Biology subjects

Palma, C. S. D.

Publications and source records attributed to Palma, C. S. D..

6 recordsLinked to original sources

bioRxiv2025

Modeling σE biochemical network reveals context-dependent feedback control and kinetic constraints in the envelope stress response

The bacterial cell envelope is essential for mechanical stability, barrier function, defining cell size and shape, and supporting cellular processes. Because external or internal stressors can challenge its integrity, bacteria have evolved stress response systems to maintain envelope homeostasis. For example, under stress, E. coli activates {sigma}E to induce a regulon that restores envelope homeostasis. The molecular mechanisms underlying the transcriptional and post-translational regulation of {sigma}E and its anti-sigma factor RseA have been well-mapped. However, how these regulatory layers function at the systems level to regulate {sigma}E activity remains unclear. Here, we combine mathematical modeling with quantitative gene expression measurements to determine how the interplay of transcription and post-translational regulation affects the dynamics of the {sigma}E response. The results suggest that the {sigma}E activity is determined by the balance between the release from RseA, resulting from its degradation, and slow binding of free {sigma}E to the intact RseA. Notably, the combined action of transcriptional and post-translational regulation reveals that autoregulation, traditionally assumed to be a positive feedback, transitions from negative to positive feedback under extreme stress, enabling a finely tuned response that prevents premature activation while ensuring a robust response under severe envelope stress. In summary, our findings elucidate the mechanisms of {sigma}E regulation, thereby advancing our understanding of how alternative sigma factor bacterial networks control stress-response pathways.

systems biology↗

bioRxiv2025

Changes in Spo0A~P pulsing frequency control biofilm matrix deactivation

Under starvation conditions, B. subtilis survives by differentiating into one of two cell types: biofilm matrix-producing cells or sporulating cells. These two cell-differentiation pathways are activated by the same phosphorylated transcription factor - Spo0A~P. Despite sharing the activation mechanism, these cell fates are mutually exclusive at the single-cell level. This decision has been shown to be controlled by the effects of growth rate on gene dosage and protein dilution in the biofilm matrix production network. In this work, we explore an alternative mechanism of growth rate-mediated control of this cell fate decision. Namely, using deterministic and stochastic modeling, we investigate how the growth-rate-dependent pulsing dynamics of Spo0A~P affect biofilm matrix deactivation and activation. Specifically, we show that the Spo0A~P pulsing frequency tunes the biofilm matrix deactivation probability without affecting the probability of biofilm matrix activation. Interestingly, we found that DNA replication is the cell cycle stage that most substantially contributes to the deactivation of biofilm matrix production. Finally, we report that the deactivation of biofilm matrix production is not primarily regulated by the effects of growth rate on gene dosage and protein dilution. Instead, it is driven by changes in the pulsing period of Spo0A~P. In summary, our findings elucidate the mechanism governing biofilm deactivation during the late stages of starvation, thereby advancing our understanding of how bacterial networks interpret dynamic transcriptional regulatory signals to control stress-response pathways. Author SummaryBacteria have evolved various adaptation mechanisms to survive under challenging environmental conditions. For instance, under mild starvation, B. subtilis bacteria form biofilms -- communities of cells encapsulated in a protective extracellular matrix. On the other hand, these bacteria differentiate into highly resistant spores under severe starvation. Interestingly, sporulating cells are often found within biofilm communities, but they do not contribute to biofilm matrix production. This is thought to be an energy-conservation strategy, as biofilm formation is an energy-intensive process and is therefore halted before sporulation begins. Though previous work has focused on the mechanisms driving biofilm disassembly, few studies have explored the regulatory processes that B. subtilis employs to halt matrix production prior to starting sporulation. In this study, we use mathematical models to demonstrate that the temporal dynamics of the biofilm master regulator Spo0A~P control the deactivation of matrix production. Understanding the regulation of biofilm, a common lifestyle in bacteria, can lead to the development of synthetic strategies to either enhance or disrupt biofilm formation, with potential applications in medicine and industry.

systems biology↗

bioRxiv2025

Biophysical modeling reveals the transcriptional regulatory mechanism of Spo0A, the master regulator in starving Bacillus subtilis

