Estimating RNA numbers in single cells by RNA fluorescent tagging and flow cytometry

Bahrudeen, Mohamed; Chauhan, Vatsala; Palma, Cristina; Oliveira, Samuel M. D.; Kandavalli, Vinodh; Ribeiro, André S.

doi:10.1016/j.mimet.2019.105745

Cited by 8 publications

(6 citation statements)

References 41 publications

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“…The correlation between two data sets with known uncertainties (standard error of the mean (SEM) in each data point) was obtained by performing linear regression fitting using Ordinary Least Squares. The best fitting line along with its 68.2% confidence interval/bounds (CB) and statistics was obtained as described in Supplementary Materials and Methods 1.4 of (52). In short, the uncertainty of each of the N empirical data points was represented by m points, resulting in n = N×m points.…”

Section: Methodsmentioning

confidence: 99%

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The transcription factor network of E. coli steers global responses to shifts in RNAP concentration

Almeida

Bahrudeen

Chauhan

et al. 2022

Preprint

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The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the gene’s numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

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Section: Methodsmentioning

confidence: 99%

“…The best fitting line along with its 68.2% confidence interval/bounds (CB) and statistics was obtained as described in Supplementary Materials and Methods 1.4 of (52). In short, the uncertainty of each of the N empirical data points was represented by m points, resulting in n = N×m points.…”

Section: Correlations Between Data Setsmentioning

confidence: 99%

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration

Almeida

Bahrudeen

Chauhan

et al. 2022

Preprint

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“…We measured RNA and protein expression levels by RNA-seq (Supplementary Section I) and by Flow-cytometry (Supplementary Section II), respectively. We used pulse width data from flow-cytometry as a proxy for cell volume [Bahrudeen MNM et al, 2019; Cunningham et al, 1990; Traganos et al, 1984], which assisted the estimation of protein concentrations. We verified these results using microscopy data and image analysis (Supplementary Section III).…”

Section: Methodsmentioning

confidence: 99%

Positive supercoiling buildup is a trigger of E. coli’s short-term response to cold shock

Dash

Palma

Baptista

et al. 2021

Preprint

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Adaptation to cold shock (CS) is a key survival skill of gut bacteria of warm-blooded animals. In E. coli, this skill emerges from a complex transcriptional program of multiple, timely-ordered shifts in gene expression. We identified short-term, cold shock repressed (CSR) genes by RNA-seq and provide evidence that their variability in evolutionary fitness is low and that their responsiveness to cold emanates from intrinsic features. Given that their single-cell variability in protein numbers increases after CS, we hypothesized that the responsiveness of a large portion of CSR genes is triggered by the high propensity for transcription locking due to positive supercoiling buildup (PSB). We then proposed a model of this phenomenon and, in support, show that nearly half of CSR genes are highly responsive to Gyrase inhibition. Also, their response strengths to CS and Gyrase inhibition correlate and most CSR genes increase their single-cell variability in protein numbers. Further, during CS, the cells' nucleoid density increases (in agreement with increased numbers of positive supercoils), their energy levels become depleted (while the resolving of positive supercoils is ATP dependent), and the colocalization of Gyrases and the nucleoid increases (in agreement with increased time length for resolving supercoils). We conclude that high sensitivity to PSB is at the core of the short-term, cold shock responsive transcriptional program of E. coli and propose that this gene feature may be useful for providing temperature sensitivity to chromosome-integrated synthetic circuits.

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“…We measured RNA and protein expression levels by RNA-seq (Supplementary Section I) and by flow cytometry (Supplementary Section II), respectively. We used pulse width data from flow-cytometry as a proxy for cell size (39)(40)(41), required to estimate protein concentrations. We verified these results using microscopy data and image analysis (Supplementary Section III).…”

Section: Bacterial Strains Growth Conditions and Gene Expression Meas...mentioning

confidence: 99%

Alteration of DNA supercoiling serves as a trigger of short-term cold shock repressed genes ofE. coli

Dash

Palma

Baptista

et al. 2022

Nucleic Acids Research

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Cold shock adaptability is a key survival skill of gut bacteria of warm-blooded animals. Escherichia coli cold shock responses are controlled by a complex multi-gene, timely-ordered transcriptional program. We investigated its underlying mechanisms. Having identified short-term, cold shock repressed genes, we show that their responsiveness is unrelated to their transcription factors or global regulators, while their single-cell protein numbers’ variability increases after cold shock. We hypothesized that some cold shock repressed genes could be triggered by high propensity for transcription locking due to changes in DNA supercoiling (likely due to DNA relaxation caused by an overall reduction in negative supercoiling). Concomitantly, we found that nearly half of cold shock repressed genes are also highly responsive to gyrase inhibition (albeit most genes responsive to gyrase inhibition are not cold shock responsive). Further, their response strengths to cold shock and gyrase inhibition correlate. Meanwhile, under cold shock, nucleoid density increases, and gyrases and nucleoid become more colocalized. Moreover, the cellular energy decreases, which may hinder positive supercoils resolution. Overall, we conclude that sensitivity to diminished negative supercoiling is a core feature of E. coli’s short-term, cold shock transcriptional program, and could be used to regulate the temperature sensitivity of synthetic circuits.

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Estimating RNA numbers in single cells by RNA fluorescent tagging and flow cytometry

Cited by 8 publications

References 41 publications

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration

Positive supercoiling buildup is a trigger of E. coli’s short-term response to cold shock

Alteration of DNA supercoiling serves as a trigger of short-term cold shock repressed genes ofE. coli

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