Communications Biology

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

Communication between the brain and cardiovascular system is increasingly recognized as a fundamental determinant of health, yet the mechanisms coordinating neural and cardiac rhythms remain poorly understood. Sleep provides a unique physiological state for investigating neurovisceral integration because cortical slow oscillations (SOs), thalamocortical sleep spindles, and autonomic activity undergo profound reorganization during non-rapid eye movement (NREM) sleep. We analyzed overnight high-density electroencephalography and electrocardiography recordings from 30 healthy adults and quantified interactions among SOs, sleep spindles, cardiac R-peaks, heart rate variability (HRV), spectral slope, and Lempel-Ziv complexity. To assess integrated brain-heart coordination, we developed a Joint Phase-Amplitude Coupling (JPAC) metric that quantifies the corelation between SO phase-dependent modulation of spindle amplitude and cardiac timing. SO-spindle coupling showed the expected fronto-central distribution but was not significantly related to autonomic or cortical state markers. In contrast, SO-R-peak coupling and JPAC systematically tracked physiological state. Stronger JPAC was associated with higher high-frequency HRV, steeper spectral slopes, and lower neural complexity, indicating enhanced coupling during synchronized, parasympathetically dominated NREM sleep. These findings demonstrate coordinated organization of cortical, thalamocortical, and cardiac rhythms within a common oscillatory framework and identify NREM sleep as a state and potential treatment target of large-scale neurovisceral integration.

The twisted-wing parasites (order Strepsiptera) are well-known for their unusual life histories as obligate endoparasites of a wide variety of other insect orders. Here, we present the first high-quality genome assembly of a leafhopper-parasitizing strepsipteran species. We sequenced the genome of Elenchus koebelei using PacBio HiFi technology, yielding a primary assembly of 63.7Mb in 96 contigs, with an N50 of 1.60Mb and a BUSCO completeness score of 82.3%. In total, we recovered 8,167 protein-coding gene annotations, and 34.2% of the genome annotated as repeats. For the first time, we compare the genome content of three strepsipteran species and find that they are remarkably consistent in broad structure, with approximately 600 insect universal single-copy orthologs that appear to be completely missing from the order. These similarities imply a conserved genomic blueprint for strepsipteran parasitic strategy and/or differences that must be investigated at the post-translational level.

High-sugar diets in mammals are associated with a number of metabolic diseases including diabetes and obesity. However, frugivorous and nectivorous bats specialize in consuming diets high in simple sugars while remaining healthy. To investigate the metabolic adaptations in bats with high-sugar diets, we conducted targeted metabolomics on blood samples collected during the first ten minutes after glucose or fructose feeding in five bat species whose natural diets vary in sugar content. By comparing changes in blood metabolite levels over time both between feeding treatments and across dietary guild, we have been able to identify shifts in sugar processing that likely support the healthy metabolism of simple sugars mammals with high-sugar diets. Notably, despite both sugars being readily absorbed, only fructose feeding induced significant changes in other metabolites within ten minutes after feeding. Species who naturally consume more sugar showed both faster production of metabolic intermediates and lowered production of common deleterious byproducts of fructose metabolism than species with comparatively lower-sugar diets. The prioritization and refinement of fructose metabolism is therefore likely a more key aspect of how bats with high-sugar diets are able to avoid the diseases typically associated with excessive sugar consumption than modifications to glucose metabolism.

