Biology subjects

Falquet, L.

Publications and source records attributed to Falquet, L..

15 recordsLinked to original sources

bioRxiv2026

An Integrated Miniaturized Metabologenomics Workflow for High-Throughput Actinomycetes Prioritization

Antibiotic resistance is a growing global health threat, while the discovery of new antimicrobial agents remains constrained by labor-intensive cultivation, extraction, and screening workflows. We developed a miniaturized, high-throughput metabologenomics workflow integrating Duetz system-based microfermentation, extraction-free UHPLC-HRMS/MS profiling, antibacterial screening, and genome mining. A collection of 80 soil-derived actinomycetes was screened against clinically relevant pathogens, identifying seven isolates active against methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, or both. All isolates were cultivated in Duetz system-based microfermentation plates, and cell-free culture filtrates were analyzed directly by UHPLC-HRMS/MS without solvent extraction. Compared with a conventional workflow, the approach reduced culture-medium consumption by approximately 99%, decreased recurring consumable costs by approximately 96%, and reduced the time required to obtain LC-MS-ready samples from typically 5 days to approximately 20 min. Integration of antibacterial, metabolomic, and genomic datasets supported the annotation of diverse metabolite families, including actinomycins, polyether ionophores, ansamycins, angucyclines, hydroxamate siderophores, ectoines, tambromycin-associated metabolites, and putative lydicamycin metabolites. Selected annotations were validated using commercial standards. Overall, this study demonstrates that integrating miniaturized Duetz system-based cultivation, extraction-free metabolomics, antibacterial screening, and genome mining provides a scalable, robust, and cost-effective platform for high-throughput strain prioritization and natural product discovery.

microbiology↗

bioRxiv2026

Arbuscular mycorrhizal symbiosis increases drought resistance in the xerophytic argan tree ( Sideroxylon spinosum )

The xerophyte argan (Sideroxylon spinosum) has great ecological and socioeconomic importance for Morocco. However, it is endangered due to climate change and human overexploitation. We assess drought resistance traits of argan and test the potential of arbuscular mycorrhizal (AM) symbiosis to promote its growth and mitigate the consequences of drought. We compare ten endemic Moroccan mycorrhizal inocula with the model AM fungus Rhizophagus irregularis and with the drought-adapted isolate Diversispora omaniana. We integrated physiological phenotyping and RNA sequencing to investigate the stress resistance mechanisms of argan against drought. We show that AM symbiosis significantly mitigates drought effects on plant growth, mainly by improving water relations and photosynthetic efficiency, resulting in increased growth rates. Taken together, physiological and transcriptomic analyses show that stress markers were moderatly induced during severe drought stress irrespective of mycorrhizal status, suggesting that argan adopts a drought-coping strategy that involves both, stress avoidance and stress tolerance. Argan is highly AM-responsive, both at the phenotypic and transcriptomic level, suggesting that AM has great potential to promote argan growth under drought stress.

Plant Biology↗

bioRxiv2026

Chromosome-level genome assembly of the swallowtail butterfly Parides eurimedes mylotes is a valuable resource for studying wing coloration

High-quality chromosome-scale assemblies are scarce in Papilionidae. This limits comparative genomics to model species, Lepidoptera, Bombyx mori. Here, we present a phased, chromosome-level genome assembly of Parides eurimedes mylotes. We generated this assembly using 125[x] PacBio HiFi sequencing and assembled it with hifiasm. The final haplotype assemblies (Hap1 and Hap2) span 274 Mb and 270 Mb, respectively. These assemblies are organized into 31 near-telo-mere-to-telomere chromosomes, with scaffold N50 values of 9.72 Mb and 9.22 Mb, respectively. BUSCO analysis revealed assembly completeness of 96.6 % and 96.4 % for Hap1 and Hap2, respectively. Repeats annotation identified 18-19 % repetitive content, with Helitron elements being the dominant class of transposable elements. We identified the W and Z sex chromosomes and completely assembled the mitochondrial genome. Compared to the previously available Parides photinus draft assembly, our genome exhibits an 11,000-fold reduction in scaffold fragmentation and nearly complete gene assembly. This assembly provides a robust genomic reference for functional, evolutionary, and multi-omics investigation in Papilionidae. In addition to serving as a high-quality genomic reference for Papilionidae, this assembly is essential for linking the genetic architecture of butterfly wings to the hierarchical nanostructures underlying structural coloration. By identifying the genes and regulatory networks involved in scale morphogenesis, we can correlate the butterflys genotype with its photonic function. This insight into the evolutionary origin of the biological photonic systems informs the design of biomimetic, structurally colored materials.

