Biology subjects

Pilizota, T.

Publications and source records attributed to Pilizota, T..

9 recordsLinked to original sources

bioRxiv2026

Metabolic feedback during bacterial fermentation is a motility brake

We study an unexpectedly fast decay of motility in dense suspensions of Escherichia coli bacteria supplied with excess glucose under anaerobic conditions. The decrease in swimming speed occurs on a timescale inversely proportional to the cell concentration, and is associated with the secretion of organic acids by the bacteria. We show that the decay is driven by the progressive accumulation of non-ionised organic acids in the medium, and develop a chemical kinetic model that successfully predicts the swimming speed variations over a range of conditions in the presence of these acids. We further measure the internal pH of E. coli cells exposed to organic acids, and find that the speed decay coincides with sharp declines in internal pH and metabolic rate. Our findings identify an additional layer of motility control that can arise in complex environments even when motility genes are expressed and energy sources are abundant. This mechanism is likely relevant for understanding bacterial motility in habitats such as the human gut, where high densities of bacteria and organic acids are common.

microbiology↗

bioRxiv2024

Nonlinear dependency of the bacterial flagellar motor speed on proton motive force and its consequences for swimming

The bacterial flagellar motor enables bacteria to swim by rotating helical flagellar filaments that form a bundle at the back of the cell. Escherichia colis motor uses the energy stored in the electrochemical gradient of protons, the proton motive force (PMF), to generate the torque driving this rotation. Until now, motor speed was thought to be proportional to the PMF, irrespective of the viscous load on the motor, and across the physiological range of PMF values. Here, we show that the PMF-speed relationship is non-linear in the high-torque regime. Because saturation in the relationship occurs in the physiologically relevant range, it challenges all current models of motor function that assume a tight coupling between the motor rotation and PMF across the entire physiological range. Furthermore, we experimentally determine the load on the motor experienced by swimming cells, and show that free swimming occurs close to or within the saturation regime, making the observed limiting torque evolutionary relevant.

microbiology↗

bioRxiv2024

A minimalist model lipid system mimicking the biophysical properties of Escherichia coli membrane

Biological membrane are highly complex systems that are of fundamental importance to the development and survival of organisms. Native membranes typically comprise different types of lipids, biomolecules and proteins assembled around a lipid bilayer structure. This complexity can render investigations challenging, with many studies relying on model membranes such as artificial vesicles and supported lipid bilayers (SLBs). The purpose of a model system is to capture the desired dominant features of the native context while remaining uniquely defined and simpler. Here, we search for a minimal lipid-only model system of the Escherichia coli inner membrane. We aim to retain the main lipidomic components in their native ratio while mimicking the membrane thermal and mechanical properties. We design a collection of candidate model systems reflecting the main aspects of the known native lipidomic composition and narrow down our selection based on the systems phase transition temperature. We further test our candidate model systems by independently measuring their elastic properties. We identify 3 ternary model systems able to form stable bilayers that closely mimic E. colis inner membrane lipid composition and mechanical properties. These model systems are made of commercially available synthetic 16:0-18:1 phosphatidylethanolamine (POPE), 16:0-18:1 phosphatidylglycerol (POPG), and 16:0-18:1 Cardiolipin (CL). We anticipate our results to be of interest for future studies making use of E. coli models, for example investigating membrane proteins function or macromolecule-membrane interactions. Significance StatementEscherichia Coli membranes serve as model systems for numerous fundamental and technological applications in the field of membrane biophysics. Being a Gram-negative bacterium, E. Coli is characterized by a thin cell wall that separates two phospholipid membranes: the inner and outer membranes. These membranes are complex, comprising many different lipids, proteins and other biomolecules. Here we develop a minimalist system to mimic the biophysical properties and lipidic composition of E. Colis inner membrane. Using only commercially available lipids, we develop a model membrane that can be used for studies where simplicity is needed to aid interpretation of the results, for example to investigate protein-induced mechano-transduction across E. Coli membranes.

biophysics↗

bioRxiv2021

The proton motive force determines Escherichia coli's robustness to extracellular pH

Maintaining intracellular homeostases is a hallmark of life, and key physiological variables, such as cytoplasmic pH, osmotic pressure, and proton motive force (PMF), are typically interdependent. Using a mathematical model, we argue that near neutral pH homeostasis implies that cells must export ions other than protons to generate physiological electrical potential across their plasma membrane. For Escherichia coli, proton:ion antiporters are the only known cation efflux pumps, and we therefore predict that principal function of antiporters is to generate an out-of-equilibrium plasma membrane potential and so maintain the PMF at the constant levels observed. Consequently, the strength of the PMF determines the range of extracellular pH over which the cell is able to preserve its near neutral cytoplasmic pH, and the non-zero PMF is needed to maintain membrane potential. In support, we concurrently measure the PMF and cytoplasmic pH in single cells and demonstrate both that decreasing the PMFs strength impairs E. colis ability to maintain its pH and that artificially collapsing the PMF destroys the out-of-equilibrium plasma membrane potential. We further predict the observed ranges of extracellular pH for which three of E. colis antiporters are expressed, through defining their cost by the rate at which they divert protons from being imported to generate ATP. Taken together, our results suggest a new perspective on bacterial electrophysiology, where cells regulate the plasma membrane potential to maintain

microbiology↗

bioRxiv2021

Coordination of gene expression with cell size enables Escherichia coli to efficiently maintain motility across conditions