The posterior parietal cortex integrates somatosensory information with signals supporting action, spatial cognition, and multisensory processing, yet the fine-scale topographic relationship between the intraparietal sulcus (IPS) and primary somatosensory cortex (S1) remains poorly understood. Here, we used resting-state fMRI in 10 awake rhesus macaques to characterize functional connectivity between four S1 subregions (areas 3a, 3b, 1, and 2) and 223 single-voxel seeds densely sampling an IPS-centered parietal territory. Across S1 subregions and hemispheres, IPS-S1 connectivity exhibited a highly structured spatial organization characterized by systematic anteroposterior and mediolateral gradients and a prominent semi-oval topology following the geometry of the intraparietal sulcus. Connectivity was generally stronger toward the cortical banks and convexities and weaker near the sulcal fundus, a pattern that could not be explained by reduced local temporal signal-to-noise ratio. Unsupervised hierarchical clustering independently recovered this spatial organization and identified connectivity-defined subdivisions that only partially corresponded to classical cytoarchitectonic boundaries. Along the S1 mediolateral axis, prominent troughs in cluster-averaged connectivity profiles corresponded to several previously described somatotopic transitions, allowing to consistently map putative boundaries between tongue and face representations and between hand and trunk-leg representations. Based on these boundaries, individual IPS clusters exhibited connectivity with multiple S1 body-part sectors, with the pattern of connectivity across these sectors varying across IPS clusters and S1 subregions. Together, our findings reveal that IPS-S1 functional connectivity is organized by a large-scale sulcal topology while preserving fine-grained features of S1 somatotopic organization. These findings suggest that the functional organization of the IPS is shaped by multiple spatial dimensions, including large-scale sulcal topology and the organization of S1 subregions and body-part representations, which may together contribute to the functional specialization of individual IPS regions.

Background: Positive selection shapes protein function by favoring amino acid substitutions that increase fitness. Although numerous studies have identified positively selected genes (PSGs), the structural principles underlying residue-level positive selection remain incompletely understood and previous studies have produced conflicting conclusions, particularly regarding the role of intrinsically disordered regions. In order to resolve these issues and to better understand the driving forces underlying positive selection in human proteins in general, we performed a comprehensive analysis on a manually curated, high quality collection of PSGs and positively selected residues (PSRs) as well as on a large-scale PSR dataset restricted to proteins with experimental structures. Results: Human PSRs were significantly enriched in secreted and cell membrane proteins and preferentially localized to extracellular regions, solvent-exposed surfaces, coil structures, and protein-protein interaction interfaces, whereas neither domains nor intrinsically disordered regions showed an enrichment. Spatial clustering analysis reinforced that PSRs accumulate within localized surface patches. Integration of host-pathogen interaction data highlighted that adaptive changes preferentially affect extracellular molecular recognition surfaces, recurrently appearing in certain pathogen-interacting membrane protein families and particular domain types. Additionally, PSRs showed an enrichment in the residues of human-pathogen interaction interfaces that are in direct contact with pathogenic proteins, implying that neutralizing pathogen attacks is one of the driving forces behind the adaptive evolution of human proteins. By analysing human variation data we found that PSRs are enriched in benign but depleted in pathogenic substitutions, extending previous observations that positively selected genes contain elevated levels of missense variation. Notably, PSGs of the large-scale dataset were also enriched among clinical-stage drug targets, suggesting a potential link between positive selection and pharmacological relevance. Conclusion: Together, our results provide a refined structural model of adaptive evolution in human proteins and identify extracellular exposed molecular recognition surfaces and human-pathogen interaction interfaces as recurrent hotspots of positive selection. Our results point to the direction that precise identification of human PSRs could delineate important host-pathogen interfaces that exert evolutionary selection pressure on humans and appoint novel targets with therapeutic potential.

Signals of positive selection around the SLC30A9 gene have been reported in human populations outside Africa. Selection likely acted on a highly differentiated single-nucleotide polymorphism, rs1047626, leading to a non-synonymous substitution in the encoded zinc transporter. Because of the striking similarity between the putatively selected SLC30A9 haplotype observed in several current human populations and the Denisovan individual, previous work has proposed adaptive introgression. Yet alternative explanations, including ancient human variation, and the precise archaic source -Neanderthal or Denisovan- remained unresolved. Considering the potentially complex evolution of SLC30A9, we applied Approximate Bayesian Computation (ABC) algorithms coupled to machine learning to investigate the most plausible evolutionary origin of this substitution. After modelling different evolutionary scenarios with forward-in-time simulations, our results highlight that the most probable scenario is a Denisovan origin of the rs1047626 polymorphism. However, the allele likely introgressed into Neanderthals first and was then passed into non-African modern humans. Moreover, the derived allele frequency for rs1047626 across several African populations is consistent with back-to-Africa migrations. Finally, our ABC analyses indicate strong positive selection in East Asian populations and other out-of-Africa populations, whereas in Neanderthal populations, the selection coefficient was probably neutral or slightly deleterious.

