Biology subjects

Bokinsky, G.

Publications and source records attributed to Bokinsky, G..

8 recordsLinked to original sources

bioRxiv2026

Conformational selection and redox-dependent destabilization in the IspG-FldA electron transfer complex

Electron transfer between proteins is mediated by transient complexes that sample many binding orientations, only a subset productive. Protein electron carriers (PECs) form such complexes with diverse enzymes across central metabolism, yet no general framework predicts which PEC-enzyme pairs will function. A representative case is IspG, the iron-sulfur enzyme catalyzing the penultimate methylerythritol phosphate pathway reaction, whose [4Fe-4S] cluster must be reduced after each turnover by the Escherichia coli flavodoxin FldA. It is not known whether FldA engages the open (substrate-free) or closed (substrate-bound) conformation of IspG, or how the cofactor redox states shape their interaction. Using atomistic molecular dynamics with custom cofactor parameters, we find that FldA preferentially engages the open conformation, implying that reduction precedes substrate binding. A positively charged arginine patch anchors FldA, and a single residue, Tyr58, provides a short tunneling bridge to the cluster. Electron transfer then weakens this interface, driving the complex toward dissociation -- pointing to an intrinsic mechanism by which a PEC binds its targets transiently yet productively: conformational selection recruits the partner, and electron transfer releases it.

biophysics↗

bioRxiv2025

Coordination of membrane synthesis with cell growth: localization of the Escherichia coli phospholipid synthesis enzyme PlsB responds to membrane abundance in a manner consistent with filamentation-mediated inhibition

Cell viability demands tight coordination between growth and the synthesis of new membrane. The mechanisms coordinating cell growth with membrane synthesis in any organism are unclear. In Escherichia coli, initiation of phospholipid synthesis by the glycerol-3-phosphate acyltransferase PlsB is regulated by cell growth via an unknown allosteric mechanism. Previous studies have established that PlsB assembles into enzymatically inactive, membrane-bound filaments when overexpressed. We propose that PlsB filamentation regulates PlsB activity and coordinates membrane synthesis with growth. Here, we test our hypothesis by observing the localization of fluorescently labelled PlsB using live-cell fluorescence microscopy. During growth, PlsB localizes as discrete foci, consistent with formation of inactive filaments. Reducing cellular membrane content eliminates PlsB foci and delocalizes PlsB into the cytoplasm. Restoring membrane synthesis causes PlsB foci to reform after a delay. These results are consistent with our hypothesis and suggest a model in which PlsB reversibly assembles into inactive filaments in response to phospholipid abundance. This mechanism establishes a negative feedback loop that controls initiation of phospholipid synthesis by PlsB and effectively coordinates membrane synthesis with growth.

microbiology↗

bioRxiv2025

Transcriptional competition biases the effects of second messengers in E. coli

Cells regulate gene expression by balancing transcriptional resources across different functional groups of genes. In Escherichia coli, second messengers such as ppGpp and cAMP control ribosome biogenesis and metabolic gene expression, respectively. While these regulators are typically studied in isolation, we provide a theory showing that their effects are intertwined due to global transcriptional competition. Using experimental data from RelA overexpression and a mechanistic modeling framework, we show that ppGpp-mediated repression of ribosomal genes competes for transcriptional resources with cAMP-driven activation of catabolic genes. This competition reshapes proteome allocation in a way that transcends individual regulators. Our findings challenge common modeling assumptions about transcription factor action and revive classical ideas suggesting that large-scale gene regulation should be studied within the broader context of resource availability, with implications for understanding cellular regulation across diverse biological systems, beyond bacterial physiology.

systems biology↗

bioRxiv2024

Exogenous fatty acids inhibit fatty acid synthesis through competition between endogenously- and exogenously-generated substrates for phospholipid synthesis in Escherichia coli

Exogenous fatty acids are directly incorporated into bacterial membranes, heavily influencing bacterial ecology and antibiotic susceptibility. We use liquid chromatography/mass spectrometry to characterize how exogenous fatty acids impact the Escherichia coli fatty acid synthesis pathway. We find that acyl-CoA synthesized from exogenous fatty acids rapidly increases long-chain acyl-ACP levels while depleting malonyl-ACP, indicating inhibition of fatty acid synthesis. Contrary to previous assumptions, acyl-CoA does not inhibit FabI in vivo; instead, substrate competition between acyl-CoA and acyl-ACP for phospholipid synthesis enzymes causes long-chain acyl-ACP to accumulate, inhibiting fatty acid synthesis initiation. Furthermore, changes in the acyl-ACP pool driven by acyl-CoA amplify the effects of exogenous fatty acids on the balance between saturated and unsaturated membrane lipids. Transcriptional regulation rebalances saturated and unsaturated acyl-ACP by adjusting FabA and FabB expression. Remarkably, all other fatty acid synthesis enzymes remain at stable levels, maintaining a fixed synthesis capacity despite the availability of exogenous fatty acids. Since all bacterial pathways for exogenous fatty acid incorporation characterized so far converge with endogenous synthesis pathways in a common substrate pool, we propose that the substrate competition-triggered feedback mechanism identified here is ubiquitous across bacterial species.

molecular biology↗

bioRxiv2023

A temperature-sensitive metabolic valve and a transcriptional feedback loop drive rapid homeoviscous adaptation in Escherichia coli

