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High-throughput quantification of microbial birth and death dynamics using fluorescence microscopy.

ABSTRACT: Background:Microbes live in dynamic environments where nutrient concentrations fluctuate. Quantifying fitness in terms of birth rate and death rate in a wide range of environments is critical for understanding microbial evolution and ecology. Methods:Here, using high-throughput time-lapse microscopy, we have quantified how Saccharomyces cerevisiae mutants incapable of synthesizing an essential metabolite (auxotrophs) grow or die in various concentrations of the required metabolite. We establish that cells normally expressing fluorescent proteins lose fluorescence upon death and that the total fluorescence in an imaging frame is proportional to the number of live cells even when cells form multiple layers. We validate our microscopy approach of measuring birth and death rates using flow cytometry, cell counting, and chemostat culturing. Results:For lysine-requiring cells, very low concentrations of lysine are not detectably consumed and do not support cell birth, but delay the onset of death phase and reduce the death rate compared to no lysine. In contrast, in low hypoxanthine, hypoxanthine-requiring cells can produce new cells, yet also die faster than in the absence of hypoxanthine. For both strains, birth rates under various metabolite concentrations are better described by the sigmoidal-shaped Moser model than the well-known Monod model, while death rates can vary with metabolite concentration and time. Conclusions:Our work reveals how time-lapse microscopy can be used to discover non-intuitive microbial birth and death dynamics and to quantify growth rates in many environments.

SUBMITTER: Hart SFM

PROVIDER: S-EPMC6785046 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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High-throughput quantification of microbial birth and death dynamics using fluorescence microscopy.

Hart Samuel F M SFM Skelding David D Waite Adam J AJ Burton Justin C JC Shou Wenying W

Quantitative biology (Beijing, China) 20190104 1

<h4>Background</h4>Microbes live in dynamic environments where nutrient concentrations fluctuate. Quantifying fitness in terms of birth rate and death rate in a wide range of environments is critical for understanding microbial evolution and ecology.<h4>Methods</h4>Here, using high-throughput time-lapse microscopy, we have quantified how <i>Saccharomyces cerevisiae</i> mutants incapable of synthesizing an essential metabolite (auxotrophs) grow or die in various concentrations of the required met ...[more]

PMID: 31598381

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Project description:In this work, we used our recently developed method Spec-seq to characterize the binding specificity of Glucocorticoid receptor in vitro. This GR Spec-seq experiment has been run twice separately (Dec 2014 and Mar 2015) with different library compositions. The basic workflow is the same as our previous work for lac repressor published in Genetics 198.3 (2014): 1329-1343. Recombinant human GR protein was used to facilitate in vitro DNA-binding and separation experiments. Bound and Unbound DNA fragments were separated in EMSA gels, purified, barcoded for further Illumina sequencing. An initial experiment was performed using the putative consensus sequence: AGAACA GGG TGTTCT; randomized library 1: AGAACN NSN NGTTCT [diversity =512]; randomized library 2: AGAANN GGG NNNTCT [diversity = 1024]; randomized library 3: AGAACA GGG TGNNNN [diversity =256]; randomized library 4: AGAACA GGGC NNNTCT [diversity = 64]. The initial total library diversity was ~1853 with a library composition of 10% positive control sequence + randomized libraries 1-4 + 5% negative control sequence. A 5th library containing 2304 sequences based on DDNACW KKN KGTTCT, where D="not C", N="any base", W="A or T" and K="G or T" was subsequently prepared and analyzed using similar ratios of control sequences. Binding conditions for EMSA were 100ng FAM-labelled dsDNA+ 0/0.5/1/2/4uM GR DBD protein for each lane, 1X NEB buffer 4. GR DBD was prepared as previously described. The EMSA was performed using a 9% 33:1 acrylamide gel and TB buffer, and was run at 200V for 30mins @ 0 degrees C. The 2uM protein lane used for final sequencing. Bound/unbound fractions resulting from EMSA of these libraries and conditions were used to generate PWMs as described. The GR PWM that was generated through this analysis was used to define relative binding energies using the patser program (35), which can be accessed online at (http://stormo.wustl.edu/consensus/cgi-bin/Server/Interface/patser.cgi ). Derived binding affinities are proportional to the inverse of the natural log of the calculated energies.

Dataset Information

High-throughput quantification of microbial birth and death dynamics using fluorescence microscopy.

Publications

High-throughput quantification of microbial birth and death dynamics using fluorescence microscopy.

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