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of the ABC-core of SUR2B. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular KATP channels.

Fossil central nervous systems (CNS) have illuminated several facets of total-group euarthropod evolution, including their phylogenetic affinities, the origin of the head, and their ancestral neuroanatomical organization. However, the CNS remains unknown in trilobites, the most diverse and earliest group of Palaeozoic euarthropods, but can potentially clarify fundamental questions that remain disputed despite thousands of described species and countless studies spanning over two centuries, such as their affinities relative to modern representatives. Here, we describe the CNS of the cheirurid Anacheirurus adserai from the Lower Ordovician Fezouata Shale. Anacheirurus features a brain with well-developed proto, deuto- and tritocerebral neuromeres, an anteriomedian protocerebral protrusion, a central complex, an esophageal foramen, and a ganglionated ventral nerve cord. The CNS is replicated in framboidal iron that differs in texture and composition from the exoskeleton and rock matrix. The overall organization of Anacheirurus's CNS most closely resembles that of Cambrian stem-group mandibulates, and critically shares the presence of a central complex with a protocerebral bridge and lateral accessory lobes with extant pancrustaceans. Our findings provide new morphological support for the hypothesis that Trilobita belongs to the mandibulate stem lineage and bring new insights into the ancestral morphology and evolutionary history of the euarthropod central complex. More broadly, our findings offer direct anatomical evidence of the key sensorimotor system innovations that originated during the Cambrian Information Revolution.

Macrophages, alongside mammary epithelial cells, are major contributors to the innate immune response triggered by bacterial invasion of the mammary gland. To investigate breed-specific immune mechanisms, we stimulated monocyte-derived macrophages from Holstein and Normande cattle with Escherichia coli and Streptococcus uberis, two major mastitis pathogens. We monitored bacterial survival using [methode] and profiled gene expression via RNA-seq. E. coli exhibited significantly higher survival 6 hours post-infection compared to S. uberis (p < 0.05). Moreover, E. coli induced a robust transcriptional response, with 4403 differentially expressed genes (FDR < 0.05, |log2FC| > 1), versus only 813 for S. uberis. Qualitative differences were also observed: genes such as CCL2 and CSF1 were uniquely induced by S. uberis. Additionally, macrophages from Holstein and Normande cattle showed distinct expression profiles: 185, 45, and 174 genes were differentially expressed at steady state or after E. coli or S. uberis stimulation, respectively. These genes were enriched in immune-related pathways. Overall, our results demonstrate a stronger response to E. coli, although S. uberis specifically induces a subset of genes. The observed breed-specific differences may reflect underlying immune mechanisms that could influence disease susceptibility, warranting further functional validation.

Host specialization in herbivorous insects is often associated with divergence in chemosensory abilities. Here, we investigated the possible contribution of odorant receptors (ORs) in host plant restriction in the lily moth Spodoptera picta, a species specialized on Amaryllidaceae. Manual annotation of S. picta ORs in its genome revealed a repertoire similar in size and composition to those of its polyphagous sister species, S. littoralis and S. litura, suggesting that specialization did not involve major gene loss or expansion in the lily moth. To assess functional divergence beyond gene number, we applied a large scaled structure-based virtual screening approach to the entire OR repertoires of these three Spodoptera species, generating ligand-binding profiles for 120,591 volatile compounds. Among 69 1:1:1 OR orthologs, 24 exhibited divergent predicted binding spectra. We pinpointed OR29 that we also found to be highly expressed in both male and female antennae of S. picta through a RNAseq approach. Functional assays demonstrated that S. picta OR29 acquired heightened sensitivity to limonene enantiomers, volatiles emitted by host Amaryllidaceae inflorescences. Site-directed mutagenesis revealed that a single amino acid substitution within the predicted binding region underlies this shift in sensitivity. These results show that host specialization in S. picta has not been accompanied by significant OR repertoire remodeling, but rather by subtle molecular changes that fine-tune receptor sensitivity to host-derived volatiles.

