Biology subjects

Schmidt, K.

Publications and source records attributed to Schmidt, K..

At least 19 recordsLinked to original sources

bioRxiv2026

A Normative Model Reveals Functional Optimization of Synaptic Nano-Architecture

Super-resolution microscopy approaches have revealed that synaptic proteins are not randomly distributed within individual synapses. Rather, they form sub-synaptic clusters that may be coordinated between the synaptic compartments, thereby forming transsynaptic nanocolumns. However, the functional significance of this nano-architecture remains under debate. In particular, it is unclear whether the positions of synaptic proteins optimize a specific cost function. Here, we present a normative modeling framework that integrates synaptic nano-architecture imaged with STED and dSTORM microscopy with synaptic transmission recorded with electrophysiology at the Drosophila neuromuscular junction (NMJ). Model-based quantification showed that clusters of Unc13A, which likely demark presynaptic release sites, and postsynaptic glutamate receptors (GluRs) adopt aligned ring-like arrangements in individual synapses. We developed an analytical description of GluR activation relative to presynaptic release position, enabling to link super-resolution imaging data with recorded postsynaptic current amplitudes without relying on costly Markov chain Monte Carlo (MCMC) simulations. Our model predicts that glutamate released from a single vesicle spreads across the entire receptor population. Given this spread, the postsynaptic current amplitude is maximized when glutamate is released close to the active zone (AZ) center, in contradiction to the observed offset of Unc13A clusters. However, the observed relative positions of Unc13A and GluR clusters maximize the postsynaptic current amplitude and minimize the delay between vesicle release and receptor activation during multivesicular release. Hence, transsynaptically-aligned Unc13A-GluR rings optimize for multivesicular but not univesicular release. Given that multivesicular release is mainly observed during enhanced presynaptic release at the Drosophila NMJ, transsynaptic nano-architecture may be adapted to elevated presynaptic release states.

neuroscience↗

bioRxiv2026

Beyond pain: a brain-based biomarker predicts individual pain relief

Effective pain relief is a central goal of medical care, yet objective biomarkers of pain relief are lacking. Using task-based functional MRI and a capsaicin-induced tonic heat pain model, we experimentally elicited both pain exacerbation and pain relief within the same individuals. Existing brain-based signatures, including the Neurologic Pain Signature (NPS), reliably detected pain increases, but failed to capture pain relief. We therefore developed the PAin and RElief Signature (PARES), a multivariate brain signature trained to predict bidirectional changes in pain perception in n = 61 healthy controls. PARES robustly predicted both pain increases and relief and generalized to an independent cohort of people with chronic back pain (n = 58), who underwent the same experimental procedures. Together these findings establish a neural signature of pain relief and provide a potential biomarker for treatment stratification and analgesic development.

neuroscience↗

bioRxiv2026

Arteriolar GMP signaling revealed by FRET-based real-time measurement in vivo

cGMP evokes arteriolar vasorelaxation and is generated in smooth muscle by the soluble guanylyl cyclase (sGC) stimulated by endothelial NO. Phosphodiesterases (PDEs) degrade cGMP and may contribute in diameter regulation. We examined arteriolar cGMP levels in real-time in vivo upon NO and acetylcholine (ACh) and effects of PDE inhibition. Changes in cGMP were measured by FRET using the indicator protein cGi500. Arterioles in the cremaster muscle of anesthetized mice expressing cGi500 were exposed for imaging. They were stimulated with various NO donors (DEA/NO, SNP, SNAP) and ACh with or without PDE inhibition (non-specific: IBMX; PDE-5 specific: sildenafil). Furthermore, dilations induced by ACh and SNP were studied in sGC-deficient mice. DEA/NO and SNAP led to a fast, concentration-dependent rise of fluorescence ratio of cGi500 (by 4.0% at 10{micro}M), that declined on removal slowly. These ratio changes indicated cGMP increases as verified by inhibition of the sGC using ODQ. PDE inhibition increased cGMP levels slowly despite NO synthase inhibition. However, it did not amplify NO-induced ratio increases, but the decline after drug removal was decelerated. Interestingly, ACh did not modify cGMP levels and dilations were not impaired in sGC-deficient mice. FRET-based imaging allows reliable real-time assessment of arteriolar cGMP levels in vivo. PDE activity does not limit the amplitude, but the duration of cGMP signaling after stimulation. Furthermore, we conclude that ACh does not release endothelial NO in murine arterioles. Preliminary experiments demonstrate that simultaneous measurement of cGMP and diameter is feasible using mice with expression of cGi500 in smooth muscle cells.

