Plant Carbohydrate Scavenging through TonB-Dependent Receptors: A Feature Shared by Phytopathogenic and Aquatic Bacteria

TonB-dependent receptors (TBDRs) are outer membrane proteins mainly known for the active transport of iron siderophore complexes in Gram-negative bacteria. Analysis of the genome of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc), predicts 72 TBDRs. Such an overrepresentation is common in Xanthomonas species but is limited to only a small number of bacteria. Here, we show that one Xcc TBDR transports sucrose with a very high affinity, suggesting that it might be a sucrose scavenger. This TBDR acts with an inner membrane transporter, an amylosucrase and a regulator to utilize sucrose, thus defining a new type of carbohydrate utilization locus, named CUT locus, involving a TBDR for the transport of substrate across the outer membrane. This sucrose CUT locus is required for full pathogenicity on Arabidopsis, showing its importance for the adaptation to host plants. A systematic analysis of Xcc TBDR genes and a genome context survey suggested that several Xcc TBDRs belong to other CUT loci involved in the utilization of various plant carbohydrates. Interestingly, several Xcc TBDRs and CUT loci are conserved in aquatic bacteria such as Caulobacter crescentus, Colwellia psychrerythraea, Saccharophagus degradans, Shewanella spp., Sphingomonas spp. or Pseudoalteromonas spp., which share the ability to degrade a wide variety of complex carbohydrates and display TBDR overrepresentation. We therefore propose that TBDR overrepresentation and the presence of CUT loci designate the ability to scavenge carbohydrates. Thus CUT loci, which seem to participate to the adaptation of phytopathogenic bacteria to their host plants, might also play a very important role in the biogeochemical cycling of plant-derived nutrients in marine environments. Moreover, the TBDRs and CUT loci identified in this study are clearly different from those characterized in the human gut symbiont Bacteroides thetaiotaomicron, which allow glycan foraging, suggesting a convergent evolution of TBDRs in Proteobacteria and Bacteroidetes

Reconstitution of a functional IS608 single-strand transpososome: role of non-canonical base pairing

Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5′ to the foot of each IP, guide (G) sequences, using a network of canonical (‘Watson–Crick’) base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences†

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpAIS200/IS605). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpAREP) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpAREP is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpAREP was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpAREP activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpAREP shared the same general organization and similar catalytic characteristics with TnpAIS200/IS605 transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpAREP may therefore be one of the first examples of transposase domestication in prokaryotes

The stb Operon Balances the Requirements for Vegetative Stability and Conjugative Transfer of Plasmid R388

The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation) and a propagation mode (conjugation). The consequences of this novel concept in plasmid physiology will be discussed

Irradiation-Induced Deinococcus radiodurans Genome Fragmentation Triggers Transposition of a Single Resident Insertion Sequence

Stress-induced transposition is an attractive notion since it is potentially important in creating diversity to facilitate adaptation of the host to severe environmental conditions. One common major stress is radiation-induced DNA damage. Deinococcus radiodurans has an exceptional ability to withstand the lethal effects of DNA–damaging agents (ionizing radiation, UV light, and desiccation). High radiation levels result in genome fragmentation and reassembly in a process which generates significant amounts of single-stranded DNA. This capacity of D. radiodurans to withstand irradiation raises important questions concerning its response to radiation-induced mutagenic lesions. A recent study analyzed the mutational profile in the thyA gene following irradiation. The majority of thyA mutants resulted from transposition of one particular Insertion Sequence (IS), ISDra2, of the many different ISs in the D. radiodurans genome. ISDra2 is a member of a newly recognised class of ISs, the IS200/IS605 family of insertion sequences

Genome-Wide Analysis of the “Cut-and-Paste” Transposons of Grapevine

Background: The grapevine is a widely cultivated crop and a high number of different varieties have been selected since its domestication in the Neolithic period. Although sexual crossing has been a major driver of grapevine evolution, its vegetative propagation enhanced the impact of somatic mutations and has been important for grapevine diversity. Transposable elements are known to be major contributors to genome variability and, in particular, to somatic mutations. Thus, transposable elements have probably played a major role in grapevine domestication and evolution. The recent publication of the complete grapevine genome opens the possibility for an in deep analysis of its transposon content. Principal Findings: We present here a detailed analysis of the ‘‘cut-and-paste’ ’ class II transposons present in the genome of grapevine. We characterized 1160 potentially complete grapevine transposons as well as 2086 defective copies. We report on the structure of each element, their potentiality to encode a functional transposase, and the existence of matching ESTs that could suggest their transcription. Conclusions: Our results show that these elements have transduplicated and amplified cellular sequences and some of them have been domesticated and probably fulfill cellular functions. In addition, we provide evidences that the mobility o