genomics↗

bioRxiv2026

Potato foliar infection with Phytophthora infestans drives strong, cultivar-specific shifts in rhizosphere communities

BackgroundPotato is an important crop worldwide, yet its production is severely threatened by Phytophthora infestans, the causal agent of late blight. Alternatives to the current control strategies are needed, as these rely heavily on environmentally harmful treatments. The recruitment of beneficial microbes by plants upon stress ("cry-for-help" mechanism) may represent an opportunity to find new biocontrol agents but this has not yet been reported for potato. The aim of this study was to analyse whether foliar late blight infection induces shifts in the phyllosphere, rhizosphere and soil bacterial communities associated with two potato cultivars of differing sensitivity to late blight. Moreover, we aimed at isolating members of the plant microbiota to test whether bacteria putatively recruited upon infection would be particularly active in protecting the plant against late blight. ResultsControlled foliar infection triggered substantial, cultivar-specific shifts in the rhizosphere communities across two successive generations. Despite the number of differentially abundant ASVs detected being ten times higher in the second generation than in the first one, the same taxonomic groups were concerned by the shifts: Burkholderiales, Flavobacteriales, and Bacillales. Furthermore, the communities linked to the susceptible cultivar consistently shifted more strongly than the communities linked to the resistant cultivar. The obtained ASV sequences were used to identify 163 corresponding isolates. The inhibition potential of these strains against P. infestans spores was assessed through biological assays, which revealed the biocontrol potential of strains otherwise not yet known to inhibit phytopathogenic organisms, such as Advenella, Nocardioides and Phyllobacterium strains. Although we found no correlation between the relative abundance shift of the ASVs upon infection and the activity of the corresponding strains, we observed that the overall activity of strains isolated from the resistant cultivar was higher than that of the strains isolated from the susceptible one. ConclusionTaken together, the higher activity of the strains isolated from the resistant cultivar, along with its comparatively modest microbiome shifts upon infection suggest that the investigated resistant cultivar might harbour specific microbiota enriched in strains with efficient protective abilities against their host plants pathogens, which possibly contribute to its higher resistance against P. infestans.

microbiology↗

bioRxiv2025

Chromosome-level phased genome assembly of the Argan tree Sideroxylon spinosum L.

Argan (Sideroxylon spinosum L. previously known as Argania spinosa) is an endemic tree of Morocco, primarily used for seed oil extraction. Increasing interest in its biology and in genes relevant for oil quality and stress resistance demands for high-quality models of its genome and transcriptome. We integrated sequencing data from PacBio HiFi long-reads and Illumina Hi-C to generate independently assembled phased genome models for both parental haplotigs with lengths of 636Mb and 655 Mb, respectively, and with a BUSCO completeness score of >97.8%. Both haplotigs consist of 11 independently assembled fully resolved telomere-to-telomere chromosomes, similar to other species of the Sapotaceae (n = 10-13), and they both contain approximately [~]60% of highly repetitive regions. Comparative analysis with other Sapotaceous genomes suggests overall chromosome conservation, with repeat expansion and chromosome fusion for the two largest chromosomes (chr1, chr2). We also independently assembled the chloroplast genome. This high-quality genome assembly provides a valuable resource for advancing future research on argan biology, its genetic diversity, and traits relevant for fitness and oil production.

genomics↗

bioRxiv2025

Genome-Resolved Metagenomics Analysis of Rice Straw Degradation Experiments Unveils MAGs with High Potential to Decompose Lignocellulosic Residues

