Biology subjects

Sourjik, V.

Publications and source records attributed to Sourjik, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Antagonistic small proteins enable magnesium-dependent tuning of two-component signaling

Small proteins are emerging regulators of bacterial virulence via signaling pathways. However, how multiple small proteins coordinate to control a single pathway remains unclear. Here we show that two antagonistic small proteins, SafA and MgrB, forming a magnesium-dependent control module, enhance the signaling sensitivity of the PhoQ/PhoP two-component system in E. coli. Leveraging conditions that enable sequential expression of SafA and MgrB, we dissect and characterize their regulatory dynamics. Our results reveal magnesium as the central control parameter modulating the abundance and affinities of the small proteins for the sensor kinase PhoQ. This coupling together with the competitive binding of the small proteins enables self-adjustment of the signaling network and enhances its sensitivity to environmental magnesium changes. Functionally, loss of either small proteins influences the ability of enteropathogenic E. coli to evade macrophage phagocytosis, linking this regulatory scheme to host interaction. Our findings establish a framework for achieving a high level of input sensitivity through antagonistic regulations in a signaling network.

microbiology↗

bioRxiv2026

Repurposing a chromosome segregation ParB-CTPase fold into an ATPase toxin for contact-dependent growth inhibition in plant and animal pathogens

Bacterial competition drives the evolution of antibacterial mechanisms, yet how new activities arise remains poorly understood. A major route to innovation is the reuse of pre-existing genetic systems, whereby conserved protein modules are repurposed in new biological contexts to generate new capabilities. Here, we show that the ParB-CTPase fold, a conserved nucleotide-binding module best known for its role in chromosome segregation, can be functionally repurposed as an antibacterial toxin. We identify ToxB, a ParB-like domain embedded within the polymorphic toxin region of contact-dependent inhibition systems and show that it functions as a potent antibacterial effector. Structural and biochemical analyses reveal that ToxB retains the core architecture of the ParB-CTPase fold but lacks DNA-binding capability and preferentially binds ATP. This shift in nucleotide specificity underpins a distinct mode of action, in which ATP binding and hydrolysis trigger rapid nucleoid compaction, chromosome segregation defects, oxidative stress, cell chaining, and ultimately cell lysis. ToxB also exhibits toxic activity in plant cells, suggesting that it targets conserved cellular processes. Together, these findings provide direct experimental evidence that the ParB-NTPase fold is biologically versatile and can be repurposed for biological roles fundamentally distinct from its ancestral function in DNA segregation.

microbiology↗

bioRxiv2026

Western diet suppresses canonical intestinal stem cells and reprograms c-Kit+ reserve stem cells via proinflammatory dysbiosis

While colorectal cancer (CRC) is thought to originate primarily from resident stem cells, diet heavily influences intestinal stem cell homeostasis and disease onset. In this study, we demonstrate that a Western-style diet (WSD) alters intestinal homeostasis by uncoupling canonical stemness from tumorigenesis. WSD exposure suppresses canonical Lgr5 stem cells while activating an alternative pool of facultative c-Kit secretory cells. Although these reprogrammed stem-like cells exhibit genotoxic stress, they remain proliferative under prolonged dietary exposure, suggesting increased susceptibility to tumor-initiating mutations. We identify diet-induced microbial shifts and the expansion of enterotoxigenic Bacteroides fragilis (ETBF) as the upstream driver. ETBF and its toxin fragilysin autonomously trigger multipotency in c-Kit+ cells through Wnt/{beta}-catenin and YAP signaling. Importantly, these alterations are fully reversible upon dietary intervention. Together, our results highlight a targetable dietary-microbial axis that shapes epithelial stemness and underscores the role of facultative stem cells in diet-induced CRC onset.

cell biology↗

bioRxiv2025

Effects of multiple cell regulators on curli gene expression in Escherichia coli

Curli amyloid fibers is the key protenacious component of extracellular matrix in Escherichia coli. Regulation of curli expression is highly complex and depends on the cellular response to diverse environmental conditions. Although many genetic determinants of curli production are known, previous studies were performed under diverse conditions and using different strains of E. coli or Salmonella. Furthermore, the origin of bimodal curli expression in bacterial population remains unknown. Here, we systematically investigated the role of multiple cell factors in expression of curli structural genes csgBA in unstructured planktonic E. coli culture. We observed that multiple regulators involved in regulation of stress response, cell motility, cell physiology and metabolism, as well as in formation of extracellular matrix and maintenance of DNA architecture modulated expression of curli by either promoting or repressing activity of csgBA. We further elucidated which regulators act upstream of the master transcription factor CsgD and which are crucial for bimodality of curli gene expression. We also investigated the impact of individual diguanylate cyclases on the regulation of the csgBA activity. This study provides an overview of regulation of curli gene expression in planktonic E. coli culture in the absence of any microenvironmental gradients.