In starving Bacillus subtilis bacteria, the initiation of two survival programs - biofilm formation and sporulation - is both controlled by the same phosphorylated master regulator Spo0A[~]P. Its gene, spo0A, is transcribed from two promoters, Pv and Ps, that are respectively regulated by RNAP holoenzymes bearing {sigma}A and {sigma}H. Notably, transcription is directly autoregulated by Spo0A[~]P binding sites known as 0A1, 0A2, and 0A3 box, located in between the two promoters. It remains unclear whether, at the onset of starvation, these boxes activate or repress spo0A expression, and whether the Spo0A[~]P transcriptional feedback plays a role in the increase in spo0A expression. Based on the experimental data of the promoter activities under systematic perturbation of the promoter architecture, we developed a biophysical model of transcription regulation of spo0A by Spo0A[~]P binding to each of the 0A boxes. The model predicts that Spo0A[~]P binding to its boxes does not affect the RNA polymerase recruitment to the promoters but instead affects the transcriptional initiation rate. Moreover, the effects of Spo0A[~]P binding to 0A boxes are mainly repressive and saturated early at the onset of starvation. Therefore, the increase in spo0A expression is mainly driven by the increase in RNAP holoenzyme levels. Additionally, we reveal that Spo0A[~]P affinity to 0A boxes is strongest at 0A3 and weakest at 0A2 and that there are attractive forces between the occupied 0A boxes. Our findings, in addition to clarifying how the sporulation master regulator is controlled, offer a framework to predict regulatory outcomes of complex gene-regulatory mechanisms. ImportanceCell differentiation is often critical for survival. In bacteria, differentiation decisions are controlled by transcriptional master regulators under transcriptional feedback control. Therefore, understanding how master regulators are transcriptionally regulated is required to understand differentiation. However, in many cases, underlying regulation is complex with multiple transcription factor binding sites and multiple promoters, making it challenging to dissect the exact mechanisms. Here, we address this problem for the B. subtilis master regulator Spo0A. Using a biophysical model, we quantitatively characterize the effect of individual transcription factor binding sites on each spo0A promoter. Further, the model allows to identify the specific transcription step that is affected by transcription factor binding. Such model is promising for the quantitative study of a wide range of master regulators involved in transcriptional feedback.

biophysics↗

bioRxiv2022

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

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 genes 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.

systems biology↗

bioRxiv2021

A general model of the dynamics of genes with dual σ factor preference

Escherichia coli uses {sigma} factors to quickly control large gene cohorts during stress conditions. While most of its genes respond to a single {sigma} factor, approximately 5% of them have dual {sigma} factor preference. The most common are those responsive to both {sigma}70, which controls housekeeping genes, and {sigma}38, which activates genes during stationary growth and stresses. Using RNA-seq and flow-cytometry measurements, we show that {sigma}70+38 genes are nearly as upregulated in stationary growth as {sigma}38 genes. Moreover, we find a clear quantitative relationship between their promoter sequence and their response strength to changes in {sigma}38 levels. We then propose and validate a sequence dependent model of {sigma}70+38 genes, with dual sensitivity to {sigma}38 and {sigma}70, that is applicable in the exponential and stationary growth phases, as well in the transient period in between. We further propose a general model, applicable to other stresses and {sigma} factor combinations. Given this, promoters controlling {sigma}70+38 genes (and variants) could become important building blocks of synthetic circuits with predictable, sequence-dependent sensitivity to transitions between the exponential and stationary growth phases.

systems biology↗

bioRxiv2021

Analytical kinetic model of native tandem promoters in E. coli

Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites dTSS, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS [≤] 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs. Author SummaryTandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs. Thus, their regulation and potentially unique dynamics remain unexplored. We first performed a comprehensive exploration of the conservation of genes regulated by these promoters in E. coli and the properties of their input transcription factors. We then measured protein and RNA levels expressed by 30 Escherichia coli tandem promoters, to establish an analytical model of the expression dynamics of genes controlled by such promoters. We show that start site occlusion and downstream RNAP occupancy can be realistically captured by a model with RNAP binding affinity, the time length of open complex formation, and the nucleotide distance between transcription start sites. This study contributes to a better understanding of the unique dynamics tandem promoters can bring to the dynamics of gene networks and will assist in their use in synthetic genetic circuits.

systems biology↗