20 research outputs found
Whole Genome DNA Methylation (Methylome) Analysis of an Entomopathogenic Bacterium
Background: DNA methylation is an epigenetic mechanism involved in the pathogenicity of several major bacterial pathogens. It can decrease the affinity of some transcriptional regulators to their binding site, leading to sub-populations expressing or not various genes, depending on the DNA methylation state. Dam DNA methyltransferase is widespread in Gammaproteobacteria and methylates the adenine of GATC sites. Objectives: The role of Dam was investigated in Photorhabdus luminescens during its symbiosis with a soil nematode and during its pathogenic stage in insects.Methods: SMRT sequencing (PacBio) and Bisulfite-seq were performed to identify the DNA methylation of the whole genome (methylome). In addition, RNAseq and phenotypic analysis were performed in a P. luminescens strain overexpressing Dam.Results: Dam overexpression caused a decrease in motility whereas it increased biofilm formation. While symbiosis ability of the Dam overexpressing strain was not significantly different from that of a control strain, the nemato-bacterial complex displayed an impaired pathogenicity in insect, as also observed after direct insect injection of the bacteria alone. Transcriptomic analysis revealed that the observed phenotypes were related to differences at the transcriptional level. More than 99% of the GATC sites of the genome were found methylated and DNA methylation levels did not change over growth kinetics. However, the Dam-overexpressing strain displayed more methylated GATC sites than the control and most of these sites were located in promoter regions. These sites may be involved in the observed differences in phenotypes and gene expression and provide clues to understand the involvement of Dam DNA methylation in P. luminescens life-cycle
Role of the Photorhabdus Dam methyltransferase during interactions with its invertebrate hosts
Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex
DNA methylation role in the insect pathogen bacterium Photorhabdus luminescens
Photorhabdus luminescens est une entérobactérie retrouvée en symbiose avec les nématodes du genre Heterorhabditis. Dans les sols, ce complexe némato-bactérien est pathogène d’insectes ravageurs et est utilisé en contrôle biologique. Le nématode pénètre dans l’insecte et libère la bactérie dans l’hémolymphe. Photorhabdus va ensuite se multiplier et secréter divers facteurs de virulence comme des toxines. L’insecte meurt de septicémie puis le nématode et la bactérie vont se nourrir du cadavre. Une fois les ressources épuisées, le complexe némato-bactérien va se reformer et sortir du cadavre à la recherche d’une nouvelle cible. Plusieurs exemples d’hétérogénéité phénotypique ont été décrits chez cette bactérie amenant chacun à la présence de sous-populations dans une culture bactérienne. Cette hétérogénéité phénotypique peut-être causée par des mécanismes épigénétiques et plus précisément par la méthylation de l’ADN. Chez les entérobactéries, la méthyltransférase Dam est très conservée. Elle méthyle les adénines des sites GATC et est impliquée dans la réparation des erreurs lors de la réplication de l’ADN, la régulation du cycle cellulaire mais aussi la régulation de divers gènes. Cette méthyltransférase est en compétition avec certain régulateurs transcriptionnel. Selon qui de la méthyltransférase ou du régulateur se fixera en premier, le gène sera ou non exprimé donnant naissance à deux sous-populations. Cette thèse a pour objectif de mettre en évidence les rôles de la méthyltransférase Dam chez Photorhabdus luminescens. Dans un premier temps, j’ai montré que la surexpression de Dam (Dam+) amène une diminution de la mobilité et du pouvoir pathogène de la bactérie mais à l’inverse augmente sa capacité à former des biofilms. Une analyse transcriptomique (RNAseq) a montré des différentiels d’expression de certains gènes impliqués dans les phénotypes observés. En recombinant la souche de Photorhabdus Dam+ avec les nématodes hôtes, l’effet sur la pathogénicité a été augmenté en comparaison des résultats après injection de la bactérie seule. L’établissement de la symbiose némato-bactérienne avec cette souche Dam+ n’est pas significativement impacté par rapport à la souche sauvage. Enfin l’analyse du méthylome (détection de tous les sites méthylés sur le génome grâce à la technique SMRT) de Photorhabdus dans diverses phases de croissance nous a permis de déterminer que la méthylation par Dam semble être stable aux différents temps de la croissance bactérienne testés chez