Biology subjects

Lupo, V.

Publications and source records attributed to Lupo, V..

7 recordsLinked to original sources

bioRxiv2026

A new automated pipeline for whole genome shotgun sequencing analysis and hazard characterization of microbial pesticides

Microbial pesticides are increasingly important for sustainable crop protection, yet hazard analysis and risk assessment remain challenging and require specific characterization of properties relevant to both safety and biological activity. Whole-genome sequencing (WGS) can support the identification of microorganisms at high resolution and characterize potential hazards associated with infectivity, pathogenicity, antimicrobial resistance, and toxic metabolite production. However, the routine use of WGS in this context requires accessible, reproducible, and interpretable workflows. Here, we present a publicly available web-based workflow for WGS-supported hazard analysis of microbial biocontrol agents. The workflow accepts assembled genomes as well as short-read, long-read and hybrid sequencing data, and performs genome quality assessment, taxonomic assignment, genome annotation, AMR detection, mobile-element screening, pathogenicity prediction and secondary-metabolite analysis, and compiles the results into an HTML report. For bacterial agents, we implement a transparent rule-based risk-classification module integrating taxonomic identity, PathogenFinder2 predictions, CARD/RGI resistance evidence, WHO priority taxa, and medically important antimicrobial categories. Secondary-metabolite assessment combines antiSMASH with local BLAST searches and EFSA-aligned identity/coverage thresholds to support product-level interpretation of biosynthetic gene clusters. Comparison with the currently used MOpS workflow developed by EFSA demonstrates the added benefits of the proposed workflow for a guided hazard analysis of microbial pesticides. The workflow is intended as a community-accessible pre-assessment tool that complements regulatory platforms by improving transparency, reproducibility, and early identification of potential hazards in microbial biocontrol candidates, envisioned to make WGS an integral part of the risk assessment of microbial pesticides. Key pointsO_LIThe RATION-GUI provides a public, reproducible workflow for whole-genome sequencing- supported hazard characterization of bacterial and fungal microbial pesticides. C_LIO_LIThe workflow integrates genome quality, taxonomy, annotation, antimicrobial resistance, pathogenicity-related evidence, mobile elements, and secondary-metabolite potential. C_LIO_LIDomain-specific outputs are translated into structured hazard indicators, evidence summaries, and recommended follow-up actions without replacing expert regulatory judgment. C_LIO_LICase studies demonstrate how the workflow improves transparency, traceability, and interpretation of genomic evidence for microbial pesticide risk assessment. C_LI

bioinformatics↗

bioRxiv2025

Out of Antarctica: new insights into Antarctic Subcluster 5.2 picocyanobacteria based on high-quality genomes

2.Synechococcus-like cyanobacteria are cosmopolitan unicellular picocyanobacteria that have colonized diverse aquatic and terrestrial habitats. The so-called subcluster 5.2 represents a particularly diversified subgroup, including marine and freshwater organisms adapted to extreme conditions, notably polar environments. We increased the genomic representation of polar taxa in this subcluster by reconstructing new high-quality genomes from five Antarctic lacustrine strains and one Arctic freshwater isolate using a combination of small Illumina and long Nanopore reads. A maximum likelihood (ML) phylogenomic analysis of these new assemblies combined with all publicly available good quality assemblies of the subcluster 5.2 suggests evidence of a dispersal process from Antarctica. Indeed, the topology of the phylogenomic tree indicates one basal Antarctic lineage followed by the emergence of two lineages, one Antarctic and one non-Antarctic (Spain). This finding is further supported by a 16S rRNA ML phylogenetic and a pangenomic analysis. Although secondary colonization of Antarctica by cyanobacteria following the cooling of the continent 34 million years ago has been reported, this study is the first to support an Out-of-Antarctica scenario inside subcluster 5.2. 3. Impact statementThis study provides novel insights into the evolutionary history and biogeography of Synechococcus-like picocyanobacteria. By expanding the genomic sampling of polar subcluster 5.2, we reveal that Antarctic taxa are not monophyletic and are intermixed with non-Antarctic lineages, suggesting an unprecedented Out-of-Antarctica dispersal scenario. Our results, supported by 16S rRNA, phylogenomic and pangenomic analyses, highlight the role of polar regions as both a refuge and a source of microbial diversity. This work advances our understanding of cyanobacterial adaptation, radiation and genome evolution in extreme environments. 4. Data summaryRaw sequencing reads and genome assemblies from this study have been deposited in the European Nucleotide Archive (ENA) under Project accession number PRJEB103944. Raw sequencing reads are available under accession numbers ERR15933635 to ERR15933638, ERR15933641 to ERR15933644, ERR15903357, ERR15903358, ERR15905256 and ERR15905257. The assemblies can be accessed under accession numbers GCA_977927515, GCA_977927525, GCA_977927535, GCA_977927545, GCA_977927555 and GCA_977929215. The scripts and code as well as large supplementary files generated during this study are available on Figshare and can be downloaded from https://doi.org/10.6084/m9.figshare.30772781 [1]. Supplementary data files are also provided and contains supplementary figures (Fig. S1-S4) and tables (Table S1-S6).