BackgroundRice is one of the top three crops that contribute 60% of the calories consumed by humans worldwide. Nonetheless, extensive rice harvesting yields more than 800 million tons of rice straw (RS) per year globally, generating a byproduct that is often difficult for farmers to manage efficiently without burning it. As a result, millions of tons of carbon dioxide and greenhouse gases are released, causing issues such as respiratory problems, soil degradation, and global warming. In this work, we explore the biological decomposition of RS through the application of microbial consortia from a metagenomics perspective. ResultsWe applied different treatments to RS placed in a mulching setup during experiments carried out in Colombian rice fields, using various combinations of a Trichoderma-based commercial product, the bacterial strain Bacillus altitudinis IBUN2717, inorganic nitrogen, and a mixture of potassium-reducing organic acids. Before inoculation and after 30 days of treatment, we characterized the microbial community on the RS surface and from the bulk soil by performing a reference-based compositional analysis, and reconstructing and functionally annotating Metagenome-Assembled Genomes (MAGs). High-quality MAGs with great potential to decompose RS, represented by the extensive number of carbohydrate-active enzymes, were recovered. Soil MAGs taxonomic classification indicates that they may represent potential novel microbial taxa. At the same time, the main part of the RS MAGs with superior lignocellulose-degrading capacity were affiliated under Actinomycetota and Bacteroidota phyla. Moreover, {beta}-glucosidase activity measurements indicated an increased RS degradation after the application of the treatment that included inorganic nitrogen. ConclusionsThis contribution underscores the possibility of promoting RS degradation through the application of biological strategies. Further, the newly unveiled MAGs with high RS-degrading potential provide a valuable resource for exploring the functional potential of previously uncharacterized microbial diversity in Colombian agricultural ecosystems, including microorganisms that have not been previously reported as remarkable lignocellulose decomposers. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/642948v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@bc3c35org.highwire.dtl.DTLVardef@14ce6aorg.highwire.dtl.DTLVardef@1fbb30aorg.highwire.dtl.DTLVardef@1a788ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Comparative genomics of Mycoplasma feriruminatoris, a fast-growing pathogen of wild Caprinae

Mycoplasma feriruminatoris is a fast-growing Mycoplasma species isolated from wild Caprinae and first described in 2013. M. feriruminatoris isolates have been associated with arthritis, keratoconjunctivitis, pneumonia and septicemia, but were also recovered from apparently healthy animals. To better understand what defines this species, we performed a genomic survey on 14 strains collected from free-ranging or zoo-housed animals between 1987 and 2017. The average chromosome size of the M. feriruminatoris strains was 1,040 {+/-} 0,024 kbp, with 24% G+C and 852 {+/-} 31 CDS. The core genome and pan-genome of the M. feriruminatoris species contained 628 and 1,312 protein families, respectively. The M. feriruminatoris strains displayed a relatively closed pan-genome, with many features and putative virulence factors shared with species from the M. mycoides cluster, including the MIB-MIP Ig cleavage system, a repertoire of DUF285 surface proteins and a complete biosynthetic pathway for galactan. M. feriruminatoris genomes were found to be mostly syntenic, although repertoires of mobile genetic elements, including Mycoplasma Integrative and Conjugative Elements, insertion sequences, and a single plasmid varied. Phylogenetic- and gene content analyzes confirmed that M. feriruminatoris was closer to the M. mycoides cluster than to the ruminant species M. yeatsii and M. putrefaciens. Ancestral genome reconstruction showed that the emergence of the M. feriruminatoris species was associated with the gain of 17 gene families, some of which encode defense enzymes and surface proteins, and the loss of 25 others, some of which are involved in sugar transport and metabolism. This comparative study suggests that the M. mycoides cluster could be extended to include M. feriruminatoris. We also find evidence that the specific organization and structure of the DnaA boxes around the oriC of M. feriruminatoris may contribute to drive the remarkable fast growth of this minimal bacterium.

