Rahul Jagadeesan, Suchintak Dash, Cristina S. D. Palma, Ines S. C. Baptista, Vatsala Chauhan, Jarno Mäkelä, Andre S. Ribeiro (2025) Sci. Adv. 11, eadl3570.
DOI: 10.1126/sciadv.adl3570
Data: https://datadryad.org/dataset/doi:10.5061/dryad.zgmsbccks
Bacterial gene networks have operons, each coordinating several genes under a primary promoter. Half of the operons in Escherichia coli have been reported to also contain internal promoters. We studied their role during genome-wide stresses targeting key transcription regulators, RNA polymerase (RNAP) and gyrase. Our results suggest that operons’ responses are influenced by stress-related changes in premature elongation terminations and internal promoters’ activity. Globally, this causes the responses of genes in the same operon to differ with the distance between them in a wave-like pattern. Meanwhile, premature terminations are affected by positive supercoiling buildup, collisions between elongating and promoter-bound RNAPs, and local regulatory elements. We report similar findings in E. coli under other stresses and in evolutionarily distant bacteria B subtilis, C glutamicum, and H pylori. Our results suggest that the strength, number, and positioning of operons’ internal promoters might have evolved to compensate for premature terminations, providing distal genes similar response strengths.
Baptista ISC, Dash S, Arsh AM, Kandavalli V, Scandolo CM, Sanders BC, and Ribeiro AS (2025) Bimodality in E. coli gene expression: Sources and robustness to genome-wide stresses. PLoS Comput Biol 21(2): e1012817.
https://doi.org/10.1371/journal.pcbi.1012817
Data Availability: https://doi.org/10.5061/dryad.dz08kps5n;
Software: https://doi.org/10.5281/zenodo.14746159;
Supplemental information: https://doi.org/10.5281/zenodo.14746189.
Bacterial cell populations exhibiting multiple phenotypes under the same environmental condition should have enhanced survival chances to unpredictable environmental fluctuations. This capacity could rely on genes with the ability to switch between high- and low-expression levels, under the same environment. So far, few genes are known to have such a capacity. We report on seven genes with bimodal dynamics, show that bimodality is robust to some but not all stresses, and reversibility of bimodality if returning to control conditions. We also show evidence that bimodality emerges from events in transcription initiation and propose a model of their behavior. Our findings could advance research on bacterial decision-making processes and engineering multi-stable synthetic circuits.
Dash S, Jagadeesan R, Baptista ISC, Chauhan V, Kandavalli V, Oliveira SMD, Ribeiro AS. 0. A library of reporters of the global regulators of gene expression in Escherichia coli. mSystems 0:e00065-24.
Escherichia coli have evolved hundreds of transcription factors to tune the expression of thousands of genes. Interestingly, a few transcription factors control almost half of all genes and are thus named global regulators (GRs). Tracking the numbers of these GRs over time is essential to understand phenotypic modifications, e.g., under stress conditions. We have engineered a library of strains to track GR levels. Each strain has a single-copy plasmid coding for a fast-maturing green fluorescent protein whose transcription is controlled by a copy of the natural promoter of the GR. This library should become useful in scientific research, and future applications in therapeutics and the bioindustries. For more information, please visit: 10.1128/msystems.00065-24.
V Chauhan, ISC Baptista, A Arsh, R Jagadeesan, S Dash, and AS Ribeiro (2024) Transcription attenuation in synthetic promoters in tandem formation. Biochemistry 63(16), 2009–2022. DOI: 10.1021/acs.biochem.4c00012.
We studied study transcriptional interference using engineered two pairs and one trio of synthetic promoters in nonoverlapping, tandem formation, in single-copy plasmids in E. coli cells. We show that these constructs exhibit a phenomenon of transcription attenuation due to RNAP collisions and fall-offs that can be externally regulated. Thus, they may become very useful tools in future synthetic circuits.
Dash S*, Palma CSD*, et al. (2022) Alteration of DNA supercoiling serves as a trigger of short-term cold shock repressed genes of E. coli. Nucleic Acids Research 50(15) 8512–8528. DOI: 10.1093/nar/gkac643. *Equal authorship.
