Abstract The protozoan parasite Leishmania infantum exhibits significant genetic variability among isolates, influencing disease manifestation and treatment response. Although L. infantum is classically described as the causative agent of Visceral Leishmaniasis (VL) – often associated with immune deficiency, cases of Cutaneous Leishmaniasis (CL) caused by this species in immunocompetent individuals have been reported in different countries. To investigate the molecular basis of this unusual shift in tissue tropism and pathogenicity, we applied comparative genomic and transcriptomic approaches on two canine isolates ( CanL ) and two human isolates associated with Cutaneous Leishmaniasis ( CL ) in Tunisia. While the CanL isolates showed close genetic similarity to the L. infantum reference strain (JPCM5), the CL isolates formed a separate, highly divergent cluster based on SNP localization and frequency, differing not only from JPCM5 but also from each other. Utilizing the metagenomics sequence classification tool Kraken, we revealed a complex hybrid nature of the CL isolates, showing introgression from L. donovani and L. tropica , suggesting that hybridization has played a key role in generating novel phenotypic traits. Integration of RNA-seq and DNA-seq data demonstrated that only a minority of gene expression variation within and in-between the CanL or CL groups reflected gene dosage effects due to copy number variation, while the majority of expression differences were independent of gene dosage, implying post-transcriptional regulatory mechanisms contributing to parasite adaptation. In conclusion, our study identifies hybridization, genome instability, and transcriptomic adaptation as interconnected drivers of the L. infantum evolutionary potential. These mechanisms can collectively enhance parasite fitness gain, potentially explaining the emergence of cutaneous disease forms in a species traditionally linked to visceral infection. Author Summary This research reveals that hybridization between distinct Leishmania parasite species could be a key mechanism driving the evolution of new disease forms. By demonstrating that cutaneous leishmaniasis (CL) cases are caused by hybrid L. infantum parasites whose genomes show introgression with DNA from L. donovani and L. tropica , this study reveals a molecular mechanism potentially linked to the emergence of tegumentary disease from a species traditionally known to cause visceral infection. These findings contribute to our understanding of Leishmania evolution, the emergence of atypical forms of leishmaniasis linked to hybridization, and the impact of genome instability and transcriptomic adaptation as potent forces for generating phenotypic diversity and enhancing parasite fitness.

Genome assembly from long-read sequencing data has become a standard approach for resolving complex genomic regions and producing high-contiguity assemblies. However, the diversity of available assemblers, their varying performance across species, and the need for reproducible workflows present ongoing challenges. We developed LORA, an easy-to-use and reproducible application for assembling genomes from long-read data. LORA integrates several well-established assemblers, including Canu, HiFiasm, Flye, and Unicycler, as well as more recent tools such as Necat and Pecat. It is implemented as a Snakemake pipeline to parallelize tasks and support seamless execution on both local machines and computing clusters. LORA includes multiple quality assessment steps, interactive HTML reports for interpretation, BLAST-based taxonomic identification, and completeness evaluation. Together, these features provide users with a comprehensive view of assembly quality and potential problems. We illustrate the capabilities of LORA using datasets from bacterial genomes and unicellular eukaryotes, sequenced with both PacBio and Oxford Nanopore technologies, highlighting typical outcomes and common pitfalls encountered during long-read assemblies. LORA is distributed as part of the Sequana project, an open-source framework designed for reproducibility, maintainability, and straightforward deployment across computing environments.

Segmented filamentous bacteria (SFB) describe morphologically similar gut commensals found in mammals, fish and birds. In mice, SFB intimately colonizes the ileal epithelium at the time of weaning and elicits a strong pleiotropic immune activation that fosters colonization resistance while augmenting disease severity in various disease models. SFB is therefore critical in both health and disease but information regarding SFB in humans remains limited. Here, we first identify and characterize a human SFB species with SFB-specific morphology, including the hook-like tip structure that mediates attachment, and unique genome features, including a starch and glycogen degradation module. This species, which we name Anisomitus miae and establish as the nomenclature type for the SFB genus, is within a SFB lineage common across Africa. We then bioinformatically identify, based on the 16S rRNA gene V3-V4 variable region sequence, four major, and two minor, human SFB lineages in forty-four countries distributed across all six inhabited continents. We provide evidence towards the co-colonization potential of the SFB lineages and their colonization dynamics, including a potent but short-lived colonization peak in children between one to five years of age. This study establishes the presence of multiple SFB species in the human population and SFB as a minor but wide-spread group of commensals in humans.