Photorhabdus. Le méthylome de la souche Dam+ a confirmé l’hypothèse que cette surexpression augmentait le taux de méthylation des sites GATC sur le génome. La comparaison combinée entre le RNAseq et les sites GATC différentiellement méthylés entre la souche contrôle et Dam+ a mis en évidence certains gènes candidats ressortant de ces deux analyses. En effet, certains gènes sont différentiellement exprimés dans les deux souches et ont un différentiel de méthylation au niveau de sites GATC dans leur région promotrice, l’étude détaillée de leur régulation par la méthylation fait maintenant partie des perspectives et permettront peut-être d’expliquer une partie des phénotypes observés chez Photorhabdus luminescens.Photorhabdus luminescens is an Enterobacteriaceae found in soils in symbiosis with a nematode from the genus Heterorhabditis. This nemato-bacterial complex is highly pathogenic against insect pest crops and so used in biocontrol. The nematode enters into the insect and releases Photorhabdus in the hemolymph of the insect. Photorhabdus multiplies and produces diverse virulence factors as toxins. Insect die from septicemia and both nematodes and bacteria feed on the nutrients in the cadaver. Once nutrients are lacking, the nematodes and the bacteria reassociate and exit from the cadaver to find new insects to infect. Photorhabdus is switching between pathogenic and symbiotic state. This bacterium displays phenotypic heterogeneity as we observe subpopulations coexisting in a same bacterial culture. Phenotypic heterogeneity can be explained by epigenetic mechanisms such as DNA methylation. In Enterobacteriaceae, Dam methyltransferase is broadly distributed. It methylates the adenine of GATC sites. Dam is involved in post-replicative mismatch repair, cell-cycle regulation and also gene transcription regulation. This methyltransferase can be in competition with some transcriptional regulators. Depending on which will bind first on the promoter region, gene will be expressed or not, leading to the rise of two subpopulations. This thesis aims to understand roles of Dam in Photorhabdus luminescens. Overexpression of the methyltransferase leads to a decrease in motility and pathogenicity of Photorhabdus Dam+ strain whereas it increases biofilms formation. A transcriptomic analysis (RNAseq) revealed differential expression of genes involved in the observed phenotypes. Symbiosis establishment does not seem to be strongly impacted in Dam+ strain as the only difference observed when compared to the nematode associated with the control strain is the same as with bacteria alone (a delayed virulence). A methylome analysis was also done (screening of all methylated sites in the genome using SMRT sequencing) in several growth conditions which revealed that DNA methylation is stable over growth kinetics. Dam+ strain methylome analysis confirmed the hypothesis that Dam overexpression increases GATC methylation over the genome. Comparative analysis of methylome and RNAseq experiments between control and Dam+ strains highlighted several common genes. In fact, some genes are differentially expressed between both strains and also have GATC sites differentially methylated in their promoter region. Their transcription regulation by methylation is a future aim and may give some explanation for a part of the phenotypes observed in Photorhabdus luminescens
Etude de la méthylation de l'ADN chez la bactérie pathogène d'insectes Photorhabdus luminescens
Photorhabdus luminescens is an Enterobacteriaceae found in soils in symbiosis with a nematode from the genus Heterorhabditis. This nemato-bacterial complex is highly pathogenic against insect pest crops and so used in biocontrol. The nematode enters into the insect and releases Photorhabdus in the hemolymph of the insect. Photorhabdus multiplies and produces diverse virulence factors as toxins. Insect die from septicemia and both nematodes and bacteria feed on the nutrients in the cadaver. Once nutrients are lacking, the nematodes and the bacteria reassociate and exit from the cadaver to find new insects to infect. Photorhabdus is switching between pathogenic and symbiotic state. This bacterium displays phenotypic heterogeneity as we observe subpopulations coexisting in a same bacterial culture. Phenotypic heterogeneity can be explained by epigenetic mechanisms such as DNA methylation. In Enterobacteriaceae, Dam methyltransferase is broadly distributed. It methylates the adenine of GATC sites. Dam is involved in post-replicative mismatch repair, cell-cycle regulation and also gene transcription