Adapting to cold is a key skill of gut bacteria of warm-blooded animals. We hypothesized that cold affects DNA supercoiling, which is regulated by gyrase. We performed two RNA-seqs, one after cold-shock, the other after adding Novobiocin, an antibiotic that represses gyrase. We found that many cold-shock repressed genes are similarly sensitive to Novobiocin. Next, we observed similar changes in the protein numbers of these genes to both perturbations. Moreover, under cold shock, nucleoid density increases, and gyrases and nucleoid become colocalized. Also, the cell energy decreases, which hinders the resolution of positive supercoils. We suggest that responsiveness to low negative supercoiling is a core feature of E. coli’s short-term, cold shock transcriptional response program, and could be used to regulate the temperature sensitivity of synthetic circuits.
B Almeida et al (2022) The transcription factor network of E. coli steers global responses to shifts in RNAP concentration. Nucleic Acids Research 50(12), 6801-6819. DOI: 10.1093/nar/gkac540.
The role of transcription factor networks (TFNs) on the robustness, sensitivity, plasticity, and overall adaptability of microorganisms to environmental stresses remains largely unexplored. We studied how the TFN of E. coli responds to genome-wide perturbations caused by quick changes in medium richness, known to alter RNA polymerase (RNAP) concentrations.
We found that, at the gene cohort level, the average magnitude of the mid-term transcriptional responses can be explained by the average absolute difference between the numbers of activating and repressing input TFs of a gene. Interestingly, this difference is strongly positively correlated with the average number of input TFs of a gene. Our result suggests that the global topological traits of TFNs influence their global response dynamics to genome-wide stresses. This may be a general principle by which TFNs regulate genome-wide information propagation.
Bacteria process information to bind past events to future actions, so as to adapt to environment changes. This is made possible by a timely organized execution of multiple tasks such as counting time, sensing the environment, and decision making, which are performed in parallel, by semi-independent, tuned small genetic circuits. These circuits differ in structure, which defines the function. Meanwhile, the properties of their internal components, e.g. the kinetics of transcription initiation of the promoters, define the efficiency with how functions are performed.
We constructed a single-copy repressilator (SCR) by implementing the original repressilator circuit of Elowitz et al on a single-copy F-plasmid. We studied its behaviour as a function of temperature and compared to the original low-copy-number repressilator (LCR). In optimal temperature, the dynamics of the two systems differ, but respond similarly to temperature changes. Interestingly, the SCR is more robust to lower temperatures and perturbations than the original LCR, which loses functionality as temperature increases beyond 30 C, due to the loss of functionality of one of its proteins, CI.
Selected Publications:
JG Chandraseelan, SMD Oliveira, A Häkkinen, H Tran, I Potapov, A Sala, M Kandhavelu, and AS Ribeiro (2013) Effects of temperature on the dynamics of the LacI-TetR-CI repressilator. Mol. BioSyst. 9 (12), 3117-3123. DOI:10.1039/c3mb70203k
SMD Oliveira*, JG Chandraseelan*, A Häkkinen, NSM Goncalves, O Yli-Harja, S Startceva, and AS Ribeiro (2015) Single-cell kinetics of the Repressilator when inserted into a single-copy plasmid. Mol. BioSyst. 11, 1939-1945. *Equal contributions. DOI: 10.1039/C5MB00012B
Cell division in E coli is morphologically symmetric due to, among other, their ability to place the Z-ring at midcell. At sub-optimal temperatures, this symmetry decreases at the single-cell level. Using fluorescence microscopy, we observed FtsZ-GFP and DAPI-stained nucleoids to assess the robustness of the symmetry of Z-ring formation and positioning in cells at sub-optimal and critical temperatures. We found that the Z-ring formation and positioning is robust at sub-optimal temperatures, as the Z-ring ’ s mean width, density and displacement from midcell maintain correlation to one another. However, at critical temperatures, the Z-ring displacement from midcell is greatly increased. This is due to enhanced distance between the replicated nucleoids and, thus, reduced Z-ring density, which explains the weaker precision in setting a morphologically symmetric division site. This also occurs in rich media and is cumulative, i.e. combining richer media and critically high temperatures enhances the asymmetries in division, which is evidence that the causes are biophysical. To support this, we showed that the effects are reversible, i.e. shifting cells from optimal to critical, and then to optimal again, reduces and then enhances the symmetry in Z-ring positioning, respectively. Overall, we found that Z-ring positioning in E. coli is a robust biophysical process under sub-optimal temperatures, and that critical temperatures cause significant asymmetries in division.