Translation initiation signals shape gene expression across all domains of life. In eukaryotes, nu-cleotide constraints surrounding the start codon are commonly described by the Kozak Consensus Sequence (KCS), whereas in bacteria and archaea, initiation frequently involves Shine–Dalgarno ribosome-binding motifs. Although these signals have been extensively characterized in model or-ganisms, their large-scale diversity and evolutionary distribution remain incompletely explored. We present KozakExplorer, a reproducible framework for quantitative and comparative analysis of translation initiation contexts from genome assemblies and annotations. The software per-forms strand-aware extraction of start codon environments from FASTA and GFF3 files and ap-plies information-theoretic metrics—including Kullback–Leibler (KL) divergence and information content (IC)—to measure positional nucleotide constraints relative to a background model. Derived summary statistics (Kozak Strength Index [KSI], maximum information content, peak position) con-vert motif patterns into interpretable per-genome signatures suitable for cross-species comparison. Our primary analysis covers 2,282 eukaryotic reference genomes, producing a standardized dataset of translation initiation metrics. Dimensionality reduction via t-SNE on per-position KL divergence, information content, and motif nucleotide frequencies reveals a structured eukaryotic KCS land-scape with kingdom-level clustering and continuous variation in signal strength. A dedicated case study of 216 Apicomplexa genomes shows genus-level structure consistent with host range and phylogeny. An extended analysis across 25,344 reference genomes (22,253 bacteria, 809 archaea) places eukaryotic patterns in a global comparative framework, revealing transitions between sharply localized Kozak motifs and distributed Shine–Dalgarno-type signatures. Implemented within the open-source Sequana ecosystem, KozakExplorer is distributed as a Python module and an interactive web application that accepts local annotated assemblies, GenBank records, or NCBI RefSeq accessions, and exports all computed metrics, embeddings, and coordi-nates for downstream comparative and evolutionary genomics. Availability Implemented in Python within the Sequana framework. Source code available at https://github.com/sequana/sequana and https://github.com/sequana/webapp_kozak . Contact thomas.cokelaer@pasteur.fr

Hybridization, the merging of distinct genomes, is increasingly recognized as a major evolutionary force among eukaryotic pathogens, including facultatively sexual protist parasites like Leishmania and Trypanosoma. While it may contribute to pathogen virulence and drug resistance, hybridization remains poorly characterized, particularly how genetic distance between parental cells influences genomic compatibility and which compensatory mechanisms ensure hybrid viability. Here, we report the in vitro generation of an unusual sexual hybrid between Leishmania species infecting mammals (L. infantum) and reptiles (the Sauroleishmania L. tarentolae), and used this unique genetic model system to address these open questions. Our data provide evidence of genomic compatibility between even highly divergent Leishmania species, offering new insights into the evolutionary potential of Leishmania and related pathogens. We demonstrate that the genomic shock caused by the fusion of distinct genomes can be mitigated by two key mechanisms: (i) at the genomic level, chromosome loss allows the establishment of mosaic aneuploidy in the newly formed hybrid, and (ii) at the post-transcriptional level, preferential mono-parental allelic expression acts as a secondary compensatory mechanism. Our findings establish genome instability and post-transcriptional regulation as central processes in Leishmania hybridization, which may be of broad relevance to other biological systems undergoing genetic exchange.

Dioxygen (O2) is vital for aerobic life, but its utilization leads to the inevitable production of superoxide, a toxic oxidant. The prevailing theory of oxygen toxicity postulates that superoxide-scavenging enzymes (SOSEs), such as superoxide dismutases (SODs), are crucial for most aerobes and play a key role in the virulence of pathogens. However, our knowledge of superoxide adaptation primarily stems from the study of SOSE-encoding bacteria. Here, we investigated the evolution of a naturally SOSE-deficient pathogen (Leptospira spp.) and its alternative mechanisms to combat superoxide stress. We demonstrated that SOD was ancestral in the genus Leptospira but lost by pathogenic species, and heterologous expression of a SOD in this pathogen did not improve superoxide tolerance. In L. interrogans, inheritable increased expression of a genetic locus, including a MFS transporter, mediated a long-lasting adaptation to superoxide, independently of any permanent genetic modification. Using a multi-omics approach, we identified a leuA2-encoded isopropylmalate synthase, the enzyme catalyzing the first step of leucine biosynthesis, as the most upregulated factor by superoxide. Interestingly, LeuA2 lacks the canonical domain for feedback inhibition by leucine and is the only upregulated factor of leucine biosynthesis, suggesting a moonlighting activity for LeuA2 in the adaptation to superoxide. Moreover, the cysteine biosynthesis pathway was significantly upregulated in response to superoxide, and we demonstrated the importance of sulfur metabolism in adaptation to superoxide. This study revisits our conventional understanding of the oxygen toxicity theory and proposes a new model of superoxide adaptation through redox-based metabolic rewiring in SOSE-deficient aerobic bacteria.IMPORTANCESuperoxide is a toxic reactive oxygen species produced as an inevitable byproduct during oxygen respiration. It is therefore assumed that aerobic bacteria require superoxide scavenging enzymes (SOSEs), such as superoxide dismutases. Recent studies estimate that around 10% of all living organisms lack SOSEs. However, we ignore how these organisms survive superoxide stress when confronted with oxygen. Here, using Leptospira interrogans, a naturally SOSE-deficient aerobic pathogen, we address the evolutionary path and defense mechanisms leading to the adaptation to superoxide in the absence of any SOSE. We demonstrate that a SOD was ancestral in this genus but was lost with the emergence of pathogenic species. In addition, we show that pathogenic Leptospira induce metabolic pathways to fight superoxide, such as cysteine biosynthesis and isopropylmalate synthase. Thus, our study reveals that redox-based metabolic reprogramming may compensate for the loss of SOSEs in pathogenic bacteria.