physiology↗

bioRxiv2026

Cyst-type epithelial heterogeneity shapes therapeutic responsiveness in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) exhibits substantial interpatient variability in disease course and therapeutic response, but the cellular basis for this variability remains poorly understood. Here, we combine single-nucleus RNA sequencing of human cyst epithelia with machine learning-based histological analysis of >1,800 cysts to resolve three epithelial cyst types--proximal tubule-like, collecting duct-like, and mixed. These cyst types display distinct injury states, metabolic programs, and stromal microenvironments, including a mixed-cyst niche enriched for CCL2-associated inflammatory signaling. Expression of key therapeutic targets was highly cell-type specific with CFTR enriched in proximal-like epithelia, whereas AVPR2 expression was confined to AQP2-positive collecting duct-like cells. Cyst-type composition varied widely across patients and in an orthologous mouse model (Pkd1RC/RC) in which the burden of AQP2-positive cysts correlated with responsiveness to tolvaptan. These findings identify cyst-type heterogeneity as a major determinant of molecular pathway activation and predictability of therapeutic response in ADPKD.

molecular biology↗

bioRxiv2026

Mlp1 and Mlp2 cooperate to build a stoichiometric nuclear pore basket in budding yeast

The nuclear pore complex is the only gateway between the nucleus and the cytoplasm in eukaryotic cells. Its nucleoplasmic face is decorated by the nuclear basket, a filamentous structure with important roles in mRNA export and chromatin organization. In contrast to major parts of the nuclear pore scaffold, the architecture of the nuclear basket remains poorly defined. Here, we investigate the interactions required for formation and maintenance of the nuclear basket in vivo using budding yeast. While previous work has often focused on Mlp1, the largest and most abundant nuclear basket protein, we demonstrate that its paralogue, Mlp2, also plays a central role in nuclear basket architecture. Mlp2 can interact with the NPC scaffold independently of Mlp1, and interactions between the coiled-coil regions of both proteins stabilize their binding. Furthermore, the N-termini of both Mlp1 and Mlp2 are required for recruitment of Pml39. In addition, we show that Pml39 uses its N- and C-terminal helices to recruit additional Mlp1 subunits. We propose a refined model of nuclear basket architecture with a stoichiometry of 4:2:1 per spoke for Mlp1:Mlp2:Pml39. Summary statementBased on experiments in budding yeast, a refined model for nuclear pore basket architecture is proposed with a stoichiometry of 4:2:1 per spoke for the major components Mlp1:Mlp2:Pml39.

cell biology↗

bioRxiv2025

Dynamic protrusions mediate unique crawling motility in Asgard Archaea (Promethearchaeota)

Crawling motility is a hallmark of eukaryotic cells and requires a dynamic actin cytoskeleton, regulated adhesion, and spatially organized signalling pathways1-3. Asgard archaea (phylum Promethearchaeota) which are considered the closest known prokaryotic relatives of eukaryotes potentially encode these functions within their large set of eukaryotic signature proteins4-9. The few cultivated members show a complex cell morphology, consisting of a central cell body from which several protrusions extend, filled with an actin-based cytoskeleton10,11. Here, live cell microscopy of two organisms of the Loki- and Hodarchaea lineages10,12 showed that they dynamically and drastically change their cell shape on a minute time scale and grow and retract their extensive protrusions with a speed of 1.5 to 5.3 {micro}m/min, respectively. After adhering to a glass surface, cells employ their protrusions to undergo active crawling motion. In the presence of selected actin inhibitors however, the observed dynamics were arrested, suggesting a central role of actin in these processes. The observed cellular plasticity and motility are unique features among prokaryotes and might have been crucial for the emergence of the first eukaryotic cells that are thought to have formed through the association of a member of the Promethearchaeota and an alphaproteobacterium, the ancestor of mitochondria.