microbiology↗

bioRxiv2025

Response of Escherichia coli chemotaxis pathway to pyrimidine deoxyribonucleosides

Nucleosides are essential components of all living cells. Bacteria use salvage pathways to import nucleosides from their environment and to utilize them for nucleic acid biosynthesis, but also as alternative sources of carbon, nitrogen and energy. Motile bacteria commonly show chemoattraction towards nutritionally valuable compounds, and in this work, we demonstrate that the chemotaxis pathway of Escherichia coli exhibits specific attractant response to pyrimidine nucleosides. Particularly sensitive response, in the sub-micromolar range, was observed for deoxyribonucleosides thymidine and deoxycytidine. In contrast, ribonucleosides elicited weaker and less sensitive response, and no response to pyrimidine nucleobases was observed in the micromolar range of concentrations. Our subsequent analysis revealed that the pathway response to pyrimidine deoxyribonucleosides is mediated by the minor E. coli chemoreceptor Tap. The observed narrow dynamic range of this response indicates that sensing of deoxyribonucleosides is indirect, likely via an unknown periplasmic binding protein that interacts with Tap.

microbiology↗

bioRxiv2025

Specificities of Chemosensory Receptors in the Human Gut Microbiota

The human gut is rich in metabolites and harbors a complex microbial community, yet the sensory repertoire of its commensal bacteria remains largely uncharacterized. Here we systematically mapped ligand specificities of extracytoplasmic sensory domains from twenty members of the human gut microbiota, with a primary focus on the abundant and physiologically important class of Clostridia. We identified diverse metabolites as specific stimuli for three major functional classes of transmembrane receptors. We further characterized novel subsets of sensors belonging to the Cache superfamily, specific for lactate, dicarboxylic acids, and for uracil and short-chain fatty acids (SCFAs), respectively, and investigated the evolution of their ligand specificity. Structural and biochemical analysis of the newly described dCache_1UR domain revealed an independent binding of uracil and SCFA at distinct modules. Altogether, we could identify or predict specificities for over a half of the Cache-type chemotactic sensors in the selected gut commensals, with the carboxylic acids representing the largest class of ligands. Among those, the most commonly found specificities were for lactate and formate, indicating particular importance of these metabolites in the human gut microbiome and consistent with their observed beneficial impact on the growth of selected bacterial species.

microbiology↗

bioRxiv2024

Collective dynamics of Escherichia coli growth under near-lethal acid stress

Many neutralophilic bacteria, including Escherichia coli, can withstand acidic conditions due to the action of several protective mechanisms. While the survival of E. coli under growth-inhibitory extreme acid stress is well understood, less is known about the physiology of E. coli growth under severe but permissive acidity. Here, we observed that growth of E. coli MG1655 in a rich medium with pH of 4.1 to 4.4 exhibits a characteristic multi-phasic growth pattern, consisting of an initial exponential elongation in the absence of cell division, followed by growth arrest and subsequent resumption of exponential growth. The duration of the growth arrest phase was strongly dependent on the pH of the medium, but also on the initial cell density of the culture, suggesting the collective nature of this phenomenon. Cell-density dependent multiphasic growth at the near-lethal pH, including the initial increase in cell volume associated with either elongation or widening of the cell body, was also observed for all tested natural E. coli isolates and for Salmonella enterica serovar Typhimurium. Such transient increase in volume apparently enables bacteria to induce acid resistance systems, including the lysine-dependent Cad system, to subsequently modify pH of the medium in the density-dependent manner. Consistent with the collective recovery, even a minor fraction of acid-tolerant cells could fully cross-protect acid-sensitive cells enabling them to resume growth in coculture. Thus, collective dynamics plays a central role in bacterial growth under near-lethal acid stress. ImportanceSince many E. coli and other Enterobacteriaceae isolates are gastrointestinal pathogens for humans, it is important to understand their growth under acidic conditions imposed by the host physiology in the stomach, colon, and inside macrophages. Here we show that E. coli growth under near-lethal acidic conditions is a collective phenomenon that critically depends on cell density. This collective behavior is favored by changes in cell morphology as a response to high acidity. The observed density dependence might have implications for pathogen proliferation in the acidic environment of the human gastrointestinal tract and possibly also for their interactions with immune cells.

microbiology↗

bioRxiv2024

A toolbox of FRET-based c-di-GMP biosensors and its FRET-To-Sort application for genome-wide mapping of the second messenger regulatory network