Estimating receptive fields in large neural populations is often limited by experimental time: stimuli with theoretically favorable statistics for receptive-field estimation may, in practice, evoke too few informative spikes in neurons with high specificity for structured features, requiring impracticably long recordings. Therefore, we asked whether a structured stimulus could broadly engage neural populations across stages of the early visual system while permitting a tractable and interpretable reverse-correlation analysis. To address this question, we developed "reverse correlation against stimulus elements (RCASE)", an analysis framework for stimuli that can be represented frame by frame through the presence or absence of specific stimulus elements. We then designed such a stimulus consisting of random moving objects (RMO) and compared RMO/RCASE with conventional spike-triggered averaging using a dense binary white noise (WN/STA) stimulus across different stages of the early visual system of the mouse, namely the retina, the nucleus of the optic tract, the superior colliculus and the primary visual cortex, and in the primate retina. To distinguish the contributions of spatial and temporal scales, motion, stimulus structure, and analysis choice, we additionally compared against WN/STA with different square sizes and frame rates, spike-triggered covariance analysis, as well as random static objects and spatially correlated cloud stimuli. RMO evoked stronger and more reliable responses than WN in the mouse retina and across the investigated mouse visual areas. RMO/RCASE yielded discernible receptive fields for a substantially larger proportion of neurons and in a fraction of the recording time. Although the stimulus objects featured localized moving contrast edges, this advantage was not restricted to direction-selective cell populations. In the primate retina, in contrast, WN/STA approached the performance of RMO/RCASE. RMO/RCASE also supported simultaneous estimation of stimulus direction, speed, and color tuning. Our approach enables rapid and interpretable functional characterization of large populations of visual neurons, enabling to simultaneously recover receptive fields and feature tuning, whenever a low-dimensional feature space can be specified beforehand.

Prospero-related homeobox (Prox) genes encode a family of transcription factors that play essential roles in the development of several organs and systems, including the central nervous system, lymphatic endothelium, musculature, and liver. Despite their developmental importance, the evolutionary history of the vertebrate Prox gene family remains poorly understood. In this study we combined phylogenetic and synteny analysis to characterise the evolution of the Prox family in vertebrates. Our results reveal that two to three Prox subfamilies were already present in the last common ancestor of jawed vertebrates. We clarify the identity and evolutionary relationships of well-studied members of this family and identify multiple independent losses of Prox2 and Prox3 genes in specific vertebrate lineages. Furthermore, we uncover evidence for the existence of a fourth Prox gene in the ancestral vertebrate genome, which was subsequently lost. Overall, this study provides the first comprehensive analysis of the evolutionary history of the vertebrate Prox gene family and establishes a foundations for future studies on the functional roles of these genes.

Mycobacterium bovis causes tuberculosis (TB) in cattle, badgers (Meles meles), other wildlife, and humans, posing major animal and public health challenges in Britain and Ireland. Shared genotypes between cattle and badgers have been reported at regional scales, but whether this concordance holds at finer spatial scales, where control interventions operate, and whether it varies by genotype combination, remains unknown. Resolving this is critical for interpreting cross-species transmission risk and designing targeted control measures. We analyzed 1,248 M. bovis isolates from cattle and badgers (Northern Ireland, 1986-2018), including samples from a small, densely sampled intervention research area, using Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA) and kernel discriminant analysis (KDA) across multiple spatial scales (25-95% density contours). We identified 36 MLVA types with four shared between species and one (MLV06) endemic to the area. Spatial concordance varied by type combination and scale: MLVA004-MLVA297 showed strong, bidirectional, scale-robust concordance; MLVA006-MLVA297 was concordant only at fine spatial scales; MLVA004-MLVA006 showed weak, asymmetric concordance; and no concordance was detected when all three types were analysed together. Our findings reveal spatial concordance between shared M. bovis genotypes varied by genotype combination and scale, indicating genotype-specific dynamics within this multi-host system.

