Biology subjects

Mergaert, P.

Publications and source records attributed to Mergaert, P..

12 recordsLinked to original sources

bioRxiv2025

Tiarins, a diverse family of natural Trojan-horse aminoacyl-tRNA synthetase inhibitors discovered by genome mining

Trifolitoxin (TFX) is a ribosomally synthesized and post-translationally modified peptide antibiotic produced by Rhizobium anhuiense T24. Although discovered more than half a century ago, its mechanism of action has remained elusive. Here we demonstrate that TFX inhibits arginyl-tRNA synthetase (ArgRS), an essential translation enzyme. TFX acts as a Trojan-horse antibiotic: it enters cells via the inner membrane oligopeptide transporter YejABEF and then undergoes partial proteolysis by the aminopeptidase PepN to release a "warhead" that binds ArgRS and arrests translation. A systematic analysis of prokaryotic genomes revealed that TFX belongs to a widespread family of modified peptides that we designate the tiarins. In addition to ArgRS inhibitors, the tiarin family includes compounds that specifically target at least seven other aminoacyl-tRNA synthetases. These findings define a previously unrecognized family of natural antibiotics and provide a framework for developing a new class of inhibitors targeting various tRNA synthetases.

molecular biology↗

bioRxiv2025

Structure-based phylogenetic analysis reveals multiple events of convergent evolution of cysteine-rich antimicrobial peptides in legume-rhizobium symbiosis

Nitrogen is essential for plant growth, yet its availability often limits agricultural productivity. Some legumes have evolved a unique ability to form symbiotic relationships with nitrogen-fixing soil bacteria called rhizobia, enabling them to thrive in nitrogen-deficient soils. In five legume clades, an exploitive strategy has evolved in which rhizobia undergo Terminal Bacteroid Differentiation (TBD), where the bacteria become larger, polyploid, and have a permeabilized membrane. Terminally differentiated bacteria are associated with higher N2-fixation and, thus, a higher return on investment to the plant. In several members of the IRLC (Inverted Repeat-Lacking Clade) and the Dalbergioid clades of legumes, this differentiation process is triggered by a set of apparently unrelated plant antimicrobial peptides with membrane-damaging activity, known as Nodule-specific Cysteine-Rich (NCR) peptides. However, whether NCR peptides are also implicated in symbiotic TBD in other legume clades and whether they are evolutionarily related remains unknown. Here, to address the molecular identity of NCR peptides and their evolution in different legume clades, we performed inter- and intra-clade comparisons of NCR peptides in representative species of four TBD-inducing legume clades. First, we collected genomic and proteomic data of species for which NCR peptides are known (1523 NCR peptides). We then used sequence similarity-based clustering to regroup the NCR peptides, resulting in over 400 different NCR clusters, each clade-specific. We obtained Hidden Markov Models for each cluster and used them to predict NCR peptides in 21 legume genomes (6 clades), including newly generated deep-sequenced root and nodule RNA-seq data of Indigofera argentea (Indigoferoid clade) and newly assembled high-quality transcriptomes of Lupinus luteus and Lupinus mariae-josephae (Genistoid clade), using tailored gene prediction pipeline and transcriptome matching. This resulted in 3710 NCR peptides in species that induce TBD. To date, the rapid diversification of NCR peptides that reduces the sequence similarities has masked the origin of NCR peptide evolution. We obtained high-confidence structural models for one sequence of each cluster. We performed structure-based clustering and phylogenetics, which resulted in 23 superclusters (14 inter-clade and nine clade-specific) that we represent in a structural distance-based tree. Our study revealed that the evolution of NCR peptides is a mix of divergent and convergent processes within each clade. We further chose nine independently evolved NCR peptides to test in vitro whether they are functional analogs in symbiosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=145 HEIGHT=200 SRC="FIGDIR/small/675119v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8d1698org.highwire.dtl.DTLVardef@c65b98org.highwire.dtl.DTLVardef@a75994org.highwire.dtl.DTLVardef@ea1a73_HPS_FORMAT_FIGEXP M_FIG Overview of the experimental and computational workflow for NCR peptide detection, characterization, and structural analysis. Nodule and root samples from Indigofera argentea (8 weeks post-inoculation) were collected and subjected to RNA extraction, library preparation, and Illumina PE150 sequencing. Raw RNA-seq reads from two Lupinus species were also included (Lupinus luteus and Lupinus mariae-josephae). Bacteroid differentiation of I. argentea was assessed by flow cytometry and confocal microscopy. Transcriptomes were assembled de novo and analyzed for differential gene expression between root and nodule tissues. NCR peptides were identified from them and other legume genomes and transcriptomes using the SPADA pipeline and HMM profiles from NCR clusters of the known NCR peptides. The putative NCR peptides were filtered based on conserved cysteine motifs, length, and nodule expression to build an exhaustive NCR peptide database. 3D structural predictions of NCR clusters were performed using AlphaFold2 (pLDDT >70), followed by structural clustering (Foldseek) and phylogenetic analysis (Foldtree). Functional validation involved flow cytometry and antimicrobial assays (against Eschericha coli, Sinorhizobium meliloti, and Bacillus subtilis), enabling structural and evolutionary characterization of NCR peptides. The green box at the top represents the experimental analysis, the blue box represents the sequence-based computational pipeline, the red box represents the structure-based computational pipeline, and the grey box at the bottom left represents the functional validation and interpretation of the results. C_FIG

