Pyphe: A python toolbox for assessing microbial growth and cell viability in high-throughput colony screens

Kamrad, Stephan; Rodríguez-López, Maria; Cotobal, Cristina; Correia‐Melo, Clara; Ralser, Markus; Bähler, Jürg

doi:10.1101/2020.01.22.915363

Cited by 11 publications

(14 citation statements)

References 31 publications

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“…Growth assays were performed on SC, SM and YPD by time-course imaging of colonies, using our Pyphe pipeline (Kamrad et al, 2020), as described in (Kamrad et al, 2022). Library plates were grown from cryostocks in 384 format for three days on agar media.…”

Section: Methodsmentioning

confidence: 99%

The Proteomic Landscape of Genome-Wide Genetic Perturbations

Messner

Demichev

Muenzner

et al. 2022

Preprint

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SummaryFunctional genomic strategies help to address the genotype phenotype problem by annotating gene function and regulatory networks. Here, we demonstrate that combining functional genomics with proteomics uncovers general principles of protein expression, and provides new avenues to annotate protein function. We recorded precise proteomes for all non-essential gene knock-outs in Saccharomyces cerevisiae. We find that protein abundance is driven by a complex interplay of i) general biological properties, including translation rate, turnover, and copy number variations, and ii) their genetic, metabolic and physical interactions, including membership in protein complexes. We further show that combining genetic perturbation with proteomics provides complementary dimensions of functional annotation: proteomic profiling, reverse proteomic profiling, profile similarity and protein covariation analysis. Thus, our study generates a resource in which nine million protein quantities are linked to 79% of the yeast coding genome, and shows that functional proteomics reveals principles that govern protein expression.Highlights-Nine million protein quantities recorded in ~4,600 non-essential gene deletions in S. cerevisiae reveal principles of how the proteome responds to genetic perturbation-Genome-scale protein expression is determined by both functional relationships between proteins, as well as common biological responses-Broad protein expression profiles in slow-growing strains can be explained by chromosomal aneuploidies-Protein half-life and ribosome occupancy are predictable from protein abundance changes across knock-outs-Functional proteomics annotates missing gene function in four complementary dimensions

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Section: Methodsmentioning

confidence: 99%

The Proteomic Landscape of Genome-Wide Genetic Perturbations

Messner

Demichev

Muenzner

et al. 2022

Preprint

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“…Plates were incubated for 2 days at 30 °C until patterns of colonies appeared. Images of agar plates were acquired with Pyphe-scan 90 using an Epson V700 scanner in transmission mode. Plate image analysis and quantification of the number of CFUs in the ageing culture, based on the colony patterns observed, were performed using the R package, DeadOrAlive 89 .…”

Section: Methodsmentioning

confidence: 99%

“…Plates were incubated for 2 days at 30 °C until patterns of colonies appeared. Images of agar plates were acquired with Pyphe-scan 90 colony patterns observed, were performed using the R package, DeadOrAlive 89 . In both conventional-and HTP-CFU assays, survival of the different strains was normalised, first to biomass at time of sample collection, as measured by OD600, and then to the survival of the respective wild-type controls at the beginning of the stationary phase (48 hours from the start of culture).…”

Section: Conventional and High-throughput Colony Forming Unit (Cfu) A...mentioning

confidence: 99%

Cell-cell metabolite exchange creates a pro-survival metabolic environment that extends lifespan

Correia‐Melo

Kamrad

Messner

et al. 2022

Preprint

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Metabolism is fundamentally intertwined with the ageing process. We here report that a key determinant of cellular lifespan is not only nutrient supply and intracellular metabolism, but also metabolite exchange interactions that occur between cells. Studying chronological ageing in yeast, we observed that metabolites exported by young, exponentially growing, cells are re-imported during the stationary phase when cells age chronologically, indicating the existence of cross-generational metabolic interactions. We then used self-establishing metabolically cooperating communities (SeMeCos) to boost cell-cell metabolic interactions and observed a significant lifespan extension. A search for the underlying mechanisms, coupling SeMeCos, metabolic profiling, proteomics and genome-scale metabolic modelling, attributed a specific role to methionine consumer cells. These cells were enriched over time, adopted glycolytic metabolism and increased export of protective metabolites. Glycerol, in particular, accumulated in the communal metabolic environment and extended the lifespan of all cells in the community in a paracrine fashion. Our results hence establish metabolite exchange interactions as a determinant of the ageing process, and show that metabolically cooperating cells shape their metabolic environment to achieve lifespan extension.

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“…Plates were grown for 2 days at 32°C, and images were acquired by transmission scanning (Epson V800 Photo). Colony sizes were determined with gitter (Wagih & Parts, ) and corrected for spatial biases using reference‐grid normalisation (Zackrisson et al , ), as implemented in our freely available pipeline (preprint: Kamrad et al , ). Further, strains which did not grow at all (colony size < 10 pixels at 600 dpi scanning resolution) or showed abnormal circularity values (> 1.1 or < 0.85) were excluded from further analysis.…”

Section: Methodsmentioning

confidence: 99%

Pyruvate kinase variant of fission yeast tunes carbon metabolism, cell regulation, growth and stress resistance

Kamrad

Großbach

Rodríguez-López

et al. 2020

Molecular Systems Biology

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Cells balance glycolysis with respiration to support their metabolic needs in different environmental or physiological contexts. With abundant glucose, many cells prefer to grow by aerobic glycolysis or fermentation. Using 161 natural isolates of fission yeast, we investigated the genetic basis and phenotypic effects of the fermentationrespiration balance. The laboratory and a few other strains depended more on respiration. This trait was associated with a single nucleotide polymorphism in a conserved region of Pyk1, the sole pyruvate kinase in fission yeast. This variant reduced Pyk1 activity and glycolytic flux. Replacing the "low-activity" pyk1 allele in the laboratory strain with the "high-activity" allele was sufficient to increase fermentation and decrease respiration. This metabolic rebalancing triggered systems-level adjustments in the transcriptome and proteome and in cellular traits, including increased growth and chronological lifespan but decreased resistance to oxidative stress. Thus, low Pyk1 activity does not lead to a growth advantage but to stress tolerance. The genetic tuning of glycolytic flux may reflect an adaptive trade-off in a species lacking pyruvate kinase isoforms.

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Pyphe: A python toolbox for assessing microbial growth and cell viability in high-throughput colony screens

Cited by 11 publications

References 31 publications

The Proteomic Landscape of Genome-Wide Genetic Perturbations

The Proteomic Landscape of Genome-Wide Genetic Perturbations

Cell-cell metabolite exchange creates a pro-survival metabolic environment that extends lifespan

Pyruvate kinase variant of fission yeast tunes carbon metabolism, cell regulation, growth and stress resistance

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