Biology subjects

Buda, K.

Publications and source records attributed to Buda, K..

8 recordsLinked to original sources

bioRxiv2026

Short-term effects of experimental artificial light at night on nocturnal vocal activity in a temperate bird community

Artificial light at night (ALAN) can alter the daily timing of birds activity, but the immediate effects of brief nocturnal illumination on vocal behaviour remain poorly understood. We experimentally examined whether short-term exposure to a local artificial light source affected nocturnal vocalisation rates and whether responses differed among species and between stages of the breeding season. Experiments were conducted at ten sampling points in western Poland, during nautical night in the early and late stages of the breeding season. Bird vocalisations were recorded continuously for 30 min before illumination, for 38 min during gradual and full illumination, and for 30 min after the lights were switched off. We recorded 28,839 vocalisations from 13 species. Of the five species analysed formally, only the Eurasian coot showed a significant illumination effect, with a higher vocalisation rate after illumination than before or during illumination. The sedge warbler showed higher estimated rates during and after illumination than before illumination, but the overall phase effect was not significant after false-discovery-rate correction. No phase effects were detected in the common nightingale, common quail, or Savis warbler. Descriptive patterns show increased vocal activity during or after illumination in the Eurasian skylark, common cuckoo, and corn crake, but limited spatial replication prevented formal testing. Overall, brief illumination did not produce a clear and consistent response across the recorded species. Short-term ALAN exposure may affect nocturnal vocal activity with some delay, but responses appear to be strongly species-specific. The observed patterns identify candidate species for further controlled experimental study.

ecology↗

bioRxiv2026

Species-specific responses of diurnal birds to nocturnal conspecific song playback

The vast majority of birds are diurnal and concentrate their vocal activity during daylight hours. However, some diurnal birds can also be vocally active at night, although the functions of this phenomenon remain poorly understood. We conducted playback experiments in the Warta Landscape Park (central Poland) to determine whether nocturnal singing by diurnal birds serves breeding-related functions by analysing responses to playback of songs from unfamiliar conspecific males. The sedge warbler (Acrocephalus schoenobaenus) was selected as the focal species because it exhibits relatively high levels of nocturnal song activity, while nine additional diurnal species detected near focal sedge warbler territories were included to explore whether responsiveness to nocturnal conspecific song extended across a broader taxonomic range. Playback experiments were conducted during the early and late stages of the breeding season and during the early and late parts of the nautical night. Out of 10 species tested, three responded vocally: sedge warbler, Savi s warbler (Locustella luscinioides), and common snipe (Gallinago gallinago). Sedge warblers did not modify song rate and song duration but increased flight activity after nocturnal playback. Savi s warblers and common snipes produced more vocalisations after playback than before in May, a pattern consistent with territorial defence function. General nocturnal vocal activity was higher at the beginning of the season, suggesting that birds motivation to establish territories and form pairs can extend into the night, providing additional benefits. Moreover, the probability of singing by the sedge warbler was higher in the latter part of the night. Our study demonstrates that nocturnal stimulation of foreign male playback of diurnal birds can elicit vocal responses from conspecifics, suggesting that nocturnal singing can occur in the absence of obvious artificial light pollution, but in the case of some species and environmental conditions, it may contribute to nocturnal social communication, especially in the early breeding season.

animal behavior and cognition↗

bioRxiv2026

Substrate-dependent epistasis probes active site intramolecular wiring

Enzyme adaptation toward novel substrates involves the rewiring of intramolecular residue networks, yet how this rewiring differs across multiple substrates, and how it underpins functional trade-offs and promiscuity, remains poorly understood. Here, we profile all 64 combinations of six key mutations in a phosphotriesterase across nine structurally diverse substrates spanning three chemical classes (organophosphates, esters, and lactones), thus generating a multi-dimensional map of epistasis and promiscuity within the phosphotriesterases active site. We developed a statistically robust reference-based analysis pipeline incorporating error propagation and significance testing to move beyond global epistatic trends and resolve idiosyncratic, substrate-dependent intramolecular wiring in specific genetic backgrounds. Simulations confirm that this pipeline reliably identifies genuine higher-order epistatic interactions while minimizing false positives. We reveal that intramolecular network wiring varies substantially between substrates - even within the same chemical class - with notable divergences between the adaptive target substrate 2-naphthyl hexanoate and its shorter-chain ester analogs. Key higher-order networks, including d233E/h254R/l271F and l271F/f306I/i313F, exhibit substrate-specific epistatic signatures that discriminate between subtle structural features such as acyl chain length, leaving group identity, and heteroatom substitution. These substrate-dependent rewiring events account for observed functional trade-offs, particularly the strong anti-correlation between the adaptive and native substrates. Collectively, these findings demonstrate that comprehensive cross-substrate epistatic profiling, paired with rigorous statistical analysis, provides a powerful framework for dissecting the molecular basis of enzyme promiscuity and the trade-offs that define adaptive evolution. Significance of the studyEnzymes are biological catalysts whose function depends on precisely coordinated networks of interacting amino acids. Understanding how these networks change as enzymes evolve to recognize new molecules is fundamental to understanding and predicting evolution. Here, we developed a statistical framework to map these networks across multiple molecules simultaneously, revealing how subtle differences in molecular shape drive functional trade-offs during evolution. These insights could inform the rational design of enzymes with tailored specificities for industrial and therapeutic applications.