microbiology↗

bioRxiv2025

A neoantigen-microbead platform for personalized T cell cancer vaccination

Colorectal cancer (CRC) is a leading cause of cancer mortality and is characterized by a high tumor mutational burden, making it responsive to immunotherapy. We developed a bioinformatic and manufacturing pipeline for personalized cancer vaccines and evaluated it in the MC38 colon adenocarcinoma mouse model. Thirty-six high-scoring neoantigens (NAGs) were identified by exome and transcriptome analysis, produced as six purified polypeptides, and coupled to paramagnetic beads. Intralymphatic vaccination of C57BL/6 mice with NAG beads induced robust NAG-specific T cell and antibody responses, resulting in significant inhibition of MC38 tumor growth. Treated tumors displayed increased necrosis and CD8+ T cell infiltration. Compared with soluble peptides, bead-coupled antigens elicited superior protection. Studies in T cell-deficient and antibody-depleted mice confirmed that both CD4+ and CD8+ T cells mediated the antitumor effect. These findings highlight the potential of NAG bead vaccination as an effective immunotherapy for CRC.

cancer biology↗

bioRxiv2025

Cryptic multicellularity in wild yeast brings selective advantages under stress

Multicellularity has evolved dozens of times in many branches across the Tree of Life, in each case producing a new kind of individual that has the potential for division of labor among its constituent cells. In many of these instances, nascent multicellularity has remained facultative, manifesting only under conditions where it provides a decisive fitness advantage to the genome in which it has arisen. Here we investigate the mechanistic basis for, and the selective advantages of, cryptic multicellularity in wild strains of the yeast Saccharomyces cerevisiae. Meiotic purification of this trait, followed by genome sequencing, indicates that the genome of diploid champagne yeast DB146 is homozygous at the AMN1 locus (Antagonist of Mitotic exit Network) for an allele that dysregulates post-mitotic cell separation. Expression of this trait in haploid derivatives of DB146 results in clonal multicellular clusters in which daughters remain attached to mother cell walls. Systematic analysis of viability in haploid and diploid DB146 derivatives subjected to benign conditions as well as to starvation, desiccation, and low temperature at different cell titers demonstrates that haploid multicellular variants exhibit higher survivorship under conditions that wild yeast likely experience when they overwinter. Examining a collection of other wild strains, we find that [~]20% exhibit ploidy-dependent expression of clonal multicellularity. Altogether, our data suggest that in yeast that sporulate when starved, cryptic multicellularity may come under balancing selection because haploid multicellular progeny enjoy a transient survival advantage that fades once favorable growth conditions resume.

microbiology↗

bioRxiv2025

Understanding Virtual Staining with generative adversarial networks for Osteoclast Imaging

Virtual staining with generative adversarial networks is an efficient, non-invasive and scalable alternative to conventional cell staining, minimizing the need for destructive and time-consuming protocols. In this study, we investigate the explainability of a network trained to virtually stain osteoclast cultures, using intensity-based label-free input images. The model enables analysis of cell cultures without immunostaining. Explainability assessments, including receptive field and feature map analyses, show that the background in input images significantly influences staining predictions within cellular regions and the trained network performs an internal segmentation during the image transformation process. This suggests that contextual cues beyond cell boundaries are implicitly learned and integrated during training. By eliminating repetitive staining procedures, virtual staining enables longitudinal studies, allows multiplexing of individual samples, and reduces reagents and laboratory waste. Our findings enhance understanding of the virtual staining process and highlight its potential for biomedical research applications.

bioengineering↗

bioRxiv2025

Targeting the translation machinery: The RNase Y specificity factor Y-complex coordinates ribosome degradation

Limiting the synthesis and activity of ribosomes is crucial for adaptation to stresses, such as heat or nutrient starvation. In Bacillus subtilis, this can be achieved through the coordinated action of the alarmones (p)ppGpp and the transcription factor Spx. Here, we performed a genetic screen to uncover novel factors contributing to the heat shock response of B. subtilis. We identified the Y-complex, which confers specificity to the endonuclease RNase Y, as a critical player during stress conditions, such as heat or transition into stationary phase. This protein complex is required for processing diverse RNAs, notably the maturation of mRNAs encoding proteins involved in translation and metabolism. We further demonstrate that the Y-complex and RNase Y initiate the degradation of rRNAs of mature ribosomes, lowering their abundance. We propose that the Y-complex is a regulatory hub that modulates gene expression, adjusts protein synthesis and resource allocation.