All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. A quantitative description of how organisms maintain constant fluidity at all growth temperatures has not been achieved. By quantifying both enzymes and metabolic intermediates of the Escherichia coli fatty acid and phospholipid synthesis pathways, we discover how E. coli measures steady-state temperature and restores optimal membrane fluidity within a single generation after temperature shocks. The first element of the system is a temperature-sensitive metabolic valve that allocates flux between the saturated and unsaturated fatty acid synthesis pathways. The second element is a transcription-based negative feedback loop that counteracts the temperature-sensitive valve. The combination of these elements accelerates membrane adaptation by causing a transient overshoot in the synthesis of saturated or unsaturated fatty acids following temperature shocks. This overshoot strategy accelerates membrane adaptation, and is comparable to increasing the temperature of a water bath by adding water that is excessively hot rather than adding water at the desired temperature. These properties are captured in a quantitative model, which we further use to show how hard-wired parameters calibrate the system to generate membrane compositions that maintain constant fluidity across a wide range of temperatures. We hypothesize that core design features of the E. coli system will prove to be ubiquitous features of homeoviscous adaptation systems.

microbiology↗

bioRxiv2021

Resolving phylogenetic and biochemical barriers to functional expression of heterologous iron-sulphur cluster enzymes

Many of the most promising applications of synthetic biology, including engineering of microbes for renewable chemical production, relies upon the ability of genetically-tractable hosts to express heterologous enzymes from foreign species. While countless methods for facilitating heterologous enzyme expression have been developed, comparable tools for facilitating heterologous enzyme activity are generally lacking. Such tools are needed to fully exploit the biosynthetic potential of the natural world. Here, using the model bacterium Escherichia coli, we investigate why iron-sulphur (Fe-S) enzymes are often inactive when heterologously expressed. By applying a simple growth complementation assay with collections of Fe-S enzyme orthologs from a wide range of prokaryotic diversity, we uncover a striking correlation between phylogenetic distance and probability of functional expression. Moreover, co-expression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be functionally expressed. On the other hand, we find that heterologous Fe-S enzymes that require specific electron carrier proteins within their natural host are rarely functionally expressed unless their specific reducing partners are identified and co-expressed. We demonstrate in vitro that such selectivity in part derives from a need for low-potential electron donors. Our results clarify how phylogenetic distance and electron transfer biochemistry each separately impact functional heterologous expression and provide insight into how these barriers can be overcome for successful microbial engineering involving Fe-S enzymes.

synthetic biology↗

bioRxiv2020

ppGpp is a bacterial cell size regulator

Growth and division are central to cell size. Bacteria achieve size homeostasis by dividing when growth has added a constant size since birth, termed the "adder" principle, by unknown mechanisms [1-4]. Growth is well known to be regulated by ppGpp, which controls anything from ribosome production to metabolic enzyme activity and replication initiation, and whose absence or excess can induce the stress response, filamentation, and yield growth-arrested miniature cells [5-8]. These observations raise unresolved questions about the relation between ppGpp and size homeostasis mechanisms during normal exponential growth. Here, to untangle effects of ppGpp and nutrients, we gained control of cellular ppGpp by inducing the synthesis and hydrolysis enzymes RelA and Mesh1. We found that ppGpp not only exerts control over the growth rate, but also over cell division and hence the steady state cell size. The added size responds rapidly to changes in the ppGpp level, aided by transiently accelerated or delayed divisions, and establishes its new constant value while the growth rate still adjusts. Moreover, the magnitude of the added size and resulting steady-state birth size correlate consistently with the ppGpp level, rather than with the growth rate, which results in cells of different size that grow equally fast. Our findings suggest that ppGpp serves as a critical regulator that coordinates cell size and growth control.

microbiology↗

bioRxiv2019

Membrane synthesis in Escherichia coli is coordinated with growth via post-translational regulation

Every cell must produce enough membrane to contain itself. However, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. By comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of Escherichia coli across a 3-fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, PlsB. Due to feedback regulation of the fatty acid pathway, PlsB activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. Surprisingly, concentrations of the enzyme that catalyses the committed step of lipopolysaccharide synthesis (LpxC) do not vary across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by PlsB. In contrast to steady-state regulation, we find that responses to environmental perturbations are triggered directly via changes in acetyl-CoA concentrations, which enables rapid adaptation. Adaptations are further modulated by ppGpp, which regulates PlsB activity during slow growth and growth arrest. The strong reliance of the membrane synthesis pathway upon post-translational regulation ensures both reliability and responsiveness of membrane synthesis. SignificanceHow do bacteria cells grow without breaking their membranes? Although the biochemistry of fatty acid and membrane synthesis is well-known, how membrane synthesis is balanced with growth and metabolism has remained unclear. This is partly due to the many control points that have been discovered within the membrane synthesis pathways. By precisely establishing the contributions of individual pathway enzymes, our results simplify the model of membrane biogenesis in the model bacteria species Escherichia coli. Specifically, we find that allosteric control of a single enzyme, PlsB, is sufficient to balance growth with membrane synthesis and to ensure that growing E. coli produces sufficient membrane. Identifying the signals that activate and deactivate PlsB will answer the question of how membrane synthesis is synchronized with growth.

microbiology↗