Takeuchi, R., Tamura, T., Nakayashiki, T., Tanaka, Y., Muto, A., Wanner, B. L., & Mori, H.
BMC Microbiol. 2014; 14: 171.
Precise quantitative growth measurements and detection of small growth changes in high-throughput manner is essential for fundamental studies of bacterial cell. However, an inherent tradeoff for measurement quality in high-throughput methods sacrifices some measurement quality. A key challenge has been how to enhance measurement quality without sacrificing throughput.
Gallone, B., Steensels, J., Prahl, T., Soriaga, L., Saels, V., Herrera-Malaver, B., Merlevede, A., Roncoroni, M., Voordeckers, K., Miraglia, L., Teiling, C., Steffy, B., Taylor, M., Schwartz, A., Richardson, T., White, C., Baele, G., Maere, S., … Verstrepen, K. J.
Cell. 2016 Sep 8; 166(6): 1397–1410.e16.
Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today’s industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains.
Fabian Istel, Tobias Schwarzmüller, Michael Tscherner, and Karl Kuchler
Bio Protoc. 2015 Jul 20; 5(14): e1530.
Here, we describe a method enabling the phenotypic profiling of genome-scale deletion collections of fungal mutants to detect phenotypes for various stress conditions. These stress conditions include among many others antifungal drug susceptibility, temperature-induced and osmotic as well as heavy metal or oxidative stress. The protocol was extensively used to phenotype a collection of gene deletion mutants in the human fungal pathogen Candida glabrata (C. glabrata)
Vlaming, H., Molenaar, T. M., van Welsem, T., Poramba-Liyanage, D. W., Smith, D. E., Velds, A., Hoekman, L., Korthout, T., Hendriks, S., Altelaar, A., … van Leeuwen, F.
eLife. 2016; 5: e18919.
Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features.
Rafael Hoyos-Manchado, Félix Reyes-Martín, Charalampos Rallis, Enrique Gamero-Estévez, Pablo Rodríguez-Gómez, Juan Quintero-Blanco, Jürg Bähler, Juan Jiménez, and Víctor A. Tallada
Rafael Hoyos-Manchado, Félix Reyes-Martín, Charalampos Rallis, Enrique Gamero-Estévez, Pablo Rodríguez-Gómez, Juan Quintero-Blanco, Jürg Bähler, Juan Jiménez, and Víctor A. Tallada
The synthesis, processing and function of coding and non-coding RNA molecules and their interacting proteins has been the focus of a great deal of research that has boosted our understanding of key molecular pathways that underlie higher order events such as cell cycle control, development, innate immune response and the occurrence of genetic diseases. In this study, we have found that formamide preferentially weakens RNA related processes in vivo. Using a non-essential Schizosaccharomyces pombe gene deletion collection, we identify deleted loci that make cells sensitive to formamide. Sensitive deletions are significantly enriched in genes involved in RNA metabolism. Accordingly, we find that previously known temperature-sensitive splicing mutants become lethal in the presence of the drug under permissive temperature. Furthermore, in a wild type background, splicing efficiency is decreased and R-loop formation is increased in the presence of formamide. In addition, we have also isolated 35 formamide-sensitive mutants, many of which display remarkable morphology and cell cycle defects potentially unveiling new players in the regulation of these processes. We conclude that formamide preferentially targets RNA related processes in vivo, probably by relaxing RNA secondary structures and/or RNA-protein interactions, and can be used as an effective tool to characterize these processes.
Leonie Johanna Jahn, Bethany Mason, Peter Brøgger, Tea Toteva, Dennis Kim Nielsen, and Genevieve Thon
David B. H. Barton, Danae Georghiou, Neelam Dave, Majed Alghamdi, Thomas A. Walsh, Edward J. Louis, and Steven S. Foster
Background
Microbial arrays, with a large number of different strains on a single plate printed with robotic precision, underpin an increasing number of genetic and genomic approaches. These include Synthetic Genetic Array analysis, high-throughput Quantitative Trait Loci (QTL) analysis and 2-hybrid techniques. Measuring the growth of individual colonies within these arrays is an essential part of many of these techniques but is useful for any work with arrays. Measurement is typically done using intermittent imagery fed into complex image analysis software, which is not especially accurate and is challenging to use effectively. We have developed a simple and fast alternative technique that uses a pinning robot and a commonplace microplate reader to continuously measure the thickness of colonies growing on solid agar, complemented by a technique for normalizing the amount of cells initially printed to each spot of the array in the first place. We have developed software to automate the process of combining multiple sets of readings, subtracting agar absorbance, and visualizing colony thickness changes in a number of informative ways.