ecology↗

bioRxiv2024

Identification and Characterization of Archaeal Pseudomurein Biosynthesis Genes through Pangenomics

The peptidoglycan (PG; or murein) is a mesh-like structure, which is made of glycan polymers connected by short peptides and surrounds the cell membrane of nearly all bacterial species. In contrast, there is no PG counterpart that would be universally found in Archaea, but rather various polymers that are specific to some lineages. Methanopyrales and Methanobacteriales are two orders of Euryarchaeota that harbor pseudomurein (PM), a structural analogue of the bacterial PG. Owing to the differences between PG and PM biosynthesis, some have argued that the origin of both polymers is not connected. However, recents studies have revealed that the genomes of PM-containing Archaea encode homologues of the bacterial genes involved in PG biosynthesis, even though neither their specific functions nor the relationships within the corresponding inter-domain phylogenies have been investigated so far. In this work, we devised a pangenomic bioinformatic pipeline to identify proteins for PM biosynthesis in Archaea without prior genetic knowledge. The taxonomic distribution and evolutionary relationships of the candidate proteins were studied in detail in Archaea and Bacteria through HMM sequence mining and phylogenetic inference of the Mur domain-containing family, the ATP-grasp superfamily and the MraY-like family. Our results show that archaeal muramyl ligases are of bacterial origin, but diversified through a mixture of horizontal gene transfers and gene duplications. However, in the ATP-grasp and MraY-like families, the archaeal members were not found to originate from Bacteria. Our pangenomic approach further identified five new genes potentially involved in PM synthesis and that would deserve functional characterization.

microbiology↗

bioRxiv2022

Origin and Evolution of Pseudomurein Biosynthetic Gene Clusters

The peptidoglycan (PG; or murein) is a mesh-like structure, which is made of glycan polymers connected by short peptides and surrounds the cell membrane of nearly all bacterial species. In contrast, there is no PG counterpart that would be universally found in Archaea, but rather various polymers that are specific to some lineages. Methanopyrales and Methanobacteriales are two orders of Euryarchaeota that harbor pseudomurein (PM) in their cell-wall, a structural analogue of the bacterial PG. Owing to the differences between PG and PM biosynthesis, some have argued that the origin of both polymers is not connected. However, recents studies have revealed that the genomes of PM-containing Archaea encode homologues of the bacterial genes involved in PG biosynthesis, even though neither their specific functions nor the relationships within the corresponding inter-domain phylogenies have been investigated so far. In this work, we devised a bioinformatic pipeline to identify all potential proteins for PM biosynthesis in Archaea without relying on a candidate gene approach. After an in silico characterization of their functional domains, the taxonomic distribution and evolutionary relationships of the collected proteins were studied in detail in Archaea and Bacteria through HMM similarity searches and phylogenetic inference of the Mur domain-containing family, the ATP-grasp superfamily and the MraY-like family. Our results notably show that the extant archaeal muramyl ligases are ultimately of bacterial origin, but likely diversified through a mixture of horizontal gene transfer and gene duplication. Moreover, structural modeling of these enzymes allowed us to propose a tentative function for each of them in pentapeptide elongation. While our work clarifies the genetic determinants behind PM biosynthesis in Archaea, it also raises the question of the architecture of the cell wall in the last universal common ancestor.

microbiology↗

bioRxiv2022

CRitical Assessment of genomic COntamination detection at several Taxonomic ranks (CRACOT)