To swim and navigate, motile bacteria synthesize a complex motility machinery involving flagella, motors, and a sensory system. A myriad of studies has elucidated the molecular processes involved, but less is known about the coordination of motility expression with cellular physiology: In Escherichia coli, motility genes are strongly upregulated in nutrient-poor conditions compared to nutrient-replete conditions; yet a quantitative link to cellular motility has not been developed. Here, we systematically investigate gene expression, swimming behavior, and cell growth across a broad spectrum of exponential growth condition. We establish that E. coli up-regulates the expression of motility genes at slow growth to compensate for reduction in cell size, such that the number of flagella per cell is maintained across conditions. The observed 4-5 flagella per cell is the minimum number needed to keep the majority of cells motile. This simple regulatory objective allows E. coli cells to remain motile across a broad range of growth conditions while keeping the biosynthetic and energetic demands to establish and drive the motility machinery at the minimum needed. Given the strong reduction in flagella synthesis resulting from cell size increases at fast growth, our findings also provide a novel physiological perspective on bacterial cell size control: A larger cell-size at fast growth is an efficient strategy to increase the allocation of cellular resources to the synthesis of those proteins required for fast growth, while maintaining processes such as motility which are only needed on a per-cell basis.

microbiology↗

bioRxiv2020

Environmental conditions define theenergetics of bacterial dormancy and itsantibiotic susceptibility

Bacterial cells that stop growing but maintain viability and the capacity to regrow are termed dormant and have been shown to transiently tolerate high concentrations of antimicrobials. The proposed mechanism behind the enhanced survival capabilities of these cells is the reduced energy supply. However, not all reported results are in agreement, and the exact role of energetics remains unsolved. Because dormancy merely indicates growth arrest, which can be induced by various stimuli, we hypothesise that dormant cells may exist in a range of energetic states that depend on the environment. We first establish conditions that are capable of inducing dormancy, and subsequently measure the energy profiles they elicit in single dormant cells. Our simultaneous measurements of proton motive force (PMF), cytoplasmic pH and ATP concentrations confirm that dormant cells exhibit characteristic energetic profiles that can vary in level and dynamics, depending on the stimulus leading to growth arrest. We test whether the energetic makeup is associated with survival to antibiotics of different classes and find that, while growth arrest remains the dominant mechanism enabling survival, some correlations with cellular energetics exist. Our results pave the way to a classification of dormant states based on energy profiles, support a novel relationship between environment and drug susceptibility of dormant cells and suggest that knowledge of the conditions present at the infection site is necessary to design appropriate treatments.

microbiology↗

bioRxiv2019

Comparison of Escherichia coli surface attachment methods for single-cell, in vivo microscopy

For in vivo, single-cell imaging bacterial cells are commonly immobilised via physical confinement or surface attachment. Different surface attachment methods have been used both for atomic force and optical microscopy (including super resolution), and some have been reported to affect bacterial physiology. However, a systematic comparison of the effects these attachment methods have on the bacterial physiology is lacking. Here we present such a comparison for bacterium Escherichia coli, and assess the growth rate, size and intracellular pH of cells growing attached to different, commonly used, surfaces. We demonstrate that E. coli grow at the same rate, length and internal pH on all the tested surfaces when in the same growth medium. The result suggests that tested attachment methods can be used interchangeably when studying E. coli physiology.

systems biology↗

bioRxiv2019

Escherichia coli’s physiology can turn membrane voltage dyes into actuators

The electrical membrane potential (Vm) is one of the components of the electrochemical potential of protons across the biological membrane (proton motive force), which powers many vital cellular processes, and Vm also plays a role in signal transduction. Therefore, measuring it is of great interest, and over the years a variety of techniques has been developed for the purpose. In bacteria, given their small size, Nernstian membrane voltage probes are arguably the favourite strategy, and their cytoplasmic accumulation depends on Vm according to the Nernst equation. However, a careful calibration of Nernstian probes that takes into account the trade-offs between the ease with which the signal from the dye is observed, and the dyes interactions with cellular physiology, is rarely performed. Here we use a mathematical model to understand such trade-offs and, based on the knowledge gained, propose a general work-flow for the characterization of Nernstian dye candidates. We demonstrate the work-flow on the Thioflavin T dye in Escherichia coli, and identify conditions in which the dye turns from a Vm probe into an actuator.\n\nSIGNIFICANCE STATEMENT\n\nThe phospholipid bilayer of a biological membrane is virtually impermeable to charged molecules. Much like in a rechargeable battery, cells harness this property to store an electrical potential that fuels life reactions but also transduces signals. Measuring this electrical potential, also referred to as membrane voltage, is therefore of great interest and a variety of techniques have been employed for the purpose, starting as early as the 1930s. For the case of bacteria, which are smaller in size and possess a stiffer cell wall, arguably the most popular approach to measuring membrane voltage are Nernstian probes that accumulate across the bacterial membrane according to the Nernst potential. The present study characterizes the undesired effects Nernstian probes can have on cell physiology, which can be crucial for the accurate interpretation of experimental results. Using mathematical modelling and experiments, the study provides a general, simple workflow to characterise and minimise these effects.

biophysics↗

bioRxiv2019

Dynamic clustering regulates activity of mechanosensitive membrane channels

Mechanosensitive channels regulate the flux of solutes between the cell and its environment in response to mechanical stimuli. Recent in vitro experiments have suggested that bacterial mechanosensitive channels separate into 2D liquid-like clusters, exhibiting cooperative activity. To understand the functional role of the process and its physical mechanism, we develop a coarse-grained model of mechanosensitive channels embedded in a membrane, exposed to an osmotic shock. We find that the channel aggregation into liquid-like clusters promotes the closure of individual channels. The extent of clustering is highly dependent on the channel concentration and membrane stress. We show that this behaviour yields a tightly regulated gating system, whereby at high tensions channels gate individually, and at lower tensions the channels spontaneously aggregate and inactivate. We suggest that this positive feedback protects the cell against excessive loss of cytoplasmic content. Our results point to a novel role of functional micro-phase separation in the cell membrane.

biophysics↗