Biology subjects

Gueguen, E.

Publications and source records attributed to Gueguen, E..

10 recordsLinked to original sources

bioRxiv2026

The ArcZ small RNA activates SlyA production to control contact-dependent antibacterial killing and diffusible antifungal activity in Dickeya solani

Bacterial phytopathogens deploy diverse antimicrobial systems, including diffusible secondary metabolites and contact-dependent secretion apparatuses, to compete with coinhabiting microorganisms during host colonization. How the production of these distinct weapons is coordinated remains incompletely understood. Here, we identified a two-tier regulatory cascade by which the Hfq-dependent small RNA (sRNA) ArcZ controls both antibacterial and antifungal antagonism in the phytopathogen Dickeya solani. High-throughput RNA interactome analysis (RIL-seq) shows that ArcZ interacts broadly across the transcriptome and directly base-pairs with the 5' untranslated region of the mRNA encoding the MarR-family transcription factor SlyA, activating its translation. SlyA subsequently binds directly to selected promoter regions within the solanimycin biosynthetic cluster (sol) and the type VI secretion system (T6SS) loci and activates their transcription. The ArcZ-SlyA axis is required for solanimycin mediated antifungal activity against Kluyveromyces lactis and for T6SS-dependent killing of the sympatric pathogen Pectobacterium atrosepticum, demonstrating that the D. solani T6SS mediates interbacterial competition. A compensatory base-pairing mutation between ArcZ and slyA restores both competitive phenotypes, establishing this post-transcriptional interaction as a direct upstream determinant. Together, these results reveal an sRNA controlled regulatory axis that coordinates diffusible antifungal and contact-dependent antibacterial activities.

microbiology↗

bioRxiv2025

Host infection selects for sRNA variants that drive bacterial social cheating

Single-nucleotide mutations in regulatory small RNAs (sRNAs) represent a largely unexplored route to social cheating during bacterial infection. Here we show that arcZ, encoding a conserved Hfq-dependent sRNA in the plant pathogen Dickeya solani, undergoes recurrent single-nucleotide mutations during potato tuber infection. Five distinct arcZ alleles define a graded series of ArcZ functional impairment in which antifungal and protease activities are progressively reduced while virulence is compromised only in a subset of alleles. All five variants gain a fitness advantage exclusively during co-infection with wild-type arcZ1 cells. This advantage is strongest when variants are rare and declines as cheater frequency increases, ultimately correlating with reduced total bacterial productivity, the canonical signature of social cheating and a tragedy of the commons. No fitness benefit is detected in vitro. Transcriptomic profiling of five arcZ variants identifies a conserved core of 186 downregulated genes enriched in secreted and diffusible functions. Systematic genetic dissection, supported by quantitative proteomics, establishes BudAB-dependent acetoin production as the dominant cooperative public good exploited during infection. Wild-type arcZ1cells divert pyruvate flux toward acetoin via the BudAB pathway, maintaining a less acidic tissue environment that sustains disease progression; arcZ variants, which repress this pathway, exploit the resulting pH buffering without contributing to it. These results establish pleiotropic regulatory sRNAs as a previously unrecognized class of mutational targets for the emergence of social cheaters during host infection. StatementBacterial populations are not genetically uniform: variants that exploit the cooperative activities of their neighbors, social cheaters, can emerge rapidly within infected tissue and undermine collective bacterial productivity. We show that single-nucleotide mutations in the sRNA ArcZ of Dickeya solani are recurrently selected during potato infection, generating cheater variants that cease contributing to a shared metabolic benefit. Wild-type cells divert pyruvate toward acetoin, buffering tissue pH and sustaining disease; mutant variants exploit this pH maintenance without producing acetoin themselves. Because a single arcZ mutation simultaneously represses a conserved set of 186 genes, including genes involved in secreted and diffusible functions, regulatory sRNA genes represent a previously unrecognized and unusually efficient mutational target for the evolution of social cheating during infection.

microbiology↗

bioRxiv2025

Comparative essentialome analysis of six Pectobacteriaceae strains using the TNSEEK pipeline identifies conserved and strain-specific fitness determinants