microbiology↗

bioRxiv2025

Plasticity in a bacterial global regulatory switch that drives a shift in antibiotic resistance and virulence

Antibiotic resistance and expression of virulence factors can impact the outcome of infection by Pseudomonas aeruginosa. Pathogenesis is often modelled using the P. aeruginosa PAO1 reference strain but laboratory lineages vary in the sequence and activity of MexT, a global regulator impacting virulence, biofilm formation and ciprofloxacin resistance. We defined the impact of active versus inactive MexT in PAO1 and observed global transcriptomic changes affecting the expression of 900 genes. Phenotyping revealed altered metabolism, antibiotic resistance and virulence - resulting in striking variation across a single model organism. We propose that antibiotic-resistance, introduced during work with the original PAO1 strain, has caused plasticity in mexT that accounts for variation across lineages. To test this, we introduced antibiotic resistance into clinical P. aeruginosa isolates and observed downstream mutations in mexT when selective pressure was removed, supporting the proposed evolutionary pathway. Overall, we have demonstrated the transcriptomic basis of MexT as a phenotypic switch in PAO1 and implicated antibiotic resistance as a cause of downstream changes in mexT in P. aeruginosa. Furthermore, MexS/MexT-regulated efflux is implicated in the antibiotic stress response and virulence, helping identify the mechanisms for rapid phenotypic switching through mexT and confirming that PAO1 is a very different organism compared to most isolates. Improved understanding of the regulatory changes linked to antibiotic resistance is particularly relevant to P. aeruginosa where cycles of antibiotic treatment are common.

microbiology↗

bioRxiv2025

Monolithic Shape-Shifting Absorbable Implants for Long-Term Contraception

Reversible contraceptives empower women to prevent unintended pregnancies and enable family planning. However, the need for frequent dosing with pills or injections often leads to suboptimal medication adherence and reduced effectiveness-an issue common to many chronic conditions. Long-acting drug delivery implants offer a compelling alternative by enabling autonomous, multi-year drug release, thereby improving real-world adherence and treatment outcomes. However, user acceptability and access are limited by need for invasive insertion and surgical end-of-life removal, particularly in low-resource settings, as well as by limited drug loading and suboptimal drug utilization efficiency, which constrain both the duration of therapy and the range of drugs that can be effectively delivered. To address these limitations, we developed the Monolithic Shape-shifting Absorbable Implants for Chronic Care (MoSAIC) platform-a minimally invasive, fully bioresorbable system that integrates compacted drug formulations with a space-efficient device architecture. This approach reduces implant size, eliminates the need for surgical removal, and prolongs therapeutic duration compared to existing implants. We develop compacted formulations of the contraceptive drug levonorgestrel (LNG), and other poorly water-solubility drugs, demonstrating exceptional drug loading (100% w/w) and multi-year sustained drug release via surface-mediated dissolution in rats. When incorporated into MoSAIC devices, these formulations enable high-efficiency drug loading and zero-order drug release kinetics with geometrically tunable rates and durations. As a result, MoSAIC systems can be designed to be smaller, less invasive, and/or longer lasting than current contraceptive implants such as Jadelle(R) and Nexplanon(R). The MoSAIC platform expands access to reversible contraception and supports long-term medication adherence, with the potential to improve health outcomes and quality of life. More broadly, it provides a flexible approach for delivering other potent, low-solubility therapeutics and lays the foundation for a "dose it and forget it" paradigm in chronic disease management, where adherence is designed into the therapy itself.