evolutionary biology↗

bioRxiv2023

Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts

The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a mono-specific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including novel AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that contributes to establish and control the gut microbiota.

microbiology↗

bioRxiv2023

Transposon sequencing reveals the essential gene set and genes enabling gut symbiosis in the insect symbiont Caballeronia insecticola

Caballeronia insecticola is a bacterium belonging to the Burkholderia genus sensu lato, able to colonize multiple environments like soils and the gut of the bean bug Riptortus pedestris. To identify the essential genome of a bacterium is a first step in the understanding of its lifestyles. We constructed a saturated Himar1 mariner transposon library and revealed by transposon-sequencing (Tn-seq) that 498 protein-coding genes constitute the essential genome of C. insecticola for growth in free-living conditions. By comparing essential gene sets of C. insecticola and seven related Burkholderia s.l. strains, only 120 common genes were identified indicating that a large part of the essential genome is strain-specific. In order to reproduce specific nutritional conditions that are present in the gut of R. pedestris, we grew the mutant library in minimal media supplemented with candidate gut nutrients and identified several condition-dependent fitness-defect genes by Tn-seq. To validate the robustness of the approach, insertion mutants in six fitness genes were constructed and their growth-deficiency in media supplemented with the corresponding nutrient was confirmed. The mutants were further tested for their efficiency in R. pedestris gut colonization, confirming that gluconeogenic carbon sources, taurine and inositol, are nutrients consumed by the symbiont in the gut.

microbiology↗

bioRxiv2023

Sinorhizobium meliloti FcrX coordinates cell cycle and division during free-living growth and symbiosis

Sinorhizobium meliloti is a soil bacterium that establishes a symbiosis within root nodules of legumes (Medicago sativa, for example) where it fixes atmospheric nitrogen into ammonia and obtains in return carbon sources and other nutrients. In this symbiosis, S. meliloti undergoes a drastic cellular change leading to a terminal differentiated form (called bacteroid) characterized by genome endoreduplication, increase of cell size and high membrane permeability. The bacterial cell cycle (mis)regulation is at the heart of this differentiation process. In free-living cells, the master regulator CtrA ensures the progression of cell cycle by activating cell division (controlled by the tubulin-like protein FtsZ) and simultaneously inhibiting supernumerary DNA replication, while on the other hand the downregulation of CtrA and FtsZ is essential for bacteroid differentiation during symbiosis, preventing endosymbiont division and permitting genome endoreduplication. Little is known in S. meliloti about regulators of CtrA and FtsZ, as well as the processes that control bacteroid development. Here, we combine cell biology, biochemistry and bacterial genetics approaches to understand the function(s) of FcrX, a new factor that controls both CtrA and FtsZ, in free-living growth and in symbiosis. Depletion of the essential gene fcrX led to abnormally high levels of FtsZ and CtrA and minicell formation. Using multiple complementary techniques, we showed that FcrX is able to interact physically with FtsZ and CtrA. Moreover, its transcription is controlled by CtrA itself and displays an oscillatory pattern in the cell cycle. We further showed that, despite a weak homology with FliJ-like proteins, only FcrX proteins from closely-related species are able to complement S. meliloti fcrX function. Finally, deregulation of FcrX showed abnormal symbiotic behaviors in plants suggesting a putative role of this factor during bacteroid differentiation. In conclusion, FcrX is the first known cell cycle regulator that acts directly on both, CtrA and FtsZ, thereby controlling cell cycle, division and symbiotic differentiation.