A bacterial genome in transition - an exceptional enrichment of IS elements but lack of evidence for recent transposition in the symbiont Amoebophilus asiaticus

<p>Abstract</p> <p>Background</p> <p>Insertion sequence (IS) elements are important mediators of genome plasticity and are widespread among bacterial and archaeal genomes. The 1.88 Mbp genome of the obligate intracellular amoeba symbiont <it>Amoebophilus asiaticus </it>contains an unusually large number of transposase genes (n = 354; 23% of all genes).</p> <p>Results</p> <p>The transposase genes in the <it>A. asiaticus </it>genome can be assigned to 16 different IS elements termed ISCaa1 to ISCaa16, which are represented by 2 to 24 full-length copies, respectively. Despite this high IS element load, the <it>A. asiaticus </it>genome displays a GC skew pattern typical for most bacterial genomes, indicating that no major rearrangements have occurred recently. Additionally, the high sequence divergence of some IS elements, the high number of truncated IS element copies (n = 143), as well as the absence of direct repeats in most IS elements suggest that the IS elements of <it>A. asiaticus </it>are transpositionally inactive. Although we could show transcription of 13 IS elements, we did not find experimental evidence for transpositional activity, corroborating our results from sequence analyses. However, we detected contiguous transcripts between IS elements and their downstream genes at nine loci in the <it>A. asiaticus </it>genome, indicating that some IS elements influence the transcription of downstream genes, some of which might be important for host cell interaction.</p> <p>Conclusions</p> <p>Taken together, the IS elements in the <it>A. asiaticus </it>genome are currently in the process of degradation and largely represent reflections of the evolutionary past of <it>A. asiaticus </it>in which its genome was shaped by their activity.</p