Candida albicans is a commensal of the human microbiota that can form biofilms on implanted medical devices. These biofilms are tolerant to antifungals and to the host immune system. To identify novel genes modulating C. albicans biofilm formation, we performed a large-scale screen with 2,454 C. albicans doxycycline-dependent overexpression strains and identified 16 genes whose overexpression significantly hampered biofilm formation. Among those, overexpression of the ZCF15 and ZCF26 paralogs that encode transcription factors and have orthologs only in biofilm-forming species of the Candida clade, caused impaired biofilm formation both in vitro and in vivo. Interestingly, overexpression of ZCF15 impeded biofilm formation without any defect in hyphal growth. Transcript profiling, transcription factor binding, and phenotypic microarray analyses conducted upon overexpression of ZCF15 and ZCF26 demonstrated their role in reprogramming cellular metabolism by regulating central metabolism including glyoxylate and tricarboxylic acid cycle genes. Taken together, this study has identified a new set of biofilm regulators, including ZCF15 and ZCF26, that appear to control biofilm development through their specific role in metabolic remodeling.

ABSTRACT Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis ( Mtb ) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens , Mycobacterium lacus , Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent “missing links” between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb . We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C–35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb , was present only in M. decipiens , which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms. IMPORTANCE In this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis ( Mtb ) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum . We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb .

Heme and iron metabolic pathways are highly intertwined, both compounds being essential for key biological processes, yet becoming toxic if overabundant. Their concentrations are exquisitely regulated, including via dedicated two-component systems (TCSs) that sense signals and regulate adaptive responses. HemKR is a TCS present in both saprophytic and pathogenic Leptospira species, involved in the control of heme metabolism. However, the molecular means by which HemKR is switched on/off in a signal-dependent way, are still unknown. Moreover, a comprehensive list of HemKR-regulated genes, potentially overlapped with iron-responsive targets, is also missing. Using the saprophytic species Leptospira biflexa as a model, we now show that 5-aminolevulinic acid (ALA) triggers the shutdown of the HemKR pathway in live cells, and does so by stimulating the phosphatase activity of HemK towards phosphorylated HemR. Phospho~HemR dephosphorylation leads to differential expression of multiple genes, including of heme metabolism and transport systems. Besides the heme-biosynthetic genes hemA and the catabolic hmuO, which we had previously reported as phospho~HemR targets, we now extend the regulon identifying additional genes. Finally, we discover that HemR inactivation brings about an iron-deficit tolerant phenotype, synergistically with iron-responsive signaling systems. Future studies with pathogenic Leptospira will be able to confirm whether such tolerance to iron deprivation is conserved among Leptospira spp., in which case HemKR could play a vital role during infection where available iron is scarce. In sum, HemKR responds to abundance of porphyrin metabolites by shutting down and controlling heme homeostasis, while also contributing to integrate the regulation of heme and iron metabolism in the L. biflexa spirochete model.

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated with a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context. Through experimental evolution, we find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate a nice interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs.