microbiology↗

bioRxiv2022

The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria

Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer Rhizobium sp. Pop5. Using genetic and biochemical techniques, we here identified BacA and YejABEF as two importers of PHZ in a sensitive model strain Sinorhizobium meliloti Sm1021. BacA and YejABEF are members of SLiPT and ABC transporter families of non-specific peptide importers, respectively. The uptake of PHZ by two distinct families of transporters dramatically decreases the naturally occurring rate of resistance. Moreover, since both BacA and YejABEF are essential for the development of functional symbiosis of rhizobia with leguminous plants, the acquisition of PHZ resistance via the inactivation of transporters is further disfavoured since single bacA or yejABEF mutants are unable to propagate in root nodules. Crystal structures of the periplasmic subunit YejA from S. meliloti and Escherichia coli revealed fortuitous bound peptides, suggesting a non-specific peptide-binding mechanism that facilitates the uptake of PHZ and other antimicrobial peptides. SIGNIFICANCEMany bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides kill bacteria by either membrane disruption or inhibiting essential intracellular processes. The Achilles heel of the latter type of antimicrobials is their dependence on transporters to enter the susceptible bacteria since mutations in such transporters result in resistance. We describe here how the ribosome-targeting peptide phazolicin, produced by Rhizobium sp. Pop5, uses two different transporters, BacA and YejABEF, to get into the cells of the symbiotic bacterium Sinorhizobium meliloti. This dramatically reduces the probability of resistance acquisition. Both transporters need to be inactivated for phazolicin resistance acquisition. Since these transporters are also crucial in S. meliloti for its symbiotic association with host plants, their inactivation in biological settings is highly unlikely. This makes PHZ an attractive lead for the development of a biocontrol agent with potential for use in agriculture.

molecular biology↗

bioRxiv2022

Differential genetic strategies of Burkholderia vietnamiensis and Paraburkholderia kururiensis for root colonization of Oryza sativa ssp. japonica and ssp. indica, as revealed by Tn-seq

Burkholderia vietnamiensis LMG10929 (Bv) and Paraburkholderia kururiensis M130 (Pk) are bacterial rice growth-promoting models. Besides this common ecological niche, species of the Burkholderia genus are also found as opportunistic human pathogens while Paraburkholderia are mostly environmental and plant-associated species. Here, we compared the genetic strategies used by Bv and Pk to colonize two subspecies of their common host, Oryza sativa ssp. japonica (cv. Nipponbare) and ssp. indica (cv. IR64). We used high-throughput screening of transposon insertional mutant libraries (Tn-seq) to infer which genetic elements have the highest fitness contribution during root surface colonization at 7 days post inoculation. Overall, we detected twice more genes in Bv involved in rice roots colonization compared to Pk, including genes contributing to the tolerance of plant defenses, which suggests a stronger adverse reaction of rice towards Bv compared to Pk. For both strains, the bacterial fitness depends on a higher number of genes when colonizing indica rice compared to japonica. These divergences in host pressure on bacterial adaptation could be partly linked to the cultivars differences in nitrogen assimilation. We detected several functions commonly enhancing root colonization in both bacterial strains e.g., Entner-Doudoroff (ED) glycolysis. Less frequently and more strain-specifically, we detected functions limiting root colonization such as biofilm production in Bv and quorum sensing in Pk. The involvement of genes identified through the Tn-seq procedure as contributing to root colonization i.e., ED pathway, c-di-GMP cycling and cobalamin synthesis, was validated by directed mutagenesis and competition with WT strains in rice root colonization assays. ImportanceBurkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth. However, the closely related Burkholderia genus hosts both beneficial and phytopathogenic species, as well as species able to colonize animal hosts and cause disease in humans. We need to understand to what extent the bacterial strategies used for the different biotic interactions differ depending on the host and if strains with agricultural potential could also pose a threat towards other plant hosts or humans. To start answering these questions, we used here transposon sequencing to identify genetic traits in Burkholderia vietnamiensis and Paraburkholderia kururiensis that contribute to the colonization of two different rice varieties. Our results revealed large differences in the fitness gene sets between the two strains and between the host plants, suggesting a strong specificity in each bacterium-plant interaction.