Mécanismes de transferts de gènes chez les bactéries

My research is oriented towards the understanding of how MGEs operate, i.e. their intimate propagation mechanisms (modes of transfer, integration, and regulation), in vitro as well as in vivo, in the cell and more recently within bacterial communities. I have had the opportunity to study several types of elements, combining various and multi-scale approaches, such as biochemical and structural studies, genetics, genome-wide molecular genetics, bioinformatics, fluorescence microscopy and more recently a eucaryotic model microbiota. My PhD (2004-2008) was dedicated to the characterization of IS608, the model element of a widespread family of atypical bacterial transposons. We identified an entirely novel transposition pathway, involving exclusively single stranded DNA, associated with a new class of transposases (Y1). My post-doctorate in F. de la Cruz’s group (2008-2011) was devoted to study another major process of bacterial genetic diversity, conjugation. I initiated work on the stb operon and have shown that it controls the balance between two modes of transmission of plasmid R388: vertical transmission by segregation to daughter cells, and horizontal transmission by conjugation. In 2011, I joined M. Chandler’s group as a Chargée de Recherche at the CNRS, where I participated in different aspects of IS608 transposition regulation, from the choice of target sites to detailed conformational changes in the transposase during strands exchange leading to IS excision and integration. During this period, I also contributed to a project led by B. Ton-Hoang devoted to the study of the dissemination of REP sequences in bacterial genomes, and on which I co-supervised Alix Corneloup's thesis (2012-2016). REP are short palindromic DNA sequences present in large numbers in many bacterial genomes. This work, via the development of an assay to visualize and characterize TnpAREP transposase activity in vitro for the first time, has enabled us to propose a model for REP dissemination/amplification. I then joined J.-Y. Bouet’s team in 2016, which merged later with the team of F. Cornet in 2019 (LMGM) to develop projects focused on deciphering spatiotemporal dynamics of secondary replicons (plasmids). In particular, I am interested in understanding how conjugation events are integrated into the maintenance functions of the plasmid (replication, segregation), which is one important remaining question in the field. We have shown that the Stb system, present in over 15% of plasmids in enterobacteria, mechanistically links these two processes. It involves the StbA protein, of which we have characterized the DNA-binding domain, and whose activities make it both a new type of segregation system, through its role in positioning plasmid molecules in the bacterium, and an inhibitor of conjugation. This work is part of Valentin Quèbre's PhD (co-supervised 2019-2023), and I co-supervise Charlotte Hall's PhD that on this project since December 2023. I also aim to enlarge our knowledge on horizontal gene transfer to a more complex ecosystem by investigating the dynamic feature of conjugative plasmids and transposons both at the population and cellular levels within the simple eukaryotic intestine model Caenorhabditis elegans. Romane Dusfour-Castan's thesis work (2021-now) shows that Lelliottia amnigena influences the lifespan, fertility and development of C. elegans compared with other CeMBio bacteria, with these effects depending on the composition and complexity of the microbiota.Mes recherches sont orientées vers la compréhension du fonctionnement des Éléments Génétiques Mobiles (EGMs), c'est-à-dire leurs mécanismes intimes de propagation (modes de transfert, intégration et régulation), in vitro et in vivo, dans la cellule et plus récemment au sein des communautés bactériennes. J'ai eu l'opportunité d'étudier plusieurs types d'éléments, en combinant diverses approches multi-échelles, telles que des études biochimiques et structurales, la génétique, la génétique moléculaire à l'échelle du génome, la bioinformatique, la microscopie à fluorescence et plus récemment un modèle de microbiote eucaryote.Durant ma thèse (2004-2008), j'ai participé à la caractérisation d'IS608, l'élément modèle d'une famille répandue de transposons bactériens atypiques. Nous avons identifié une voie de transposition entièrement nouvelle, impliquant exclusivement l'ADN simple brin, associée à une nouvelle classe de transposases (Y1). Mon post-doctorat dans le groupe de F. de la Cruz (2008-2011) était consacré à l'étude d'un autre processus majeur de la diversité génétique bactérienne, la conjugaison. J'ai initié des travaux sur l'opéron stb, et nous avons montré qu'il contrôle l'équilibre entre deux modes de transmission du plasmide R388 : la transmission verticale par ségrégation aux cellules filles, et la transmission horizontale par conjugaison.En 2011, j'ai rejoint le groupe de M. Chandler en tant que Chargée de Recherche au CNRS, où j'ai participé à différents aspects de la régulation de la transposition d'IS608, depuis le choix des sites cibles jusqu'aux changements conformationnels de la transposase pendant l'échange de brins conduisant à l'excision et à l'intégration de l'IS. Pendant cette période, j'ai également contribué à un projet dirigé par B. Ton-Hoang visant à étudier la dissémination des séquences REP dans les génomes bactériens, et sur lequel j'ai co-encadré la thèse d'Alix Corneloup (2012-2016). Les REP sont de courtes séquences d'ADN palindromiques présentes en grand nombre dans de nombreux génomes bactériens. Ce travail, via le développement d'un test pour visualiser et caractériser pour la première fois l'activité de la transposase TnpAREP in vitro, nous a permis de proposer un modèle pour la dissémination/amplification des REP.J'ai ensuite rejoint l'équipe de J.-Y. Bouet en 2016, qui a fusionné plus tard avec l'équipe de F. Cornet en 2019 (LMGM) pour développer des projets axés sur le déchiffrement de la dynamique spatio-temporelle des réplicons secondaires (plasmides). Mes projets ont notamment pour but de comprendre comment les événements de conjugaison sont intégrés aux fonctions de maintenance du plasmide (réplication, ségrégation), ce qui constitue une question importante encore non résolue dans ce domaine. Nous avons montré que le système Stb, présent dans plus de 15% des plasmides chez les entérobactéries, lie mécaniquement ces deux processus. Il implique la protéine StbA, dont nous avons caractérisé le domaine de liaison à l'ADN, et dont les activités en font à la fois un nouveau type de système de ségrégation, par son rôle dans le positionnement des molécules de plasmide dans la bactérie, et un inhibiteur de la conjugaison. Ce travail fait partie de la thèse de Valentin Quèbre (co-encadrée 2019-2023), et je co-encadre la thèse de Charlotte Hall sur ce projet depuis décembre 2023. Mon objectif est également d'élargir nos connaissances sur le transfert horizontal de gènes à un écosystème plus complexe en étudiant la dynamique des plasmides conjugatifs et des transposons à la fois au niveau de la population et au niveau cellulaire dans le modèle intestinal eucaryote simple Caenorhabditis elegans. Les travaux de thèse de Romane Dusfour-Castan (2021-maintenant) montrent que Lelliottia amnigena influence la durée de vie, la fertilité et le développement de C. elegans par rapport à d'autres bactéries CeMBio, avec des effets dépendant de la composition et de la complexité du microbiote