Bacteroides thetaiotaomicron is a prominent member of the human gut microbiota contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm lifestyle, and it was recently shown that B. thetaiotaomicron biofilm formation is promoted by the presence of bile. This process also requires a B. thetaiotaomicron extracellular DNase, which is not, however, regulated by bile. Here, we showed that bile induces the expression of several Resistance-Nodulation-Division (RND) efflux pumps and that inhibiting their activity with a global competitive efflux inhibitor impaired bile-dependent biofilm formation. We then showed that, among the bile-induced RND-efflux pumps, only the tripartite BT3337-BT3338-BT3339 pump, re-named BipABC [for Bile Induced Pump A (BT3337), B (BT3338), and C (BT3339)], is required for biofilm formation. We demonstrated that BipABC is involved in the efflux of magnesium to the biofilm extracellular matrix, which leads to a decrease of extracellular DNA concentration. The release of magnesium in the biofilm matrix also impacts biofilm structure, potentially by modifying the electrostatic repulsion forces within the matrix, reducing interbacterial distance and allowing bacteria to interact more closely and form denser biofilms. Our study therefore, identified a new molecular determinant of B. thetaiotaomicron biofilm formation in response to bile salts and provides a better understanding on how an intestinal chemical cue regulates biofilm formation in a major gut symbiont.IMPORTANCEBacteroides thetaiotaomicron is a prominent member of the human gut microbiota able to degrade dietary and host polysaccharides, altogether contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm community lifestyle, providing protection against environmental factors that might, in turn, protect the host from dysbiosis and dysbiosis-related diseases. It was recently shown that B. thetaiotaomicron exposure to intestinal bile promotes biofilm formation. Here, we reveal that a specific B. thetaiotaomicron membrane efflux pump is induced in response to bile, leading to the release of magnesium ions, potentially reducing electrostatic repulsion forces between components of the biofilm matrix. This leads to a reduction of interbacterial distance and strengthens the biofilm structure. Our study, therefore, provides a better understanding of how bile promotes biofilm formation in a major gut symbiont, potentially promoting microbiota resilience to stress and dysbiosis events.

Defenses against oxidants are crucial for the virulence of pathogens, with superoxide scavenging enzymes (SOSEs) playing a vital role for most aerobes. However, our knowledge of superoxide adaptation primarily stems from the study of SOSE-encoding bacteria. Here, we investigated the evolution of a naturally SOSE-deficient pathogen ( Leptospira spp.), along with the alternative mechanisms it recruits to combat superoxide stress. We demonstrate that emergence of pathogenic Leptospira correlated with SOD loss, but that a long-lasting adaptation to superoxide remains possible. We reveal that cysteine and leucine biosynthesis are the most induced pathways in response to superoxide and demonstrate the importance of sulfur metabolism in superoxide adaptation in this SOSE-deficient model. We also propose cysteine oxidation as a key mediator of superoxide toxicity in the absence of SOSEs. This study challenges our conventional understanding of the oxygen toxicity theory and proposes a new model of superoxide adaptation through metabolic rewiring in bacteria.

ABSTRACTBacteroides thetaiotaomicronis a prominent member of the human gut microbiota contributing to nutrient exchange, gut function, and maturation of the host’s immune system. This obligate anaerobe symbiont can adopt a biofilm lifestyle and it was recently shown thatB. thetaiotaomicronbiofilm formation is promoted by the presence of bile, a process also requiring aB. thetaiotaomicronextracellular DNase, which is not, however, regulated by bile. Here we showed that bile induces the expression of several Resistance-Nodulation-Division (RND) efflux pumps and that inhibiting their activity with a global competitive efflux inhibitor impaired bile-dependent biofilm formation. We then showed that, among the bile-induced RND-efflux pumps, only the tripartite BT3337-BT3338-BT3339 pump, re-named BipABC (for Bile Induced Pump A (BT3337), B (BT3338) and C (BT3339), is required for biofilm formation. We demonstrated that BipABC is involved in the efflux of magnesium to the biofilm extracellular matrix, which leads to a decrease of eDNA concentration. The release of magnesium in the biofilm matrix also impacts biofilm structure, potentially by modifying the electrostatic repulsion forces within the matrix, reducing interbacterial distance and allowing bacteria to interact more closely and form denser biofilms. Our study therefore identifies a new molecular determinant ofB. thetaiotaomicronbiofilm formation in response to bile salts and provides a better understanding on how an intestinal chemical cue regulates biofilm formation in a major gut symbiont.IMPORTANCEBacteroides thetaiotaomicronis a prominent member of the human gut microbiota able to degrade dietary and host polysaccharides, altogether contributing to nutrient exchange, gut function, and maturation of the host’s immune system. This obligate anaerobe symbiont can adopt a biofilm community lifestyle, providing protection against environmental factors that might, in turn, protect the host from dysbiosis and dysbiosis-related diseases. It was recently shown thatB. thetaiotaomicronexposure to intestinal bile promotes biofilm formation. Here we reveal that a specificB. thetaiotaomicronmembrane efflux pump is induced in response to bile, leading to the release of magnesium ions, potentially reducing electrostatic repulsion forces between components of the biofilm matrix. This leads to a reduction of interbacterial distance and strengthens the biofilm structure. Our study therefore provides a better understanding of how bile promotes biofilm formation in a major gut symbiont, potentially promoting microbiota resilience to stress and dysbiosis events.

