemical mutagen EMS (Ethyl Methane Sulfonate) to increase the genetic diversity of the initial population to which selection would be applied. This mutagenized culture was cultivated at increasing phenylethanol concentrations in the culture medium along with the wild type to determine the initial stress level to be applied. Phenylethanol stress was then applied to this mutagenized culture. The phenylethanol concentration was increased gradually for each successive population. The first population was obtained upon 1.5 mL/L exposure to phenylethanol and the final 56th population was obtained upon exposure to 3.6 mL/L PEA. The final population was used for randomly selecting ten individual mutants. Those ten individual mutants, wild type and the final population were tested for phenylethanol resistance and it was observed that the evolved strain and the final population could grow at high phenylethanol concentrations at which the wild type could not show any sign of survival. One of the individual mutants which showed highest phenylethanol-resistance was chosen and genetic stability assay was applied. It was shown that phenylethanol-resistance was a genetically stable trait in the mutant tested. This evolved strain was termed C9. In this study, PEA-resistant strain C9 was analyzed according to its cross-resistance abilities against various metals and organic compounds and compared with the wild type. Different concentrations of phenylethanol (2.5 mL/L and 3 mL/L), ethanol (8%, 10% and 12% v/v), acetate (0.004% v/v), cobalt (1 mM and 3 mM), boron (80 mM), copper (0.5 mM), hydrogen peroxide (0.5 mM) and nickel (0.2 mM) were used. It was observed that, phenylethanol-resistant mutant also show had cross-resistance to ethanol. Besides, C9 had increased sensitivity to cobalt stress. To investigate the molecular mechanisms of phenylethanol resistance of the evolved strain, whole genome transcriptomic analysis was conducted for wild type and C9. Sampling for microarray analysis was carried out when the cultures were in their exponential phase of growth. The expression profile of the mutant was compared to that of the wild type. The results showed that, phenylethanol-resistant C9 strain had immense amount of upregulated and downregulated genes in its genome under control conditions without any external stress. DNA microarray analysis showed that C9 had about 1000 upregulated and 800 downregulated genes which make up about 30% of whole genome. Such large scale changes in transcription levels indicate that some global expression response was always active in C9. That genome-wide expression program resembles a highly known large-scale stress reaction called “environmental stress response” (ESR). DNA microarray analysis results indicated that there were about 1000 upregulated genes in C9 compared to wild type and majority of these genes were responsible for carbohydrate metabolism. With upregulated 166 genes, carbohydrate metabolism contributes to about 20% of all upregulated genes in C9. Following with 98 genes responsible for oxidative stress response, 63 genes for general stress response, 35 genes for cell wall reorganization and renewal, 21 genes for degradation of mitochondria and cell itself were found to be upregulated. With 20% contribution, genes in carbohydrate metabolism were shighly upregulated in phenylethanol resistant C9 strain. In addition to increased activity of genes involved in glycolysis, many other genes associated with hexose transport, alternative carbon source utilization were also over-expressed. Same cellular states were also observed under ESR conditions which may indicate that C9 strain apparently induces ESR actively and continuously. Additionally, many putative genes involved in cell wall biosynthesis, autophagy, DNA damage response were up-regulated. Same similarities were also observed in repression profile of C9 compared to wild type. Interpretation of downregulated genes showed that C9 strain selectively repressed major nucleic acid metabolism and ribosome synthesis. More than 81% of 821 downregulated genes were related to synthesis and binding of rRNA and tRNA, initiation of translation, RNA-DNA binding, and helicase activity. Additionally, similar regulations have also been observed previously during ESR in stressed-wild type strains upon initial stress exposure. C9 also showed unique stress responses against alcohol stress. In comparison with wild type, phenylethanol-resistant C9 strain showed 234-fold higher expression of ALD3 gene. This gene might be related to main resistance mechanisms against phenylethanol and ethanol. Increased ALD3 gene expression may prepare cells to overcome excess amounts of aldehyde byproducts of alcohol degradation. In this thesis study, a phenylethanol hyper-resistant S. cerevisiae mutant was obtained and characterized at transcriptomic level. Duw to the complexity and the large size of change in the transcriptomic response of the resistant mutant, it is not likely to point out one or a few genes that are crucial for phenylethanol resistance. However, it was shown that continuous induction of ESR genes may provoke specific resistance mechanisms. It could therefore be recommended to continue molecular research to enlighten the mechanism of phenylethanol resistance, for example, by overexpression/deletion of genes that were highly upregulated/downregulated according to transcriptomic analysis results.Yüksek LisansM.Sc

Stemness Related Genes Cause Resistance to SMAC mimetics in Neuroblastoma Cells.

Stemness Related Genes Cause Resistance to SMAC mimetics in NeuroblastomaCells</p

Evolutionary engineering and molecular characterization of cobalt-resistant Rhodobacter sphaeroides