Transposon sequencing (Tn-seq) is a powerful technique for defining the essential genes required for bacterial survival. However, gene essentiality can vary significantly across taxonomic levels, and comparing large Tn-seq datasets from multiple strains presents considerable analytical challenges. To address this, we developed TNSEEK, a fully automated bioinformatics pipeline for the systematic and comparative analysis of transposon sequencing experiments. We applied TNSEEK to analyze six Soft Rot Pectobacteriaceae (SRP) strains, encompassing species from the Dickeya and Pectobacterium genera, grown in a rich medium. This approach identified a core essentialome of 225 genes, primarily involved in fundamental cellular maintenance, conserved across all six strains, a set comparable in size to that of the broader Enterobacteriaceae family. Only a few genus-specific essential genes were found highlighting interesting distinct metabolic capabilities between Dickeya and Pectobacterium genera. In striking contrast, we discovered a large variable essentialome comprising 181 strain-specific genes, many of which of unknown function. A portion of these strain-specific essential genes are components of defense systems and prophage genomic regions. The unexpected essentiality of these modules suggests they form a constitutively active frontline defense. Furthermore, a comparison with the E. coli essentialome demonstrates that discrepancies in gene essentiality can often be attributed to differences in growth conditions, particularly temperature, as well as variations in genetic redundancy. In conclusion, the TNSEEK pipeline is a robust tool for exploring functional genomics across multiple strains. IMPORTANCEDickeya and Pectobacterium are two genera of the Pectobacteriaceae family that contain mainly plant pathogenic bacteria. To analyze the diversity within bacteria of this family, we performed a Tn-seq analysis on six strains representing a range of ecological niches. To this aim we developed a bioinformatics pipeline, termed TNSEEK which allows the comparative analysis of results across diverse experimental conditions and multiple strains. Applied to growth in rich medium of the tested strains, it allowed the identification of an essentialome of 225 genes at the family level, few genus-specific essential genes but a large essentialome comprising 181 strain-specific genes, many of which of unknown function. Thus, TNSEEK proved its ability to analyze a large data set coming from different Tn-seq experiments and it offers an unparalleled flexibility in handling any number of strains and experiments.

genomics↗

bioRxiv2025

Pectobacterium versatile beta-lactamase, a common good of the soft rot Pectobacteriaceae (SRP) species complex

Little is known about the role of antibiotics and associated resistance in microbial ecosystems in the absence of clinical antibiotic pressure. The Soft Rot Pectobacteriaceae (SRP) species complex, which comprises 37 bacterial species that are collectively responsible for the severe rotting of many crops, is an interesting model to analyse the role of {beta}-lactam and {beta}-lactamases in natural ecosystems. In particular, within this complex, most Pectobacterium versatile strains harbour a {beta}-lactamase called BlaPEC-1. The aim of our work was to analyse the role of BlaPEC-1 during infection. To this end, two blaPEC-1-deleted strains were constructed and compared with their wild-type counterparts in vitro and in potato tuber infections. In vitro, the BlaPEC-1 {beta}-lactamase enables P. versatile to resist ampicillin or carbapenem produced by Pectobacterium brasiliense. In mono-infections on potato tubers, blaPEC-1-deleted strains were unaffected in terms of virulence, fitness or association with commensal bacteria. In mixed infections, the BlaPEC-1 {beta}-lactamase proved necessary for the coexistence of P. versatile with the carbapenem-producing strain, and also for the protection of carbapenem-sensitive strains both in vitro and in planta. Interestingly, in planta protection was observed even if the blaPEC-1 gene was repressed and bacteria expressing BlaPEC-1 were in the minority within the symptom. These results indicate that BlaPEC-1 exerts a true {beta}-lactamase function during the infection process and acts as a public good of the SRP species complex. Finally, our results highlight the important role of {beta}-lactamase in maintaining of strains diversity in natural ecosystem. Statements relating to our ethics and integrity policiesThe data shown in this paper are available within the article and supplementary materials. The funding of the ANR, ANR-19-CE35-0016-03, is acknowledged. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. The authors declare that there are no conflicts of interest. All applicable local, national and international regulations and conventions, as well as normal scientific ethical practices, were followed in the preparation of this work. This manuscript has not been previously or simultaneously published or submitted elsewhere and was critically reviewed and approved by all co-authors before submission. The CRediT of all authors is provided.

microbiology↗

bioRxiv2025

Genetic Determinants of pOXA-48 Plasmid Maintenance and Propagation in Escherichia coli