Hepatic steatosis is the result of imbalanced nutrient delivery and metabolism in the liver and is the first hallmark of Metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is the most common chronic liver disease and involves the accumulation of excess lipids in hepatocytes, inflammation, and cancer. Mitochondria play central roles in liver metabolism yet the specific mitochondrial functions causally linked to MASLD remain unclear. Here, we identify Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, liver-specific knockout mice are protected against high fat diet-induced steatosis and metabolic dysregulation. Additionally, Mtfp1 deletion inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers additional functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for MASLD.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip, is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins.

Bat sarbecovirus BANAL‐236 is highly related to SARS‐CoV‐2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL‐236 replicates efficiently and pauci‐symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS‐CoV‐2. BANAL‐236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre‐spillover event that did not occur upon replication of a SARS‐CoV‐2‐like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS‐CoV‐2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.

Motivation The reproducibility crisis has highlighted the importance of improving the way bioinformatics data analyses are implemented, executed, and shared. To address this, various tools such as content versioning systems, workflow management systems, and software environment management systems have been developed. While these tools are becoming more widely used, there is still much work to be done to increase their adoption. The most effective way to ensure reproducibility becomes a standard part of most bioinformatics data analysis projects is to integrate it into the curriculum of bioinformatics Master's programs. Results In this article, we present the Reprohackathon, a Master's course that we have been running for the last 3 years at Universite Paris-Saclay (France), and that has been attended by a total of 123 students. The course is divided into two parts. The first part includes lessons on the challenges related to reproducibility, content versioning systems, container management, and workflow systems. In the second part, students work on a data analysis project for 3-4 months, reanalyzing data from a previously published study. The Reprohackaton has taught us many valuable lessons, such as the fact that implementing reproducible analyses is a complex and challenging task that requires significant effort. However, providing in-depth teaching of the concepts and the tools during a Master's degree program greatly improves students' understanding and abilities in this area.

Bioinformatics is a field known for the numerous standards and formats that have been developed over the years. This plethora of formats, sometimes complementary, and often redundant, poses many challenges to bioinformatics data analysts. They constantly need to find the best tool to convert their data into the suitable format, which is often a complex, technical and time consuming task. Moreover, these small yet important tasks are often difficult to make reproducible. To overcome these difficulties, we initiated BioConvert, a collaborative project to facilitate the conversion of life science data from one format to another. BioConvert aggregates existing software within a single framework and complemented them with original code when needed. It provides a common interface to make the user experience more streamlined instead of having to learn tens of them. Currently, BioConvert supports about 50 formats and 100 direct conversions in areas such as alignment, sequencing, phylogeny, and variant calling. In addition to being useful for end-users, BioConvert can also be utilized by developers as a universal benchmarking framework for evaluating and comparing numerous conversion tools. Additionally, we provide a web server implementing an online user-friendly interface to BioConvert, hence allowing direct use for the community.

Here, we report complete genome sequences of two clinical isolates of Staphylococcus aureus, namely, Xen31 and Xen36, which have been genetically modified to express an optimized Photorhabdus luminescens luciferase operon. Xen31 and Xen36 are bioluminescent strains used widely for investigation of bacterial pathogenesis, drug discovery, and development of novel therapies.

HIV elite controllers maintain a population of CD4 + T cells endowed with high avidity for Gag antigens and potent effector functions. How these HIV-specific cells avoid infection and depletion upon encounter with the virus remains incompletely understood. Ex vivo characterization of single Gag-specific CD4 + T cells reveals an advanced Th1 differentiation pattern in controllers, except for the CCR5 marker, which is downregulated compared to specific cells of treated patients. Accordingly, controller specific CD4 + T cells show decreased susceptibility to CCR5-dependent HIV entry. Two controllers carried biallelic mutations impairing CCR5 surface expression, indicating that in rare cases CCR5 downregulation can have a direct genetic cause. Increased expression of β-chemokine ligands upon high-avidity antigen/TCR interactions contributes to autocrine CCR5 downregulation in controllers without CCR5 mutations. These findings suggest that genetic and functional regulation of the primary HIV coreceptor CCR5 play a key role in promoting natural HIV control.