regulation. This methyltransferase can be in competition with some transcriptional regulators. Depending on which will bind first on the promoter region, gene will be expressed or not, leading to the rise of two subpopulations. This thesis aims to understand roles of Dam in Photorhabdus luminescens. Overexpression of the methyltransferase leads to a decrease in motility and pathogenicity of Photorhabdus Dam+ strain whereas it increases biofilms formation. A transcriptomic analysis (RNAseq) revealed differential expression of genes involved in the observed phenotypes. Symbiosis establishment does not seem to be strongly impacted in Dam+ strain as the only difference observed when compared to the nematode associated with the control strain is the same as with bacteria alone (a delayed virulence). A methylome analysis was also done (screening of all methylated sites in the genome using SMRT sequencing) in several growth conditions which revealed that DNA methylation is stable over growth kinetics. Dam+ strain methylome analysis confirmed the hypothesis that Dam overexpression increases GATC methylation over the genome. Comparative analysis of methylome and RNAseq experiments between control and Dam+ strains highlighted several common genes. In fact, some genes are differentially expressed between both strains and also have GATC sites differentially methylated in their promoter region. Their transcription regulation by methylation is a future aim and may give some explanation for a part of the phenotypes observed in Photorhabdus luminescens.Photorhabdus luminescens est une entérobactérie retrouvée en symbiose avec les nématodes du genre Heterorhabditis. Dans les sols, ce complexe némato-bactérien est pathogène d’insectes ravageurs et est utilisé en contrôle biologique. Le nématode pénètre dans l’insecte et libère la bactérie dans l’hémolymphe. Photorhabdus va ensuite se multiplier et secréter divers facteurs de virulence comme des toxines. L’insecte meurt de septicémie puis le nématode et la bactérie vont se nourrir du cadavre. Une fois les ressources épuisées, le complexe némato-bactérien va se reformer et sortir du cadavre à la recherche d’une nouvelle cible. Plusieurs exemples d’hétérogénéité phénotypique ont été décrits chez cette bactérie amenant chacun à la présence de sous-populations dans une culture bactérienne. Cette hétérogénéité phénotypique peut-être causée par des mécanismes épigénétiques et plus précisément par la méthylation de l’ADN. Chez les entérobactéries, la méthyltransférase Dam est très conservée. Elle méthyle les adénines des sites GATC et est impliquée dans la réparation des erreurs lors de la réplication de l’ADN, la régulation du cycle cellulaire mais aussi la régulation de divers gènes. Cette méthyltransférase est en compétition avec certain régulateurs transcriptionnel. Selon qui de la méthyltransférase ou du régulateur se fixera en premier, le gène sera ou non exprimé donnant naissance à deux sous-populations. Cette thèse a pour objectif de mettre en évidence les rôles de la méthyltransférase Dam chez Photorhabdus luminescens. Dans un premier temps, j’ai montré que la surexpression de Dam (Dam+) amène une diminution de la mobilité et du pouvoir pathogène de la bactérie mais à l’inverse augmente sa capacité à former des biofilms. Une analyse transcriptomique (RNAseq) a montré des différentiels d’expression de certains gènes impliqués dans les phénotypes observés. En recombinant la souche de Photorhabdus Dam+ avec les nématodes hôtes, l’effet sur la pathogénicité a été augmenté en comparaison des résultats après injection de la bactérie seule. L’établissement de la symbiose némato-bactérienne avec cette souche Dam+ n’est pas significativement impacté par rapport à la souche sauvage. Enfin l’analyse du méthylome (détection de tous les sites méthylés sur le génome grâce à la technique SMRT) de Photorhabdus dans diverses phases de croissance nous a permis de déterminer que la méthylation par Dam semble être stable aux différents temps de la croissance bactérienne testés chez Photorhabdus. Le méthylome de la souche Dam+ a confirmé l’hypothèse que cette surexpression augmentait le taux de méthylation des sites GATC sur le génome. La comparaison combinée entre le RNAseq et les sites GATC différentiellement méthylés entre la souche contrôle et Dam+ a mis en évidence certains gènes candidats ressortant de ces deux analyses. En effet, certains gènes sont différentiellement exprimés dans les deux souches et ont un différentiel de méthylation au niveau de sites GATC dans leur région promotrice, l’étude détaillée de leur régulation par la méthylation fait maintenant partie des perspectives et permettront peut-être d’expliquer une partie des phénotypes observés chez Photorhabdus luminescens