With its versatile metabolism including aerobic and anaerobic respiration, photosynthesis, photo-fermentation and nitrogen fixation, Rhodobacter sphaeroides can adapt to diverse environmental and nutritional conditions, including the presence of various stressors such as heavy metals. Thus, it is an important microorganism to study the molecular mechanisms of bacterial stress response and resistance, and to be used as a microbial cell factory for biotechnological applications or bioremediation. In this study, a highly cobalt-resistant and genetically stable R. sphaeroides strain was obtained by evolutionary engineering, also known as adaptive laboratory evolution (ALE), a powerful strategy to improve and characterize genetically complex, desired microbial phenotypes, such as stress resistance. For this purpose, successive batch selection was performed in the presence of gradually increased cobalt stress levels between 0.1–15 mM CoCl2 for 64 passages and without any mutagenesis of the initial population prior to selection. The mutant individuals were randomly chosen from the last population and analyzed in detail. Among these, a highly cobalt-resistant and genetically stable evolved strain called G7 showed significant cross-resistance against various stressors such as iron, magnesium, nickel, aluminum, and NaCl. Growth profiles and flame atomic absorption spectrometry analysis results revealed that in the presence of 4 mM CoCl2 that significantly inhibited growth of the reference strain, the growth of the evolved strain was unaffected, and higher levels of cobalt ions were associated with G7 cells than the reference strain. This may imply that cobalt ions accumulated in or on G7 cells, indicating the potential of G7 for cobalt bioremediation. Whole genome sequencing of the evolved strain identified 23 single nucleotide polymorphisms in various genes that are associated with transcriptional regulators, NifB family-FeMo cofactor biosynthesis, putative virulence factors, TRAP-T family transporter, sodium/proton antiporter, and also in genes with unknown functions, which may have a potential role in the cobalt resistance of R. sphaeroides

Transcriptomic and Physiological Meta-Analysis of Multiple Stress-Resistant <i>Saccharomyces cerevisiae</i> Strains

Meta-analysis is a beneficial approach to reevaluating the outcomes of independent previous studies in the same scope. Saccharomyces cerevisiae, or the baker’s yeast, is a commonly used unicellular and eukaryotic model organism. In this study, 12 evolved S. cerevisiae strains that became resistant to diverse stress conditions (boron, caffeine, caloric restriction, cobalt, coniferyl aldehyde, ethanol, iron, nickel, oxidative stress, 2-phenylethanol, and silver stress) by adaptive laboratory evolution were reassessed to reveal the correlated stress/stressor clusters based on their transcriptomic and stress–cross-resistance data. Principal Component Analysis (PCA) with k-means clustering was performed. Five clusters for the transcriptomic data of strains and six clusters for cross-resistance stressors were identified. Through statistical evaluations, critical genes pertinent to each cluster were elucidated. The pathways associated with these genes were investigated using the KEGG database. The findings demonstrated that caffeine and coniferyl aldehyde stressors exhibit clear distinctions from other stressors in terms of both physiological stress-cross-resistance responses and transcriptomic profiles. Pathway analysis showed that ribosome biogenesis was downregulated, and starch and sucrose metabolism was upregulated across all clusters. Gene and pathway analyses have shown that stressors lead to distinct changes in yeast gene expression, and these alterations have been systematically documented for each cluster. Several of the highlighted genes are pivotal for further exploration and could potentially clarify new aspects of stress response mechanisms and multiple stress resistance in yeast

Genome-wide identification of Chiari malformation type I associated candidate genes and chromosomal variations

Chiari malformation type I (CMI) is a brain malformation that is characterized by herniation of the cerebellum into the spinal canal. Chiari malformation type I is highly heterogeneous; therefore, an accurate explanation of the pathogenesis of the disease is often not possible. Although some studies showed the role of genetics in CMI, the involvement of genetic variations in CMI pathogenesis has not been thoroughly elucidated. Therefore, in the current study we aim to reveal CMI-associated genomic variations in familial cases.Four CMI patients and 7 unaffected healthy members of two distinct families were analyzed. A microarray analysis of the affected and unaffected individuals from two Turkish families with CMI was conducted. Analyses of single nucleotide variations (SNVs) and copy number variations (CNVs) were performed by calculation of B allele frequency (BAF) and log R ratio (LRR) values from whole genome SNV data. Two missense variations, OLFML2A (rs7874348) and SLC4A9 (rs6860077), and a 5’UTR variation of COL4A1 (rs9521687) were significantly associated with CMI. Moreover, 12 SNVs in the intronic regions of FAM155A, NR3C1, TRPC7, ASTN2, and TRAF1 were determined to be associated with CMI. The CNV analysis showed that the 11p15.4 chromosome region is inherited in one of the families. The use of familial studies to explain the molecular pathogenesis of complex diseases such as CMI is crucial. It has been sug-gested that variations in OLFML2A, SLC4A9, and COL4A1 play a role in CMI molecular pathogenesis. The CNV analysis of individuals in both families revealed a potential chromosomal region, 11p15.4, and risk regions that are associated with CMI.</jats:p

Evolutionary engineering and molecular characterization of a caffeine-resistant Saccharomyces cerevisiae strain

Caffeine is a naturally occurring alkaloid, where its major consumption occurs with beverages such as coffee, soft drinks and tea. Despite a variety of reports on the effects of caffeine on diverse organisms including yeast, the complex molecular basis of caffeine resistance and response has yet to be understood. In this study, a caffeine-hyperresistant and genetically stable Saccharomyces cerevisiae mutant was obtained for the first time by evolutionary engineering, using batch selection in the presence of gradually increased caffeine stress levels and without any mutagenesis of the initial population prior to selection. The selected mutant could resist up to 50 mM caffeine, a level, to our knowledge, that has not been reported for S. cerevisiae so far. The mutant was also resistant to the cell wall-damaging agent lyticase, and it showed cross-resistance against various compounds such as rapamycin, antimycin, coniferyl aldehyde and cycloheximide. Comparative transcriptomic analysis results revealed that the genes involved in the energy conservation and production pathways, and pleiotropic drug resistance were overexpressed. Whole genome re-sequencing identified single nucleotide polymorphisms in only three genes of the caffeine-hyperresistant mutant; PDR1, PDR5 and RIM8, which may play a potential role in caffeine-hyperresistance. Graphic abstrac