Conjugative plasmids are the main drivers of antibiotic resistance dissemination contributing to the emergence and extensive spread of multidrug resistance clinical bacterial pathogens. pOXA-48 plasmids, belonging to the IncL group, have emerged as the primary vehicle for carbapenem resistance in Enterobacteriaceae. Despite the problematic prevalence of pOXA-48, most research has focused on epidemiology and genomics, leaving gaps in our understanding of the mechanisms behind its propagation. In this study, we used a transposon sequencing approach to identify genetic elements critical for plasmid stability, replication, and conjugative transfer. Our results identified a novel type I toxin-antitoxin system, new essential maintenance factors, and components of the type IV secretion system and regulatory elements crucial for conjugation. This study advances our understanding of pOXA-48 biology, providing key insights into the genetic factors underlying its successful maintenance and spread in bacterial populations.

microbiology↗

bioRxiv2024

Blood-borne immune cells carry low biomass DNA remnants of microbes in patients with colorectal cancer or inflammatory bowel disease

The involvement of the human intestinal microbiome in the regulation of immune cell homeostasis, as well as in the pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC), are well-established1-4. Bacteria interact with immune cells at the sites of intestinal inflammation, but also in the CRC tumor microenvironment1-6. Indeed, bacterial remnants have recently been detected in human intestinal tissue in patients with IBD, at primary tumor sites and in the metastases of patients with CRC, and in whole blood3,7,8. A defective intestinal epithelial barrier is thought to promote bacterial remnant translocation and disease progression6. However, it is unknown, how bacterial remnants translocate from the intestine to sites of metastasis or into the circulation. We hypothesized that bacterial remnants translocate within peripheral blood immune cells into the circulation. Here, we thus explored the composition of the detectable microbiome in peripheral blood mononuclear cells (PBMCs) of patients with CRC or IBD compared to healthy controls. The PBMC microbiome profiles partially align with the tumor- and metastasis-derived or intestinal tissue-derived microbiome signatures obtained from the same patients with CRC or IBD, respectively. Our metagenomics data, supported by 16S-rRNA-FISH-Flow, imaging flow cytometry and species-specific qPCR, reveal the presence of translocated bacterial genetic sequences in the patients with CRC and IBD, likely due to an intestinal barrier defect. Pathway and serum metabolomics analysis connected to the metabolic potential of the PBMC-derived microbiome sequences support the onset of microbial translocation in such patients. Thus, our data suggest that in patients with intestinal barrier leakage, such as those with CRC or IBD, there is the potential for the translocation of bacterial remnants into the circulation via peripheral immune cells.

cancer biology↗

bioRxiv2024

High-throughput Tn-seq screens identify both known and novel Pseudomonas putida KT2440 genes involved in metal resistance

Chemical waste with toxic effects is released into the environment by industrial and urban activities. Pseudomonas putida, a rhizosphere bacterium, harbors a wide variety of genes capable of degrading hydrocarbons and xenobiotic compounds in its natural environment. This bacterium harbors also a large set of metal resistance genes. Most studies that identify genes involved in metal resistance in P. putida focus on over/underexpressed genes and may miss other genes important for metal resistance whose expression does not change. In this study, we used a Tn-seq approach to determine the essential genome of P. putida required for growth in the presence of an excess of metals in a culture medium. Tn-seq enables the detection of mutants with reduced or increased fitness in the presence of metal excess. We validated our screen by identifying known metal resistance gene such as czcA-1 (PP_0043), cadA-3 (PP_5139), cadR (PP_5140) and pcoA2 (PP_5380). Their mutants were underrepresented in the presence of zinc, cadmium (for cadA-3 and cadR) or copper respectively. In this study, we demonstrate by targeted mutagenesis and complementation assay that PP_5337 and PP_0887 are putative transcriptional regulators involved in copper and cadmium resistance, respectively, in P. putida. The study revealed the role of two genes, PP_1663 and PP_5002, in cadmium and cobalt resistance respectively. This is the first evidence linking these genes to metal resistance and highlights the incomplete understanding of metal resistance mechanisms in P. putida.

microbiology↗

bioRxiv2021

Dickeya solani D s0432-1 produces an arsenal of secondary metabolites with anti-prokaryotic and anti-eukaryotic activities against bacteria, yeasts, fungi, and aphids.