microbiology↗

bioRxiv2022

Direct measurement of the aerotactic response in a bacterial suspension

Aerotaxis is the ability of motile cells to navigate towards oxygen. A key question is the dependence of the aerotactic velocity with the local oxygen concentration c. Here we combine simultaneous bacteria tracking and local oxygen concentration measurements using Ruthenium encapsulated in micelles to characterise the aerotactic response of Burkholderia contaminans, a motile bacterium ubiquitous in the environment and present in living organisms. In our experiments, an oxygen gradient is produced by the bacterial respiration in a sealed glass capillary permeable to oxygen at one end, producing a bacterial band travelling towards the oxygen source. We compute the aerotactic response{chi} (c) both at the population scale, from the drift velocity in the bacterial band, and at the bacterial scale, from the angular modulation of the run times. Both methods are consistent with a power-law{chi} {propto} c-2, in good agreement with existing models based on the biochemistry of bacterial membrane receptors.

biophysics↗

bioRxiv2021

DNA methylation patterns in bacteria of the genus Ensifer during free living growth and during nitrogen-fixing symbiosis with Medicago spp.

Methylation of specific DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Using single-molecule real-time sequencing, six genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cells, methylation of GANTC motifs increased progressively from the ori to ter regions in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. In contrast, there was near full genome-wide GANTC methylation in the early stage of symbiotic differentiation. This was followed by a moderate decrease in the overall extent methylation and a progressive decrease in chromosomal GANTC methylation from the ori to ter regions in later stages of differentiation. We interpret these observations as evidence of dysregulated and constitutive CcrM activity during terminal differentiation, and we hypothesize that it is a driving factor for endoreduplication of terminally differentiated bacteroids.

microbiology↗

bioRxiv2020

Sinorhizobium meliloti functions required for resistance to antimicrobial NCR peptides and bacteroid differentiation

Legumes of the Medicago genus form symbiosis with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of the intracellular symbionts. Members of the Nodule-specific Cysteine Rich peptide (NCRs) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts, including an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes, lipopolysaccharide modifications mediated by lpsB and lpxXL as well as rpoH1, encoding a stress sigma factor. Mutants of these genes show in vitro a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions including peptide transporters, the bacterial envelope structures and stress response regulators to resist the aggressive assault of NCR peptides in the nodule cells. ImportanceThe nitrogen fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plant needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane composition and the stress response regulator RpoH1.

microbiology↗

bioRxiv2020

Bradyrhizobium diazoefficiens USDA110 nodulation of Aeschynomene afraspera is associated with atypical terminal bacteroid differentiation and suboptimal symbiotic efficiency

Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions is varying from non-fixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont but it can also establish a functional symbiotic interaction with Aeschynomene afraspera. In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy and membrane permeability leading to loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics and transcriptomics along with cytological analyses was used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establish a poorly efficient symbiosis with A. afraspera, despite the full activation of the bacterial symbiotic program. We found molecular signatures of high level of stress in A. afraspera bacteroids whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo an atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not co-evolve with such a host. ImportanceLegume-rhizobium symbiosis is a major ecological process in the nitrogen cycle, responsible for the main input of fixed nitrogen in the biosphere. The efficiency of this symbiosis relies on the coevolution of the partners. Some legume plants, but not all, optimize their return-on-investment in the symbiosis by imposing on their microsymbionts a terminal differentiation program that increases their symbiotic efficiency but imposes a high level of stress and drastically reduce their viability. We combined multi-omics with physiological analyses to show that the non-natural symbiotic couple formed by Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera is functional but displays a low symbiotic efficiency associated to a disconnection of terminal bacteroid differentiation features.

microbiology↗

bioRxiv2019

Phazolicin - a Novel Thiazole/Oxazole-Modified Peptide Inhibiting the Bacterial Ribosome in a Species-Specific Way.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding and largely untapped class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display an outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium sp. Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants belonging to the Rhizobiales. PHZ inhibits prokaryotic translation through the obstruction of the passage of the nascent peptide through the ribosome exit channel. The cryo-EM structure of the Escherichia coli ribosome with bound PHZ revealed that the drug interacts with the 23S rRNA and ribosomal proteins uL4 and uL22 and obstructs the exit tunnel in a way that is distinct from other compounds blocking the exit channel. We show that the sequence of uL4 ribosomal protein loop involved in PHZ binding determines the species-specificity of antibiotic interaction with its target. PHZ and its predicted homologs from other bacterial species expand the known diversity of LAPs and may be used in the future as biocontrol agents for the needs of agriculture.

molecular biology↗