biochemistry↗

bioRxiv2026

Mechanistic origins and evolutionary erosion of collateral sensitivity in a β-lactamase

As antibiotic discovery stalls, exploiting collateral sensitivity, where resistance to one drug increases sensitivity to another, offers a promising route to extend the lifespan of existing drugs. However, the molecular origins and robustness of such trade-offs at the level of single resistance determinants remain poorly understood. Here, we examined a previously evolved trajectory of the {beta}-lactamase OXA-48 to Q4 (A33V/F72L/T212A/S213A), which confers a 40-fold increase in ceftazidime resistance when expressed in Escherichia coli. This evolution coincided with the emergence of collateral sensitivity to piperacillin (27-fold reduction), likely caused by the introduction of F72L. We challenged the stability of this collateral sensitivity network by subjecting Q4 to directed evolution under co-selective pressure from both ceftazidime and piperacillin. This selected for the substitution V120G, which alleviated the piperacillin trade-off while maintaining elevated resistance to ceftazidime in genetic backgrounds harboring F72L. Structural and computational analyses revealed that, during evolution from OXA-48 to Q4, F72L introduced substantial conformational changes in the {Omega}-loop, likely leading to less productive piperacillin binding poses. V120G counteracted the effect of F72L by decreasing the {Omega}-loops conformational freedom, thereby partially restoring piperacillin resistance. Finally, we show that other substitutions at position 120 can exert similar mitigating effects. Taken together, our results provide a mechanistic understanding of how adaptive solutions both generate and erode collateral sensitivity, knowledge crucial for predicting the long-term stability of these networks. IMPORTANCEThe evolution of antimicrobial resistance to one drug can also increase bacterial susceptibility to other drugs, a phenomenon known as collateral sensitivity. Understanding the mechanisms underlying these effects may help clinicians design antimicrobial treatment strategies that limit the emergence of resistance. However, for clinical applications, collateral effects must be stable and robust over a certain evolutionary time. While many studies have focused on the stability of collateral effects at the bacterial population level, their stability during the evolution of a single resistance determinant has remained largely unknown. Here, we studied the molecular origins of collateral sensitivity arising during the evolution of the {beta}-lactamase OXA-48 and robustness of this network under co-selection. By linking resistance evolution to changes in protein structure, we illuminate the role of active site loop dynamics in shaping both the collateral effect and its mitigation. Such results are important for understanding collateral sensitivity-based strategies and how they may shape the evolutionary trajectories of resistance enzymes.

molecular biology↗

bioRxiv2025

Evolution tunes functional sub-state interconversion to boost enzyme function

Enzymes do not operate as static structures, but continuously fluctuate between different conformations. Enzymes therefore dynamically sample conformations with varying catalytic activity. However, it remains largely unexplored whether evolution can exploit the conformational dynamics between sub-states to improve activity. Here, we dissect the evolutionary trajectory of the {beta}-lactamase OXA-48 toward improved ceftazidime hydrolysis. Evolution relieved conformational bottlenecks by promoting alternate functional sub-states, gradually shifting the rate-limiting step from substrate binding to sub-state interconversion, and finally to the chemical step. Reorganization of the conformational landscape enhanced OXA-48s ability to hydrolyze ceftazidime and introduced a trade-off in its native activity against meropenem. This trade-off stemmed from catalytic incompatibility between the native and the evolved sub-state populations. Our findings highlight the transitions between functional sub-states as a mechanism of natural selection, shaping functional divergence and offering new strategies for enzyme and antibiotic engineering.