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster that we named ssm for solani secondary metabolite had an unknown function. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters ssm, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The secondary metabolite cluster ssm, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters ssm and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form. AUTHOR SUMMARYThe development of new antibacterial molecules is critical in tackling the emergence of new pathogens or bacterial strains resistant to already available antibiotics. Bacterial phytopathogens can potentially synthesize novel compounds capable of targeting a specific type of microorganism. Dickeya solani is the only of the twelve described Dickeya species that has the three secondary metabolic pathways ssm, ooc and zms. An investigation of the functions of these three clusters allowed us to identify the anti-yeast activity of the ssm cluster, a potential new molecule of clinical importance. By comparing the antimicrobial activity of several Dickeya solani strains, we identified the small RNA regulator ArcZ as a critical regulator in the activation of the ssm and zms clusters. Our study showed that single-nucleotide polymorphisms of sRNA encoding genes can have huge impacts on bacterial phenotypes. It is thus critical to pay attention to the allele diversity of sRNA genes.

microbiology↗

bioRxiv2019

Pseudomonas two-partner secretion toxin Exolysin contributes to insect killing

Pseudomonas chlororaphis is a promising biocontrol agent promoting plant-growth and providing protection against pest insects and phytopathogenic fungi. We have identified in the genome of P. chlororaphis PA23 an operon encoding the toxin Exolysin (ExlA) and its outer-membrane transporter, ExlB. We found that P. chlororaphis producing ExlA (ExlAPch) is cytotoxic towards murine macrophages and human epithelial cells at 30 {degrees}C. P. chlororaphis PA23 provoked shrinkage of epithelial cell, leakage of cytoplasmic components and subsequent cell death. During infection, ExlAPch incorporated into epithelial cell membranes within detergent-resistant lipid rafts, suggesting the same mechanisms of cell destruction by pore-formation as reported for P. aeruginosa toxin. ExlAPch was not involved in the capacity of the strain to kill fungi, amoeba or other bacteria. The contribution of ExlA in insecticidal activity of P. chlororaphis was evaluated in the wax moth larvae Galleria mallonella and in Drosophila melanogaster flies. The impact of the deletion of a gene encoding exlA homologue was tested in the natural fly pathogen P. entonomophila. In both models, the ExlA absence delayed killing, suggesting the contribution of the toxin in bacteria-insect pathogenic interactions.

microbiology↗

bioRxiv2017

Genome Wide Identification Of Genes Required For Bacterial Plant Infection By Tn-Seq

Soft rot enterobacteria (Dickeya and Pectobacterium) are major pathogens that cause diseases on plants of agricultural importance such as potato and ornamentals. Long term studies to identify virulence factors of these bacteria focused mostly on plant cell wall degrading enzymes secreted by the type II secretion system and the regulation of their expression. To identify new virulence factors we performed a Tn-seq genome-wide screen of a transposon mutant library during chicory infection followed by high-throughput sequencing. This allowed the detection of mutants with reduced but also increased fitness in the plant. Virulence factors identified differed from those previously known since diffusible ones (secreted enzymes, siderophores or metabolites) were not detected by this screen. In addition to genes encoding proteins of unknown function that could be new virulence factors, others could be assigned to known biological functions. The central role of the FlhDC regulatory cascade in the control of virulence was highlighted with the identification of new members of this pathway. Scarcity of the plant in certain amino acids and nucleic acids required presence of the corresponding biosynthetic genes in the bacteria. Their products could be targets for the development of antibacterial compounds. Among the genes required for full development in chicory we also identified six genes involved in the glycosylation of the flagellin FliC, glycosylation, which in other plant pathogenic bacteria contributes to virulence.\n\nAuthor summaryIdentification of virulence factors of plant pathogenic bacteria has relied on the test of individual mutants on plants, a time-consuming method. New methods like transcriptomic or proteomic can now be used but they only allow the identification of genes induced during the infection process and non-induced genes may be missed. Tn-seq is a very powerful method to identify genes required for bacterial growth in their host. We used for the first time this method in a plant pathogenic bacteria to identify genes required for the multiplication of Dickeya dadantii in chicory. We identified about 100 genes with decreased or increased fitness in the plant. Most of them had no previously described role in bacterial virulence. We unveiled important metabolic genes and regulators of motility and virulence. We showed that D. dadantii flagellin is glycosylated and that this modification confers fitness to the bacteria during plant infection. Our work opens the way to the use of Tn-seq with bacterial phytopathogens. Assay by this method of large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.

microbiology↗