bioengineering↗

bioRxiv2025

Mapping global fisheries climate risks to guide sustainable marine management

Ocean warming is projected to reduce the capacity of the global ocean to support fish biomass. The severity of projected biomass declines, however, is distributed highly unevenly at the global scale, meaning some nations face disproportionate socioeconomic risks from resultant fisheries declines. Here, we apply the IPCC climate risk framework through the lens of fishing grounds to identify global hotspots where climate-driven declines in supportable pelagic fish biomass pose the greatest threat to socioeconomic security. By integrating empirical size-spectrum projections of fish biomass declines (hazard) with present-day fishing effort (exposure) and a novel area-based index of fishing ground socioeconomic vulnerability, we identify risk hotspots around Southeast Asia, the western seaboard of Africa, and the West Pacific. Our projections are corroborated by an ensemble of ecosystem models and compared with historical observations of declining zooplankton biomass in the Northeast Atlantic, suggesting climate-driven fish biomass declines are already underway. Many of the regions we assessed as highest-risk are predominantly targeted by nations with relatively low adaptive capacity and who therefore face the greatest barriers to redistributing fishing effort to lower-risk fishing grounds. Socioeconomic risks could thus be partly mitigated by targeting risk hotspots with area-based interventions that directly support fish stocks, such as the restoration of essential fish habitats and the designation of Marine Protected Areas. Resourcing the development of regionally tailored models for high-risk regions will provide the best evidence for designing specific area-based measures that effectively reduce societal risks, but enforcing such measures will depend upon addressing illegal, unregulated and untracked fishing.

ecology↗

bioRxiv2025

Common and distinct neural mechanisms of aversive and appetitive pain-related learning

Appetitive and aversive conditioning are both fundamental to adaptive behaviour, yet there remains limited understanding of how they differ on the behavioural and neural level. We investigated the two processes during acquisition and extinction using functional magnetic resonance imaging and behavioural measures. In a within-subject differential conditioning paradigm (preregistration DRKS00027448), aversive learning was induced by pairing visual cues with a temperature increase (pain rise), while appetitive learning involved a temperature decrease (pain reduction). Valence and contingency ratings confirmed successful learning for both types of learning, though only the appetitive condition showed a return to baseline ratings during extinction, suggesting incomplete extinction in the aversive condition. On the neural level, both engaged the visual cortex during acquisition (with increased functional connectivity with the right frontal operculum) and the ventromedial prefrontal cortex (vmPFC) during extinction. However, aversive learning showed a stronger activation increase in the mediodorsal thalamus with heightened connectivity with the locus coeruleus during acquisition, as well as sustained parahippocampal activity during extinction. Moreover, incomplete extinction in the aversive condition (as indicated by contingency ratings) was associated with sustained activity in the visual cortex during pain anticipation. These results suggest that while appetitive and aversive learning share activation in regions involved in sensory processing (occipital lobe) and learning (vmPFC), aversive learning uniquely engages areas promoting rapid acquisition (mediodorsal thalamus and locus coeruleus) and cautious unlearning, in line with the notion of a better-safe-than-sorry strategy.

neuroscience↗

bioRxiv2025

Mapping the Neural Control of Force and Impedance of Wrist Movements Using Robotics and fMRI

While robots are becoming increasingly valuable tools in neurorehabilitation, our limited understanding of the brains response during human-robot interaction tasks restricts advancements in training programs to restore neural pathways after injury. Co-contraction is characteristic of several neuro-muscular disorders, such as stroke and cerebral palsy, and it is often targeted by assessments or rehabilitation programs. Despite its importance, the neural mechanisms underlying co-contraction remain poorly understood. To address this gap, this study investigates the neural substrates of muscle co-contraction via functional magnetic resonance imaging (fMRI) during a dynamic motor task with an MR-compatible wrist robot. To establish suitable fMRI experimental conditions, we first conducted a behavioral study assessing muscle activity during a wrist-pointing task with four participants. Participants reached toward a target while experiencing four main perturbation conditions (no force, divergent force, constant force up, and constant force down), designed to elicit distinct force and impedance responses. Following this behavioral validation, five additional participants performed the wrist-pointing task during fMRI. Our results suggest localization of force and impedance control within the cortico-thalamic-cerebellar network. These findings provide new insights into the neural mechanisms of co-contraction, supporting the development of neurorehabilitation paradigms.

