Biology subjects

Zietek, M.

Publications and source records attributed to Zietek, M..

4 recordsLinked to original sources

bioRxiv2025

Cis non-coding genetic variation drives gene expression changes in the E. coli and P. aeruginosa pangenomes

Bacteria use gene regulation to dynamically adapt to changes in their environment, including resistance to stress and the occupation of new niches. Gene expression is known to vary within a species pangenome, but the extent to which these changes could be explained by genetic variants in cis non-coding regions has so far been poorly investigated. Statistical genetics offers a hypothesis-free approach to this problem, as opposed to mechanistic models, which can be used only for reference isolates that are not representative of the whole species. In this study, we assembled two genomic and transcriptomic datasets for Escherichia coli (N=117) and Pseudomonas aeruginosa (N=413) and identified associations between genetic variants in cis non-coding regions and recorded gene expression variation. We identified at least one associated variant in up to 39% of the tested genes in both species. We partly validated the associations in-silico and in-vitro for E. coli, reinforcing the difficulty of identifying a single mechanism generating gene expression diversity. We then investigated the relevance of non-coding variants in explaining the variability in antimicrobial resistance in both species using two additional publicly available datasets, identifying a large number of these variants across antimicrobial compounds. This work confirms the role of genetic variation in often overlooked regions of bacterial genomes in influencing molecular and clinically relevant phenotypes.

microbiology↗

bioRxiv2025

Cell Painting in activated cells illuminates phenotypic dark space and uncovers novel drug mechanisms of action.

As drug and natural product libraries expand, assays for assessing mechanisms of action (MoA) are increasingly critical. Performing cytological profiling using the Cell Painting (CP) assay enables image-based profiling of cellular states upon treatment, yet many bioactive compounds remain uncharacterized due to undetectable cellular effects under standard conditions. To address this, we combined drug dosing with cell activation using the protein kinase C (PKC) agonist phorbol myristate acetate (PMA). Profiling A549 lung cancer cells treated with 8,387 compounds at two concentrations (1 and 10 {micro}M) in both resting and PMA-activated states allowed us to detect phenotypic effects for up to 40% of all screened compounds, effectively illuminating new phenotypic "dark space". Over 1,000 compounds exhibited phenotypes exclusively under PMA activation, establishing its advantage for MoA studies. We introduce novel quality control measures for CP screens and demonstrate that integrating phenotypic signatures enhances MoA discovery. Notably, 2-methoxycinnamaldehyde clustered with glucocorticoid receptor modulators and induced nuclear translocation, emphasizing the power of this approach in uncovering novel drug mechanisms and, therefore, aiding in improving therapeutic strategies.

systems biology↗

bioRxiv2022

Bacterial cell widening alters periplasmic size and activates envelope stress responses

The Rcs signal transduction system is a phosphorelay responsible for sensing a wide variety of enterobacterial cell envelope stresses. In Escherichia coli, the Rcs system is required to survive A22 and mecillinam treatment, two drugs that perturb cell size. To test whether cell size changes might be correlated with envelope damage and thereby sensed by the Rcs system, we tuned E. coli cell size via drug inhibition with A22, point mutations to the cell-shape determinant MreB, and mechanically confined growth. In all conditions, cell width was strongly correlated with Rcs activation, with wider cells exhibiting more activation than wild-type. In all conditions, RcsF, the outer membrane-localized upstream component of the Rcs system, was essential for responding to cell width changes. Consistently, several envelope gene deletions known to induce the Rcs system via RcsF resulted in cells that were wider than wild-type. Cryo- electron microscopy revealed that the periplasm of a wide MreB mutant was on average [~]3 nm thinner than wild-type, thereby bringing RcsF closer to the downstream components of the signaling cascade in the inner membrane. Conversely, extending the flexible linker region of RcsF by [~]3 nm increased Rcs activity in wild-type cells. In summary, we propose that the Rcs system responds to changes in cell width because of altered periplasmic thickness.

microbiology↗

bioRxiv2022

Modulation of bacterial cell size and growth rate via activation of a cell envelope stress response

Fluctuating conditions and diverse stresses are typical in natural environments. In response, cells mount complex responses across multiple scales, including adjusting their shape to withstand stress. In enterobacteria, the Rcs phosphorelay is activated by cell envelope damage and by changes to periplasmic dimensions and cell width. Here, we investigated the physiological and morphological consequences of Rcs activation in Escherichia coli in the absence of stresses, using an inducible version of RcsF that mislocalizes to the inner membrane, RcsFIM. Expression of RcsFIM immediately reduced cellular growth rate and the added length per cell cycle in a manner that was directly dependent on induction levels, but independent of Rcs-induced capsule production. At the same time, cells increased intracellular concentration of the cell division protein FtsZ, and decreased the distance between division rings in filamentous cells. Depletion of the Rcs negative regulator IgaA phenocopied RcsFIM induction, indicating that IgaA is essential due to growth inhibition in its absence. However, A22 treatment did not affect growth rate or FtsZ intracellular concentration, despite activating the Rcs system. These findings suggest that the effect of Rcs activation on FtsZ levels is mediated indirectly through growth-rate changes, and highlight feedbacks among the Rcs stress response, growth dynamics, and cell-size control.

microbiology↗