microbiology↗

bioRxiv2022

Conserved mechanism for perception of root-active CLE peptides

Precision in sensing the environmental cues and adjusting the growth and the physiology of the root system are necessary for plant robustness. Plants achieve their phenotypic plasticity by tightly controlling and buffering developmental decisions. In addition to the classical plant hormones that mediate plant development and stress responses, the CLE peptides constitute an additional crucial level of regulation. While the CLV3-CLV1 module appears to be highly conserved to control the proliferation of the shoot apical meristem stem cells, we do not yet fully understand the function of the additional CLE genes and whether they act in a similar way across the plant species, including tomato. Due to the small gene size and high sequence variability, it is extremely difficult to precisely annotate CLE genes in plant genomes. Here we present our analysis of the CLE family in tomato, based on a combination of iterative tBLASTn and Hidden-Markov-Model (HMM), which allowed us to identify thirty-seven new SlCLEs in addition to the fifteen reported previously. We could confirm the biological activities of selected SlCLEs in suppressing root meristematic cell divisions. We show that root response is mediated by SlCLAVATA2, indicating the conservation of CLE perception mechanism. One-sentence summaryUsing a combination of iterative tBLASTn and Hidden-Markov-Model approaches, we uncovered 37 new tomato CLE genes predominantly expressed in roots, and we showed a conserved effect on root meristem arrest, that was SlCLAVATA2-dependent. HighlightsO_LIWe applied a combined approach of iterative tBLASTn and Hidden-Markov-Model to identify fifty-two tomato SlCLE genes, including thirty-seven new genes C_LIO_LIAll identified genes encode for pre-propeptides with a single CLE-domain containing conserved residues similar to Arabidopsis C_LIO_LIAnalyzing the publicly available RNAseq datasets, we could confirm the expression of SlCLE genes that was often associated specifically with root or shoot, a certain developmental stage of the fruit, or with drought stress conditions C_LIO_LIRemarkably, the majority of SlCLE genes are predominantly expressed in the root tissues C_LIO_LIWe showed the conserved inhibitory effect on the root meristem and columella cells division for the selected SlCLE peptides that were SlCLAVATA2-dependent. C_LI

plant biology↗

bioRxiv2021

Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis

The biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, prevents the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

cell biology↗

bioRxiv2021

Much more than a toxin: hydrogen cyanide is a volatile modulator of bacterial behaviour

Bacterial volatile compounds have emerged as important chemical messengers between bacteria themselves as well as in their interactions with other organisms. One of the earliest examples of bioactive volatiles emitted by bacteria is hydrogen cyanide (HCN), which was long considered a mere respiratory toxin conferring competitive advantage to cyanogenic strains. Using cyanide-deficient mutants in two Pseudomonas strains and global transcriptome analysis, we demonstrate that the impact of HCN is much more global than previously thought. We first observed that the lack of cyanogenesis in emitting strains led to massive transcriptome reprogramming affecting diverse traits such as motility and biofilm formation (respectively inhibited vs. promoted by HCN), or the production of siderophores, phenazines and other antimicrobial compounds (repressed by HCN). We then exposed non-cyanogenic strains to biogenically emitted HCN from neighboring cells and observed similar transcriptome modulations and phenotypic changes, suggesting that HCN not only acts endogenously but also exogenously, remotely manipulating important traits involved in competition and virulence, e.g. siderophore production, in other organisms. Cyanogenesis in Pseudomonas has long been known to play a role in both the virulence of opportunistic pathogens and the efficient biocontrol activity of plant-beneficial strains, however this impact was so far thought to occur solely through the inhibition of respiration. We demonstrate here new ecological roles for a small and fast-diffusing volatile compound, which opens novel avenues in our understanding of and ability to interfere with important processes taking place in pathogenic and beneficial Pseudomonas strains. ImportanceBacteria communicate by exchanging chemical signals, some of which are volatile and can remotely reach other organisms. Hydrogen cyanide (HCN) was one of the first volatiles discovered to severely impact exposed organisms by inhibiting their respiration. Using HCN-deficient mutants in two Pseudomonas strains, we demonstrate that HCNs impact goes beyond the sole inhibition of respiration and affects both emitting and receiving bacteria in a global way, modulating their motility, biofilm formation and production of antimicrobial compounds. Our data suggest that bacteria could use HCN not only to control their own cellular functions, but also to remotely influence the behavior of other bacteria sharing the same environment. Since HCN emission occurs in both clinically and environmentally relevant Pseudomonas, these findings are important to better understand or even modulate the expression of bacterial traits involved in both virulence of opportunistic pathogens and in biocontrol efficacy of plant-beneficial strains.