Novel Identification of Bacterial Epigenetic Regulations Would Benefit From a Better Exploitation of Methylomic Data
DNA methylation can be part of epigenetic mechanisms, leading to cellular subpopulations with heterogeneous phenotypes. While prokaryotic phenotypic heterogeneity is of critical importance for a successful infection by several major pathogens, the exact mechanisms involved in this phenomenon remain unknown in many cases. Powerful sequencing tools have been developed to allow the detection of the DNA methylated bases at the genome level, and they have recently been extensively applied on numerous bacterial species. Some of these tools are increasingly used for metagenomics analysis but only a limited amount of the available methylomic data is currently being exploited. Because newly developed tools now allow the detection of subpopulations differing in their genome methylation patterns, it is time to emphasize future strategies based on a more extensive use of methylomic data. This will ultimately help to discover new epigenetic gene regulations involved in bacterial phenotypic heterogeneity, including during host-pathogen interactions.SCOPUS: re.jinfo:eu-repo/semantics/publishe
Dam overexpression impacts motility and virulence of the entomopathogenic bacteria Photorhabdus luminescens TT01
Bacterial DNA methylation is known to play a role in gene expression (Casades us et al., 2006). Dam is the most described DNA-methyltransferase and is widespread in gamma-proteobacteria. Dam DNA methylation is involved in pathogenicity of several bacteria.Photorhabdus luminescens is an entomopathogenic bacterium symbiotically associated with nematodes of the genus Heterorhabditis . The nemato-bacterial complex is able to kill insect larvae such as lepidoptera by bacterial septicemia (Nielsen-LeRoux et al., 2012). We identifed a Dam ortholog in P. luminescens genome and we showed that P. luminescens dam gene is functional by cloning it on a plasmid in an E. coli Dam mutant. After overexpression ofdam in P. luminescens, a decrease of about 50% in motility (p<0.02) was observed. In addition, after injection of 1000 CFU in larvae of Spodoptera littoralis, the Dam overexpressing strain showed a delayed virulence compared to that of the control strain (harboring an empty plasmid). In contrast, no di erence in growth ability was observed in vitro between the two recombinant strains. These results enhance our knowledge about Dam methylation and strengthen the hypothesis that Dam plays a major role for gene expression in proteobacteria.To go further, we plan to identify all DNA methylations in the genome of the two strains using Pacbio sequencing
Dam overexpression impacts motility and virulence of the entomopathogenic bacteria Photorhabdus luminescens TT01
Bacterial DNA methylation is known to play a role in gene expression (Casades us et al., 2006). Dam is the most described DNA-methyltransferase and is widespread in gamma-proteobacteria. Dam DNA methylation is involved in pathogenicity of several bacteria.Photorhabdus luminescens is an entomopathogenic bacterium symbiotically associated with nematodes of the genus Heterorhabditis . The nemato-bacterial complex is able to kill insect larvae such as lepidoptera by bacterial septicemia (Nielsen-LeRoux et al., 2012). We identifed a Dam ortholog in P. luminescens genome and we showed that P. luminescens dam gene is functional by cloning it on a plasmid in an E. coli Dam mutant. After overexpression of
dam in P. luminescens, a decrease of about 50% in motility (p<0.02) was observed. In addition, after injection of 1000 CFU in larvae of Spodoptera littoralis, the Dam overexpressing strain showed a delayed virulence compared to that of the control strain (harboring an empty plasmid). In contrast, no di erence in growth ability was observed in vitro between the two recombinant strains. These results enhance our knowledge about Dam methylation and strengthen the hypothesis that Dam plays a major role for gene expression in proteobacteria.
To go further, we plan to identify all DNA methylations in the genome of the two strains using Pacbio sequencing
Dam overexpression increases biofilms formation but impairs motility and pathogenicity of Photorhabdus luminescens
Dam overexpression increases biofilms formation but impairs motility and pathogenicity of Photorhabdus luminescens. 7. congress of European Microbiologists FEMS 201