C-di-GMP is a widespread second messenger, coordinating various cellular functions in bacteria. Levels of c-di-GMP can be highly dynamic and vary over a wide range of concentrations. Here we constructed a large set of FRET-based c-di-GMP biosensors, using homologues of c-di-GMP-binding effector YcgR from different bacterial species. This biosensor library was characterized using a newly established protocol to quantify FRET efficiency using flow cytometry. The resulting toolbox of 18 selected biosensors that undergo large FRET signal change upon c-di-GMP binding displays a [~]100-fold range of c-di-GMP binding affinities. We combined this toolbox with a barcoded Tn5 transposon library and cytometry-based cell sorting to develop FRET-To-Sort, a new application for systematic characterization of gene networks regulating levels of FRET-detected small molecules. Applied to planktonic E. coli cells, FRET-To-Sort identified both known and novel regulatory modules controlling c-di-GMP levels, including flagellum and fimbria biogenesis, lipid metabolism and stress response genes.

microbiology↗

bioRxiv2024

The SPFH complex HflK-HflC regulates aerobic respiration in bacteria

The bacterial HflK-HflC membrane complex is a member of the highly conserved SPFH protein family, which is found throughout all domains of life and includes eukaryotic stomatins, flotillins, and prohibitins. These proteins organize cell membranes and are involved in various processes. However, the exact physiological functions of most bacterial SPFH proteins remain unclear. Here, we report that the HflK-HflC complex in Escherichia coli is required for growth under high aeration. The absence of this complex causes an aerobic growth defect due to a reduced abundance of IspG, a crucial enzyme in the isoprenoid biosynthetic pathway. This reduction leads to lower levels of ubiquinone, reduced respiration, lower ATP levels, and misregulated expression of respiratory genes. The regulation of aerobic respiration by the HflK-HflC complex resembles the mitochondrial respiratory defects caused by prohibitin mutations in mammalian and yeast cells, suggesting a functional commonality between these bacterial and eukaryotic SPFH proteins.

microbiology↗

bioRxiv2024

Physics and physiology determine strategies of bacterial investment in flagellar motility

Regulatory strategies that allow microorganisms to balance their investment of limited resources in different physiological functions remain poorly understood, particularly for numerous cellular functions that are not directly required for growth. Here, we investigate the allocation of resources to flagellar swimming, the most prominent and costly behavior in bacteria that is not directly required for growth. We show that the dependence of motile behavior on gene expression in Escherichia coli is determined by the hydrodynamics of propulsion, which limits the ability of bacteria to increase their swimming by synthesizing more than a critical number of flagellar filaments. Together with the fitness cost of flagellar biosynthesis, this defines the physiologically relevant range of investment in motility. Gene expression in all E. coli isolates tested falls within this range, with many strains maximizing motility under nutrient-rich conditions, particularly when grown on a porous medium. The hydrodynamics of swimming may further explain the bet-hedging behavior observed at low levels of motility gene expression.

microbiology↗

bioRxiv2024

Enhanced metabolic entanglement emerges during the evolution of an interkingdom microbial community

Metabolic interactions are common in microbial communities and are believed to be a key factor in the emergence of complex life forms. However, while different stages of mutualism can be observed in nature, the dynamics and mechanisms underlying the gradual erosion of independence of the initially autonomous organisms are not yet fully understood. In this study, we conducted the laboratory evolution of an engineered microbial community and were able to reproduce and molecularly track its stepwise progression towards enhanced partner entanglement. The evolution of the community both strengthened the existing metabolic interactions and led to the emergence of de novo interdependence between partners for nitrogen metabolism, which is a common feature of natural symbiotic interactions. Selection for enhanced metabolic entanglement repeatedly occurred indirectly, via pleiotropies and trade-offs within cellular regulatory networks. This indicates that indirect selection may be a common but overlooked mechanism that drives the evolution of mutualistic communities.

evolutionary biology↗

bioRxiv2024

Experimental evolution of a reduced bacterial chemotaxis network

Chemotaxis allows bacteria to follow chemical gradients by comparing their environment over time and adjusting their swimming behavior accordingly. The chemotaxis signaling pathway is highly conserved among all chemotactic bacteria. The system comprises two modules: one for environmental sensing and signal transduction toward the flagellar motor, and the other for adapting to the constant level of background stimulation and providing short-term memory for temporal comparisons. Previous experimental analysis and mathematical modeling have suggested that all components of the paradigmatic chemotaxis pathways in Escherichia coli are essential. This indicates that it may contain a minimal set of protein components necessary to mediate gradient sensing and behavioral response. To test this assumption, here we subjected strains carrying deletions in chemotaxis genes to experimental laboratory evolution. We observed that the core components of the chemotaxis pathway are indeed essential. However, the absence of individual auxiliary pathway proteins, including the adaptation enzymes that are conserved in a vast majority of bacteria, and the phosphatase, could be compensated for to varying degrees by changes in other pathway components. Our results suggest that the experimental evolution of these deletion strains has led to the emergence of alternative strategies for bacterial chemotaxis, demonstrating the surprisingly rapid evolvability of this signaling network.