Results
The “PHENOS” pipeline (PHENotyping On Solid media), optimized for Saccharomyces yeasts, produces highly reproducible growth curves and is particularly sensitive to low-level growth. We have empirically determined a formula to estimate colony cell count from an absorbance measurement, and shown this to be comparable with estimates from measurements in liquid. We have also validated the technique by reproducing the results of an earlier QTL study done with conventional liquid phenotyping, and found PHENOS to be considerably more sensitive.
Conclusions
“PHENOS” is a cost effective and reliable high-throughput technique for quantifying growth of yeast arrays, and is likely to be equally very useful for a range of other types of microbial arrays. A detailed guide to the pipeline and software is provided with the installation files at https://github.com/gact/phenos.
Leonie Johanna Jahn, Bethany Mason, Peter Brøgger, Tea Toteva, Dennis Kim Nielsen, and Genevieve Thon
G3: GENES, GENOMES, GENETICS February 1, 2018 vol. 8 no. 2 477-489;
Chromatin structure regulates both genome expression and dynamics in eukaryotes, where large heterochromatic regions are epigenetically silenced through the methylation of histone H3K9, histone deacetylation, and the assembly of repressive complexes. Previous genetic screens with the fission yeast Schizosaccharomyces pombe have led to the identification of key enzymatic activities and structural constituents of heterochromatin. We report here on additional factors discovered by screening a library of deletion mutants for silencing defects at the edge of a heterochromatic domain bound by its natural boundary—the IR-R+ element—or by ectopic boundaries. We found that several components of the DNA replication progression complex (RPC), including Mrc1/Claspin, Mcl1/Ctf4, Swi1/Timeless, Swi3/Tipin, and the FACT subunit Pob3, are essential for robust heterochromatic silencing, as are the ubiquitin ligase components Pof3 and Def1, which have been implicated in the removal of stalled DNA and RNA polymerases from chromatin. Moreover, the search identified the cohesin release factor Wpl1 and the forkhead protein Fkh2, both likely to function through genome organization, the Ssz1 chaperone, the Fkbp39 proline cis-trans isomerase, which acts on histone H3P30 and P38 in Saccharomyces cerevisiae, and the chromatin remodeler Fft3. In addition to their effects in the mating-type region, to varying extents, these factors take part in heterochromatic silencing in pericentromeric regions and telomeres, revealing for many a general effect in heterochromatin. This list of factors provides precious new clues with which to study the spatiotemporal organization and dynamics of heterochromatic regions in connection with DNA replication.
Tea Toteva, Bethany Mason, Yutaka Kanoh, Peter Brøgger, Daniel Green, Janne Verhein-Hansen, Hisao Masai, and Geneviève Thona,
PNAS January 31, 2017. 114 (5) 1093-1098
In recent years, high-throughput studies have revealed the organization of eukaryotic genomes into chromatin domains, topologically associated domains, and replication domains, laying out important principles for the spatiotemporal organization of the nucleus. Our work shows how customized genetic screens with model organisms can help delineate functional relationships between the different layers of organization and identify the cis- and trans-acting elements underpinning them. Here, such approaches revealed that DNA elements and proteins that regulate replication-origin firing in yeasts and other eukaryotes can help partition the genome into expressed and repressed domains. We expect this insight acquired with fission yeast will inspire research in other organisms where the proteins we identified, the Shelterin component Rif1 and its effectors, are intensively studied.
Jonathan M Monk, Colton J Lloyd, Elizabeth Brunk, Nathan Mih, Anand Sastry, Zachary King, Rikiya Takeuchi, Wataru Nomura, Zhen Zhang, Hirotada Mori, Adam M Feist & Bernhard O Palsson
Nature Biotechnology volume 35, pages 904–908 (2017)
Pastor-Flores D Becker K Dick T
Interface Focus, 2017 vol: 7 (2) pp: 20160143
Mitochondrially generated oxidants are believed to play important roles in both physiology and pathophysiology. Therefore, it is of significant interest to better understand the metabolic conditions leading to enhanced mitochondrial oxidant generation. Here, we investigate the influence of oxygen and glucose availability on the redox state of peroxiredoxin-based redox probes, expressed in the cytosol and mitochondrial matrix of yeast cells. We observe that the redox state of peroxiredoxin probes reflects the balance between dioxygen-dependent peroxide generation and glucose-dependent generation of reducing equivalents. The oxidative pentose phosphate pathway appears to be the dominant source of NADPH in the system under study.