BackgroundGenome contamination is a well-known issue in (meta)genomics. Although it has received a lot of attention, with an increasing number of detection tools made available over the years, no comparison between these tools exists in the literature. ResultsHere, we report the benchmarking of six of the most popular tools using a simulated framework. Our simulations were conducted on six different taxonomic ranks, from phylum to species. The analysis of the estimated contamination levels indicates that the precision of the tools is not good, often due to large overdetection but also underdetection, especially at the genus and species ranks. Furthermore, our results show that only redundant contamination is accurately estimated. ConclusionOur results indicate that using a combination of tools, including Kraken2, is necessary to estimate the contamination level accurately. We also provide a freely available contamination simulation framework, CRACOT, which may be useful for estimating the accuracy of future algorithms.

bioinformatics↗

bioRxiv2022

The GEN-ERA toolbox: unified and reproducible workflows for research in microbial genomics .

BackgroundMicrobial culture collections play a key role in taxonomy by studying the diversity of their accessions and providing well characterized strains to the scientific community for fundamental and applied research. These microbial resource centers thus need to implement new standards in species delineation, including whole-genome sequencing and phylogenomics. In this context, the genomic needs of the Belgian Coordinated Collections of Microorganisms (BCCM) were studied, resulting in the GEN-ERA toolbox. The latter is a unified cluster of bioinformatic workflows dedicated to both bacteria and small eukaryotes (i.e. yeasts). FindingsThis public toolbox allows researchers without a specific training in bioinformatics to perform robust phylogenetic analyses. Hence, it facilitates all steps from genome downloading and quality assessment, including genomic contamination estimation, to tree reconstruction. It also offers workflows for average nucleotide identity comparisons and metabolic modeling. Technical detailsNextflow workflows are launched by a single command and are available on the GEN-ERA GitHub repository (https://github.com/Lcornet/GENERA). All the workflows are based on Singularity containers to increase reproducibility. TestingThe toolbox was developed for a diversity of microorganisms, including bacteria and fungi. It was further tested on an empirical dataset of 18 (meta)genomes of early-branching Cyanobacteria, providing the most up-to-date phylogenomic analysis of the Gloeobacterales order, the first group to diverge in the evolutionary tree of Cyanobacteria. ConclusionThe GEN-ERA toolbox can be used to infer completely reproducible comparative genomic and metabolic analyses on prokaryotes and small eukaryotes. Although designed for routine bioinformatics of culture collections, it can also be useful for other applications, as shown by our case study on Gloeobacterales.

bioinformatics↗

bioRxiv2022

An extended reservoir of class-D beta-lactamases in non-clinical bacterial strains

Bacterial genes coding for antibiotic resistance represent a major issue in the fight against bacterial pathogens. Among those, genes encoding beta-lactamases target penicillin and related compounds such as carbapenems, which are critical for human health. Beta-lactamases are classified into classes A, B, C and D, based on their amino acid sequence. Class D enzymes are also known as OXA beta-lactamases, due to the ability of the first enzymes described in this class to hydrolyze oxacillin. While hundreds of class D beta-lactamases with different activity profiles have been isolated from clinical strains, their nomenclature remains very uninformative. In this work, we have carried out a comprehensive survey of a reference database of 80,490 genomes and identified 24,916 OXA-domain containing proteins. These were deduplicated and their representative sequences clustered into 45 non-singleton groups derived from a phylogenetic tree of 1413 OXA-domain sequences, including five clusters that include the C-terminal domain of the BlaR membrane receptors. Interestingly, 801 known class D beta-lactamases fell into only 18 clusters. To probe the unknown diversity of the class, we selected ten protein sequences in ten uncharacterized clusters and studied the activity profile of the corresponding enzymes. A beta-lactamase activity could be detected for seven of them. Three enzymes were active against oxacillin and two against imipenem. These results indicate that, as already reported, environmental bacteria constitute a large reservoir of resistance genes that can be transferred to clinical strains, whether through plasmid exchange or hitchhiking with the help of transposase genes. ImportanceThe transmission of genes coding for resistance factors from environmental to nosocomial strains is a major component in the development of bacterial resistance towards antibiotics. Our survey of class D beta-lactamase genes in genomic databases highlighted the high sequence diversity of the enzymes that are able to recognize and/or hydrolyze beta-lactam antibiotics. Among those, we could also identify new beta-lactamases that are able to hydrolyze carbapenems, one of the last resort antibiotic families used in human chemotherapy. Therefore, it can be expected that the use of this antibiotic family will fuel the emergence of new beta-lactamases into clinically relevant strains.

microbiology↗