microbiology↗

bioRxiv2023

Systematic mapping of chemoreceptor specificities for Pseudomonas aeruginosa

The chemotaxis network, one of the most prominent prokaryotic sensory systems, is present in most motile bacteria and archaea. Although the conserved signaling core of the network is well characterized, ligand specificities of a large majority of diverse chemoreceptors encoded in bacterial genomes remain unknown. Here we performed a systematic identification and characterization of new chemoeffectors for the opportunistic pathogen Pseudomonas aeruginosa, which has 26 chemoreceptors possessing most of the common types of ligand binding domains. By performing capillary chemotaxis assays for a library of growth-promoting compounds, we first identified a number of novel chemoattractants of varying strength. We subsequently mapped specificities of these ligands by performing Forster resonance energy transfer (FRET) and microfluidic measurements for hybrids containing ligand binding domains of P. aeruginosa chemoreceptors and the signaling domain of the Escherichia coli Tar receptor. Direct binding of ligands to chemoreceptors was further confirmed in vitro using thermal shift assay and microcalorimetry. Altogether, the combination of methods enabled us to assign several new attractants, including methyl 4-aminobutyrate, 5-aminovalerate, L-ornithine, 2-phenylethylamine and tyramine, to previously characterized chemoreceptors and to annotate a novel purine-specific receptor PctP. Our screening strategy could be applied for the systematic characterization of unknown sensory domains in a wide range of bacterial species. ImportanceChemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands, and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.

microbiology↗

bioRxiv2022

Dynamic bimodality of curli expression in planktonic cultures of Escherichia coli is stabilized by cyclic-di-GMP regulation

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli expression occurs not only in submerged and macrocolony biofilms, but also in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells under all tested growth conditions. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of c-di-GMP regulation. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

microbiology↗

bioRxiv2022

Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions and cell growth

Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli, and, for the faster-diffusing proteins, it is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.

biophysics↗

bioRxiv2021

Construction of multicellular yeast networks using the communication toolkit with variable specificity and attenuation

The key next step in synthetic biology is to extend cellular network engineering to the multicellular level by utilizing cell-cell communication for information processing. To facilitate the implementation of multicellular networks in the most commonly used eukaryotic chassis, Saccharomyces cerevisiae, we developed the yeast communication toolkit (YCTK). This toolkit is based on the fungal mating pathway and contains five pheromone-inducible promoters (response parts), eleven pheromones (-factors; sender parts), eleven pheromone receptors (Ste2; receiver parts), as well as five Bar1 proteases (suppressor parts). All YCTK parts were thoroughly characterized and are compatible with the commonly used yeast Golden Gate cloning standard. We demonstrated the application of the YCTK by implementing several different logic gate-like population networks. Furthermore, we used this toolkit to investigate the pheromone-receptor promiscuity patterns among different yeast species. This toolkit extends currently available resources for construction of complex multicellular eukaryotic networks with varying degrees of promiscuity and attenuation.

synthetic biology↗

bioRxiv2019

BiofilmQ, a software tool for quantitative image analysis of microbial biofilm communities

Biofilms are now considered to be the most abundant form of microbial life on Earth, playing critical roles in biogeochemical cycles, agriculture, and health care. Phenotypic and genotypic variations in biofilms generally occur in three-dimensional space and time, and biofilms are therefore often investigated using microscopy. However, the quantitative analysis of microscopy images presents a key obstacle in phenotyping biofilm communities and single-cell heterogeneity inside biofilms. Here, we present BiofilmQ, a comprehensive image cytometry software tool for the automated high-throughput quantification and visualization of 3D and 2D community properties in space and time. Using BiofilmQ does not require prior knowledge of programming or image processing and provides a user-friendly graphical user interface, resulting in editable publication-quality figures. BiofilmQ is designed for handling fluorescence images of any spatially structured microbial community and growth geometry, including microscopic, mesoscopic, macroscopic colonies and aggregates, as well as bacterial biofilms in the context of eukaryotic hosts.

microbiology↗

bioRxiv2018

The protein architecture of the endocytic coat analyzed by FRET

Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast. We assessed proximities of 18 conserved coat-associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat is stratified into several functional layers situated above and below the clathrin lattice with key proteins transversing through the lattice deeply into the cytoplasm. We propose that this conserved design enables an efficient and regulated function of the endocytic coat during endocytosis.

cell biology↗