microbiology↗

bioRxiv2021

Revealing Notch-dependencies in synaptic targets associated with Alzheimer's disease

Alzheimers disease (AD) is a progressive neurodegenerative disorder and the major cause of dementia. There is evidence that synaptic dysfunction and perturbation of Excitatory/Inhibitory (E/I) balance arise at the early stages of AD, altering the normal neural network activity, and leading to cognitive decline. Recent studies have identified Notch signaling as a contributor of neurodegenerative advancement including AD pathophysiology. As part of the efforts to understand molecular mechanisms and players involved in cognitive decline, we employed transgenic mouse models with Notch1 and RBPJK loss of function (LOF) in pyramidal neurons of the CA fields. Using bulk RNAseq. We have investigated the differential expression of Notch-dependent genes either upon environmental enrichment (EE) or upon Kainate injury (KA). We found a substantial genetic diversity in absence of both Notch1 receptor or Rbpjk transcriptional activator. Among differentially expressed genes, we observed a significant upregulation of Gabra2a in both knockout models, suggesting a role for Notch signaling in the modulation of E/I balance. Upon neuroexcitotoxic stimulation, loss of Rbpjk results in decreased expression of synaptic proteins with neuroprotective effects. We confirmed Nptx2, Npy, Pdch8, TncC as direct Notch1/Rbpjk targets and Bdnf and Scg2 as indirect targets. Finally, we translate these findings into human entorhinal cortex containing the hippocampal region from Alzheimers Disease patients performing targeted transcripts analysis. We observe an increased trend for Rbpjk and the ligand DNER but not Notch1 expression. On the other hand, neuron-specific targets, Nptx2, Npy, BDNF and Gabra2a are upregulated during the mild-moderate stage, and decline in the severe phase of the disease. These findings identify Notch as a promising signaling cascade to fine-tune in order to ameliorate synaptic transmission and memory deficits that occur during early phase of the Alzheimers Disease. HighlightsO_LILoss of canonical and/or non-canonical Notch1 signaling in pyramidal neurons of the hippocampal CA field mainly affects the post-synaptic compartment. C_LIO_LIIn both RBPJKcKO and Notch1cKO mouse models there is upregulation of GABAergic receptor subunit alpha2 (Gabra2a). C_LIO_LIThe plasticity genes: Npy, Nptx2,Pcdh8 and TncC with neuroprotective functions and known association with Alzheimers Disease are direct Notch/Rbpjk targets. C_LIO_LIDuring the mild-moderate stage of AD dementia, Notch canonical signaling promotes the expression of neuroprotective proteins, in the attempt of mitigating the effect of the excitatory-inhibitory imbalance. This activity is not observed during severe stages of the disease. C_LI

neuroscience↗

bioRxiv2021

Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They lack a cell wall and have been suggested to have progressed through reductive evolution from phylogenetically closely related Clostridia. They are known to colonize the respiratory tract or the urogenital tract among other organs and can cause chronic and subclinical diseases associated with long persistence of the causative agent. Toxin-antitoxin systems (TAS) are genetic elements that have been described for several respiratory and urogenital pathogens as well as for Clostridia, but never for pathogenic mycoplasmas. Here we describe for the first-time different types of TAS in a Mycoplasma pathogen, namely M. mycoides subsp. capri. We identified candidate TAS in silico via TASmania database. Two candidate TAS identified in silico and another candidate TAS suggested in a minimal cell based on transposon mutagenesis were systematically tested for their functionality in hosts with different phylogenetic distance using heterologous expression. Phylogenetic distance of the host used for heterologous expression influenced the outcome of the functional testing. We corroborated functionality of the three candidate TAS in Mycoplasma capricolum subsp. capricolum. Moreover, we confirmed transcription and translation of molecules of the TAS investigated during in vitro growth. We sequence analyzed 15 genomes of M. mycoides subsp. capri and revealed an unequal distribution of the TAS studied pointing towards dynamic gain and loss of TAS within the species. Author summaryMycoplasmas have a minimal genome and have never been shown to possess TAS. In this work we showed the presence of different functional TAS systems in Mycoplasma mycoides subsp. capri, a caprine pathogen for the first time. Sequence analysis of a number of Mycoplasma mycoides subsp. capri strains revealed a plasticity of the genome with respect to TAS carriage. This work paves the way to investigate the biological role of TAS (e.g. persistence, stress tolerance) during infection using mycoplasmas as a simple model organism. Since most mycoplasmas lack classical virulence factors such as exotoxins and go into a kind of stealth mode to evade the immune system, TAS are likely to contribute to the parasitic lifestyle of mycoplasmas and should be investigated in that respect. The availability of synthetic genomics tools to modify a range of Mycoplasma pathogens and well-established challenge models for the latter mycoplasmas will foster future research on TAS in mycoplasmas.