molecular biology↗

bioRxiv2025

Free energy perturbations in enzyme kinetic models reveal cryptic epistasis

Epistasis--the context-dependence of mutational effects--is a key driver of protein evolution, influencing adaptive pathways and functional diversity. While specific epistasis arises from direct physical interactions between mutations, non-specific epistasis emerges when a non-linear mapping links a proteins biophysical properties to its function. Enzyme kinetic parameters are assumed to be devoid of non-specific epistasis when measured in vitro, enabling direct connection of epistasis to the enzymes structural features. Here, we show that this assumption is incorrect: enzyme catalytic parameters like kcat and KM inherently exhibit non-specific epistasis due to the multi-state nature of the catalytic cycle. Using in silico enzyme models, parameterized by free energies of ground and transition states, we simulated 1000 "mutations" or perturbations to the sub-state free energies within the kinetic ensemble. We then combined these mutations, creating one million double mutants with strictly additive free energy effects. Despite the absence of explicit mutational interactions, we observed substantial epistasis in catalytic parameters; its prevalence and complexity increasing with the number of kinetic states in the mechanism. We derived analytical conditions for the emergence of this form of epistasis in a simple kinetic model, demonstrating that non-specific epistasis depends on the relative values of key microscopic rate constants. Finally, we validated our framework by reanalyzing kinetic data for double mutants in Bacillus cereus {beta}-lactamase I and found that reported specific epistasis in catalytic efficiency was substantially stronger than previously inferred, altering mechanistic interpretations. Our results identify an intrinsic, previously unknown source of epistasis that can distort both the magnitude and sign of mutational effects in enzyme kinetics. We provide theoretical and computational tools for recognizing and correcting for this form of non-specific epistasis, enabling accurate mechanistic inference from kinetic data and improving our understanding of the links between epistasis, structure-function relationships, enzyme evolution, and protein design. Author SummaryEnzymes help organisms convert reactants to products through a series of steps; each associated with an energy that dictates how well the enzyme catalyzes a reaction. Enzymes evolve to become more efficient, or catalyze new reactions, through mutations that change the free energies of these steps. Sometimes, the effect of one mutation depends on the presence of another, a phenomenon called epistasis. Epistasis is typically studied by measuring the effect of mutations on standard enzyme parameters under the assumption that changes in these values reflect structural interactions between mutations in the protein. Our study shows that this assumption is misleading. Even when mutations act independently on the energies of steps in an enzymes reaction, when combined they can create epistasis. This phenomenon arises from the complex, non-linear relationships between the parameters that define each step in the enzyme reaction and the measurements we obtain during experiments probing enzyme functions. Using computational simulations, we mathematically derive the necessary conditions for this form of epistasis, demonstrate that epistasis increases in prevalence as the enzyme reaction becomes more complex, and apply our model to published experimental data. Our findings urge that researchers should account for these effects before drawing structural conclusions from epistasis.

evolutionary biology↗

bioRxiv2023

Epistasis Arises from Shifting the Rate-Limiting Step during Enzyme Evolution

The molecular mechanisms by which epistasis boosts enzyme activity remain elusive, undermining our ability to predict the evolution of pathogens and engineer novel biocatalysts. Here, we reveal how directed evolution of a {beta}-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects ([≤] 2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzymes conformational dynamics. Kinetic, structural, and dynamical analyses reveal that epistasis was driven by distinct effects of each mutation on the catalytic cycle. The first mutation acquired during evolution increases protein flexibility and accelerates substrate binding, which is rate-limiting in the wild-type enzyme. The ensuing mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial positive synergy boosting enzyme activity.

evolutionary biology↗

bioRxiv2022

Higher-order epistasis creates idiosyncrasy, confounding predictions in protein evolution

Epistasis shapes evolutionary outcomes during protein adaptation. In particular, when the effects of single mutations or mutational interactions are idiosyncratic, that is, unique to a genetic background, the predictability of protein evolution becomes greatly impaired. Here, we unveil a quantitative picture of the prevalence and role of idiosyncrasy in protein evolution by analysing 45 protein fitness landscapes, generated from seven enzymes. We found that mutational effects and epistasis are highly idiosyncratic across the landscapes. Idiosyncrasy obscured functional predictions of mutated proteins when using limited mutational data, and often continued to impair prediction upon incorporation of epistatic information. We show that idiosyncrasy stems from higher-order epistasis, and highlight examples where it permits, or restricts, evolutionary accessibility of certain genotypes. Our work suggests that idiosyncrasy deeply confounds predictions in protein evolution necessitating its incorporation into predictive models and in-depth exploration of its underlying molecular mechanisms.

evolutionary biology↗