bioengineering↗

bioRxiv2024

An NADH-controlled gatekeeper of ATP synthase

ATP fuels crucial cellular processes and is obtained mostly by oxidative phosphorylation (OXPHOS) at the inner mitochondrial membrane. While significant progress has been made in mechanistic understanding of ATP production, critical aspects surrounding its substrate supply logistics are poorly understood. We identify an interaction between mitochondrial apoptosis-inducing factor 1 (AIFM1) and adenylate kinase 2 (AK2) as gatekeeper of ATP synthase. This interaction is NADH-dependent and influenced by glycolysis, linking it to the cells metabolic state. Genetic interference with AIFM1/AK2 association impedes the ability of Caenorhabditis elegans animals to handle altered metabolic rates and nutrient availability. Together, the results imply AIFM1 as a cellular NADH sensor, placing AK2 next to the OXPHOS complexes for local ADP regeneration as the substrate for ATP synthesis. This metabolic signal relay balances ATP synthase substrate supply against ATP conservation, enabling cells to adapt to fluctuating energy availability, with possible implications for AIFM1-related mitochondrial diseases. HighlightsO_LIDiscovery of AIFM1/AK2 interaction as a gatekeeper of mitochondrial ATP synthase C_LIO_LIAIFM1/AK2 interaction is NADH-dependent and linked to the cells metabolic state C_LIO_LIDisrupting AIFM1/AK2 impairs metabolic adaptation in Caenorhabditis elegans C_LIO_LIAIFM1 as NADH sensor, influencing ATP synthesis with mitochondrial disease implications C_LI

biochemistry↗

bioRxiv2024

Brief sleep disruption alters synaptic structures among hippocampal and neocortical somatostatin-expressing interneurons

Study objectivesBrief sleep loss alters cognition and synaptic structures of principal neurons in hippocampus and neocortex. However, while in vivo recording and bioinformatic data suggest that inhibitory interneurons are more strongly affected by sleep loss, it is unclear how sleep and sleep deprivation affect interneurons synapses. Disruption of the SST+ interneuron population seems to be a critical early sign of neuropathology in Alzheimers dementia, schizophrenia, and bipolar disorder - and the risk of developing all three is increased by habitual sleep loss. We aimed to test how the synaptic structures of SST+ interneurons in various brain regions are affected by brief sleep disruption. MethodsWe used Brainbow 3.0 to label SST+ interneurons in the dorsal hippocampus, prefrontal cortex, and visual cortex of male SST-CRE transgenic mice, then compared synaptic structures in labeled neurons after a 6-h period of ad lib sleep, or gentle handling sleep deprivation (SD) starting at lights on. ResultsDendritic spine density among SST+ interneurons in both hippocampus and neocortex was altered in a subregion-specific manner, with increased overall and thin spine density in CA1, dramatic increases in spine volume and surface area in CA3, and small but significant changes (primarily decreases) in spine size in CA1, PFC and V1. ConclusionsOur suggest that the synaptic connectivity of SST+ interneurons is significantly altered in a brain region-specific manner by a few hours of sleep loss. This suggests a cell type-specific mechanism by which sleep loss disrupts cognition and alters excitatory-inhibitory balance in brain networks. Significance StatementChanges to the function of somatostatin-expressing (SST+) interneurons have been implicated in the etiology of psychiatric and neurological disorders in which both cognition and sleep behavior are affected. Here, we measure the effects of very brief experimental sleep deprivation on synaptic structures of SST+ interneurons in hippocampus and neocortex, in brain structures critical for sleep-dependent memory processing. We find that only six hours of sleep deprivation restructures SST+ interneurons dendritic spines, causing widespread and subregion-specific changes to spine density and spine size. These changes have the potential to dramatically alter excitatory-inhibitory balance across these brain networks, leading to cognitive disruptions commonly associated with sleep loss.

neuroscience↗

bioRxiv2024

Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. In this study, we set out to develop a comprehensive set of plasmids, promoters and reporters for S. pyogenes. We present an expansion to the current genetic toolbox that comprises new replicative and site-specific integrative plasmids. Moreover, we established a collection of constitutive promoters with a wide variety of strengths as well as a set of novel inducible regulatory elements, including a zinc-inducible promoter, an erythromycin-inducible riboswitch and an IPTG-inducible promoter that outperform previously described inducible systems in terms of tightness and inducibility. In addition, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. For this, we adapted a novel chemically defined medium called RPMI4Spy. This medium showed a highly reduced autofluorescence compared to other growth media and allowed efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which improved the generation of scarless gene deletions. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

synthetic biology↗