Memory reactivation is thought to support memory consolidation, but how ongoing brain states shape awake reactivation and its relationship to memory remain poorly understood. Here, we asked whether spontaneous hippocampal-cortical reactivation during awake rest is embedded in ongoing brain state dynamics by examining its coupling with whole-brain activity patterns and infra-slow global signal (GS) fluctuations, and whether these dynamics shape its relationship to memory retention. Reactivation preferentially occurred during an internally oriented brain state characterized by default mode network activation and dorsal attention network suppression, as well as states that reinstated encoding-related visual activity. Reactivation also clustered around the peak of infra-slow GS fluctuations and in high GS amplitude time windows, features previously associated with lower arousal. Importantly, the relationship between reactivation and memory depended on the ongoing brain state: distinct whole-brain activity patterns expressed during reactivation were associated with opposing memory outcomes, while reactivation during high GS amplitude windows was positively associated with retention. Together, these findings show that awake memory reactivation is embedded in dynamic whole-brain states that may influence its memory outcome, suggesting that recurring brain states may provide favorable windows for awake memory consolidation.

Attention is a fundamental brain function supporting perception, decision-making, and social behavior, and its dysfunction profoundly impairs daily life. It is both dynamic and stable, varying across observations and individuals, changing across the lifespan, and being shaped by social and environmental experience. Yet capturing this complexity remains a central challenge in neuroscience. Here, we integrated longitudinal behavioral assessments of semi-free-ranging macaques living in naturalistic social groups with resting-state fMRI. We quantified performance across days, ages, and social hierarchies and related it to intrinsic brain organization. Distinct attentional phenotypes emerged, including individuals with reduced attentional control. Performance followed an inverted-U lifespan trajectory, improving from childhood to adulthood before declining. Social status modulated attentional performance. Critically, nonlinear lifespan trajectories and associations with individual attentional differences were most clearly expressed in frontoparietal connectivity. Together, these findings reveal how sustained attention is organized across scales, providing a biological framework for its individual diversity, social modulation, and neural basis.

The pathogenicity of Mycobacterium tuberculosis relies on its ability to withstand hostile host environments. Coenzyme F420 is critical for redox metabolism in Mycobacterium tuberculosis an organism distinguished by its abundant use of this low-redox-potential cofactor and a repertoire of F420-dependent enzymes. While F420-dependent mechanisms are known to confer protection against antimicrobials and oxidative stress, the specific enzymes responsible remain largely unidentified. In this study, using the F420-dependent detoxification of malachite green as a phenotypic marker, we performed a genome-wide screen in Mycobacterium tuberculosis using transposon mutagenesis and whole genome sequencing. The results of transposon mutagenesis and whole genome sequencing revealed that, among the 28 genes encoding F420-dependent enzymes, only mutations in fgd (essential for F420H2 re-generation) and Rv2061c, a gene encoding a putative F420-dependent oxidoreductase, were highly susceptible to malachite green. Subsequent experiments confirmed that Rv2061c and its homolog MT2120 are required for detoxifying malachite green and crystal violet. Furthermore, the Rv2061c mutant exhibited increased susceptibility to isoniazid and pretomanid; as well as menadione-induced oxidative stress. We also identified mutations in the transcriptional repressor Rv0678 as a key determinant of resistance to malachite green and crystal violet. This study establishes Rv2061c as a pivotal F420-dependent oxidoreductase that protects Mycobacterium tuberculosis from malachite green and other antimicrobial compounds as well as oxidative stress and further supports Rv0678 as a master regulator of multidrug resistance. These findings expand our knowledge of Mycobacterium tuberculosis defense mechanisms and highlight F420-dependent pathways as a potential target for new drug development.