Selected Publication:
R Neeli-Venkata, SMD Oliveira, L Martins, S Startceva, M Bahrudeen, JM Fonseca, M Minoia and AS Ribeiro (2018) The precision of the symmetry in Z-ring placement in Escherichia coli is hampered at critical temperatures. Physical Biology 15(5):056002. DOI: 10.1088/1478-3975/aac1cb
We studied whether nucleoid exclusion contributes to the segregation and retention of Tsr chemoreceptor clusters at the cell poles. We used live time-lapse, single-cell microscopy measurements, mutant cells, etc.
Overall, we found that the single-cell spatial distributions of Tsr clusters have heterogeneities and symmetries that are consistent with nucleoid exclusion, being one of the mechanisms by which they are positioned in the cells.
R Neeli-Venkata, S Startceva, T Annila, and AS Ribeiro (2016) Polar Localization of the Serine Chemoreceptor of Escherichia coli is Nucleoid Exclusion-Dependent. Biophysical Journal 111(11), 2512–2522. DOI: 10.1016/j.bpj.2016.10.024
We have being interested in Learning and Behavior. Paw preference is a very interesting case-study since the amount of learning (information) can be quantified from measurements, such as the one shown, where the choice of which paw the mouse uses at each reach, as he learns, can be observed, and the number of possible choices (left or right) can also be quantified.
So far, some of our interesting results include:
Hand-preference behavior in mice is inherently probabilistic. Likely, this provides robustness and constant adaptability to ever-changing environments.
Strong biases in paw preference of individual mice result from weak biases that appear by chance early in training and are reinforced with training over time. Constitutive behaviors play a minor or no role.
The degree of learning with training depends on the amount of short-term and long-term memory of past choices, which are genetically determined.
The corpus callosum (CC) and the hippocampal commissure (HC) contribute heavily to memory function and formation of long-term, but not short-term, paw-preference biases.
There is a degree of randomness in paw choice that is not removed by training, which may be a critical element for behavioural plasticity in paw preference in changing environments, supplying constant adaptability in paw preferences.
Selected Publications:
AS Ribeiro, J Lloyd-Price, B Eales, and FG Biddle (2010) Dynamic Agent-Based Model of Hand-Preference Behavior Patterns in the Mouse. Adaptive Behavior 18(2), 116-131. DOI: 10.1177/1059712309339859
AS Ribeiro, BA Eales and FG Biddle, (2011) Learning of paw preference in mice is strain dependent, gradual and based on short-term memory of previous reaches, Animal Behavior, 81(1), 249-257. DOI: 10.1016/j.anbehav.2010.10.014
AS Ribeiro, B.A. Eales, and F.G. Biddle (2013) Short-term and long-term memory deficits in handedness learning in mice with absent corpus callosum and reduced hippocampal commissure. Behavioural Brain Research 245, 145–151. DOI: 10.1016/j.bbr.2013.02.021
AS Ribeiro, BA Eales, J Lloyd-Price, and FG Biddle (2014) Predictability and randomness of paw choices are critical elements in the behavioural plasticity of mouse paw preference. Animal Behavior 98, 167-176. DOI: 10.1016/j.anbehav.2014.10.008
The data is collected by our long-time collaborators Fred G. Biddle (picture below) and Brenda Eales.
Fred in his lab at the University of Calgary, Canada :-)