Dataset Information

Cytosolic Crowding Drives the Dynamics of Both Genome and Cytosol in Escherichia coli Challenged with Sub-lethal Antibiotic Treatments.

ABSTRACT: In contrast to their molecular mode of action, the system-level effect of antibiotics on cells is only beginning to be quantified. Molecular crowding is expected to be a relevant global regulator, which we explore here through the dynamic response phenotypes in Escherichia coli, at single-cell resolution, under sub-lethal regimes of different classes of clinically relevant antibiotics, acting at very different levels in the cell. We measure chromosomal mobility through tracking of fast (<15 s timescale) fluctuations of fluorescently tagged chromosomal loci, and we probe the fluidity of the cytoplasm by tracking cytosolic aggregates. Measuring cellular density, we show how the overall levels of macromolecular crowding affect both quantities, regardless of antibiotic-specific effects. The dominant trend is a strong correlation between the effects in different parts of the chromosome and between the chromosome and cytosol, supporting the concept of an overall global role of molecular crowding in cellular physiology.

SUBMITTER: Wlodarski M

PROVIDER: S-EPMC7522891 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Cytosolic Crowding Drives the Dynamics of Both Genome and Cytosol in <i>Escherichia coli</i> Challenged with Sub-lethal Antibiotic Treatments.

Wlodarski Michal M Mancini Leonardo L Raciti Bianca B Sclavi Bianca B Lagomarsino Marco Cosentino MC Cicuta Pietro P

iScience 20200915 10

In contrast to their molecular mode of action, the system-level effect of antibiotics on cells is only beginning to be quantified. Molecular crowding is expected to be a relevant global regulator, which we explore here through the dynamic response phenotypes in <i>Escherichia coli</i>, at single-cell resolution, under sub-lethal regimes of different classes of clinically relevant antibiotics, acting at very different levels in the cell. We measure chromosomal mobility through tracking of fast (< ...[more]

PMID: 33083729

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Project description:Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population called persister cells to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically repressed state. We occasionally noticed a high variation in persister levels, and to explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbrück fluctuation test. Although we kept the conditions same, the diversity in persistence level among clones was relatively consistent: varying from ~60- to 100- and ~40- to 70-fold for ampicillin and apramycin, respectively. Then, we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for approximately five generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>seven generations). Our result demonstrates (1) the existence of a small population prepared for stress ("primed cells") resulting in higher persister numbers; (2) the primed memory state is reproducible and transient, passed down for generations but eventually lost; and (3) a heterogeneous persister population is a result of a transiently primed reversible cell state and not due to a pre-existing genetic mutation. IMPORTANCE Antibiotics have been highly effective in treating lethal infectious diseases for almost a century. However, the increasing threat of antibiotic resistance is again causing these diseases to become life-threatening. The longer a bacteria can survive antibiotics, the more likely it is to develop resistance. Complicating matters is that non-genetic factors can allow bacterial cells with identical DNA to gain transient resistance (also known as persistence). Here, we show that a small fraction of the bacterial population called primed cells can pass down non-genetic information ("memory") to their offspring, enabling them to survive lethal antibiotics for a long time. However, this memory is eventually lost. These results demonstrate how bacteria can leverage differences among genetically identical cells formed through non-genetic factors to form primed cells with a selective advantage to survive antibiotics.

Project description:Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in Cys biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidposis thaliana. Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favour of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to Cd. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under non-stress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. To further elucidate the specific function(s) of the OAS-A1 isoform in the adaptation response to cadmium we extended the trasncriptome experiment to the wild type and oas-a1.1 mutant plants exposed to Cd. The comparison of transcriptomic profiles showed a higher proportion of genes with altered expression in the mutant than in the wild type, highlighting up-regulated genes identified as of the general oxidative stress response rather than metal-responsive genes. Wild type and oas-a1.1 mutant plants were grown hydroponically and, after a two-week acclimation period, the roots and shoots were harvested separately. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. We made two different comparisons to classify the differently expressed genes in the mutant plant: oas-a1.1 roots versus wild-type roots and oas-a1.1 shoots versus wild-type shoots. Hydroponically-grown wild type and oas-a1.1 mutant plants were further treated with 50µM CdCl2 and 18h-treated-roots and 24h-treated-shoots were harvested. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. Different comparisons were performed as follows: 18h Cd-treated wild type roots versus untreated wild type roots; 24h Cd-treated wild type shoots versus untreated wild type shoots; 18h Cd-treated oas-a1.1 roots versus untreated oas-a1.1 roots; 24h Cd-treated oas-a1.1 shoots versus untreated oas-a1.1 shoots; 18h Cd-treated oas-a1.1 roots versus 18h Cd-treated wild type roots; 24h Cd-treated oas-a1.1 shoots versus 24h Cd-treated wild type shoots

Dataset Information

Cytosolic Crowding Drives the Dynamics of Both Genome and Cytosol in Escherichia coli Challenged with Sub-lethal Antibiotic Treatments.

Publications

Cytosolic Crowding Drives the Dynamics of Both Genome and Cytosol in <i>Escherichia coli</i> Challenged with Sub-lethal Antibiotic Treatments.

Similar Datasets

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