The clonogenic assay is the foundational standard for evaluating cellular radiosensitivity, yet its manual quantification is limited by labor-intensive processing, high inter-observer variability, and a reductive binary scoring system. Here, we present and validate a deep learning-based segmentation pipeline for the automated, high-throughput quantification of mammalian cell survival across diverse radiation qualities (photons, protons, and carbon ions). By benchmarking against expert consensus using Bland-Altman analysis, we demonstrate that this AI framework consistently operates within natural human error margins. Crucially, transitioning to automated whole-well image-based analysis enables the extraction of continuous multidimensional data, including colony area, density, and cellularity. This phenotypic profiling successfully identified and quantified high-cellularity colonies formed by radioresistant BxPC-3 cells under particle irradiation. Coupled with rapid image acquisition platforms, this methodology significantly reduces processing bottlenecks while standardizing the derivation of relative biological effectiveness (RBE). By advancing beyond binary survival metrics to continuous morphological profiling, this framework provides a vital new analytical dimension for investigating structural mechanisms of radioresistance.

Clownfishes are a complex of 28 species that rapidly diversified after acquiring mutualism with host sea anemones around 15 million years ago. The genomic mechanisms behind this radiation are only beginning to emerge, and structural variants (SVs) have received little attention. Previous phylogenomic studies suggested two large inversions on chromosome 18, but their distribution across the clownfish phylogeny and their potential effects have not been formally explored. SVs can disrupt gene function and regulation or alter gene dosage, and they have been shown to play a central role in adaptive evolution and diversification across many taxa. Here, we characterized SVs across the clownfish radiation using long-read PacBio sequencing, generating high-quality genome assemblies for sixteen species spanning the main lineages of the genus. We identified and catalogued nearly 130,000 SVs spanning close to 570 Mb. Short insertions and deletions were the most numerous, but large inversions accounted for most of the affected sequence. No SV was consistently associated with host specialization. We nevertheless confirmed a single inversion of approximately 17 Mb on chromosome 18, clarifying earlier reports of two separate rearrangements at this locus, and identified an additional inversion on chromosome 9 whose distribution suggests a history of hybridization. This study provides the first genome-wide characterization of structural variation in clownfishes, revealing extensive SVs despite the group's rapid and recent radiation. It also delivers new, high-quality genomic resources that will support future research into this iconic group of coral reef fishes and their adaptation to sea anemones.

The sulfur mobilization (SUF) system is the only Fe-S cluster biosynthesis pathway in Mycobacterium tuberculosis (M. tb) and Mycolicibacterium smegmatis (M. smegmatis). In this pathway, SufC protein acts as an ATPase in the SufBC2D scaffold for Fe-S cluster biogenesis. This crucial role positions SufC as a promising target for the development of antimycobacterial therapeutics. However, the SufC ATPases from Mycobacterial species are not yet characterized. This study includes cloning, expression, purification, and characterization of SufC homologs from M. tb (Rv1463) and M. smegmatis (MSMEG_3124) using biochemical, biophysical, and computational approaches. Both SufC proteins were purified using affinity and size-exclusion chromatography and observed to exist as monomers in solution. Although both proteins have the conserved domains, they exhibit different ATP-dependent kinetic efficiencies, demonstrating differences in their local environment and catalytic behavior. Furthermore, CRISPR interference targeting MSMEG_3124 results led to diminished M. smegmatis growth, highlighting the critical role of SufC in cellular viability. Modeling and virtual screening led to the identification of three candidate compounds, of which Z2911048515 and Z66741142 were selected for experimental evaluation. Circular dichroism spectroscopy analysis revealed slight differences in the spectral features of Rv1463 in the presence of Z2911048515 and Z66741142, while significant spectral variations were observed for MSMEG_3124. Importantly, Z66741142 was able to cause changes in the fluorescence spectra of both SufC protein homologs. Surprisingly, both compounds showed only limited inhibition of SufC ATPase activity, exhibited distinct modes of action, and may serve as preliminary chemical scaffolds for optimization and testing as effective inhibitors. This study presents functional diversity of mycobacterial SufC proteins across pathogenic and non-pathogenic species and provides a basis for developing a framework for further mechanistic and structure-guided investigation within the indispensable SUF pathway.