microbiology↗

bioRxiv2019

Rationally designed oral vaccines can set an evolutionary trap for Salmonella Typhimurium

Introductory paragraphThe ability of gut bacterial pathogens to escape immunity by antigenic variation, particularly via changes to surface-exposed antigens, is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2, 3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here we demonstrated this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: A long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase-variation4, 5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen specific IgA and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of oral vaccines, we rationally induced IgA responses blocking all of these trajectories, which selected for Salmonella mutants carrying deletions of the O-antigen polymerase wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents, complement), predation (bacteriophages), and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

immunology↗

bioRxiv2019

Removal of a subset of non-essential genes fully attenuates a highly virulent Mycoplasma strain

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects and plants. Here, we demonstrate that highly virulent Mycoplasma mycoides subspecies capri (Mmc) can be fully attenuated via targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately ten percent of the original Mmc genome, were successively deleted using Saccharomyces cerevisiae as an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually nonessential in the JCVI-Syn3.0 minimal cell, were excised from the genome. In vitro characterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though ten percent of the genome content were removed. A novel in vivo challenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reaching endpoint criteria within seven days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of a Mycoplasma strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent in vitro or in vivo trials employing the Mycoplasma chassis will allow us to dissect the role of individual candidate Mycoplasma virulence factors and lead the way for the development of an attenuated designer vaccine.\n\nIMPORTANCEMembers of the Mycoplasma mycoides cluster cause important animal plaques in Africa and Asia, which impact animal welfare, provision of food and the life of thousands of small-scale farmers. We applied synthetic biology tools to Mycoplasma mycoides in order to design and create a fully attenuated Mycoplasma strain that was subsequently confirmed in vivo using a novel caprine infection model. This is the first time that a Mycoplasma mutant developed by applying synthetic biology tools has been tested in vivo to pin point candidate virulence traits. The mutant strain is similar to \"apathogenic E. coli K12\" strains that boosted the research on host-pathogen interactions for the genus Escherichia and other bacterial genera.

microbiology↗

bioRxiv2016

Cell Cycle Constraints and Environmental Control of Local DNA Hypomethylation in Alpha-Proteobacteria

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of -proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5-GANTC-3 sequence motifs that are methylated by he DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different -proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.\n\nAuthor SummaryDNA methylation is the post-replicative addition of a methyl group to a base by a methyltransferase that recognise a specific sequence, and represents an epigenetic regulatory mechanism in both eukaryotes and prokaryotes. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. CcrM is a conserved, cell cycle regulated adenine methyltransferase that methylates GANTC sites in -proteobacteria. N6-methyl-adenine (m6A) patterns generated by CcrM can change the affinity of a given DNA-binding protein for its target sequence, and therefore affect gene expression. Here, we combine restriction enzyme cleavage-deep sequencing (REC-Seq) with SMRT sequencing to identify hypomethylated 5-GANTC-3 (GANTCs) in -proteobacterial genomes instated by conserved cell cycle factors. By comparing SMRT and REC-Seq data with chromatin immunoprecipitation-deep sequencing data (ChIP-Seq) we show that a conserved transcriptional regulator, MucR, induces local hypomethylation patterns by occluding GANTCs to the CcrM methylase and we provide evidence that this competition occurs during S-phase, but not in G1-phase cells. Furthermore, we find that environmental signals (such as phosphate depletion) are superimposed to the cell cycle control mechanism and can override the specific hypomethylation pattern imposed by the cell cycle transcriptional circuitry.

microbiology↗