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

Plastic architecture of bacterial genome revealed by comparative genomics of Photorhabdus variants

Background: The phenotypic consequences of large genomic architecture modifications within a clonal bacterial population are rarely evaluated because of the difficulties associated with using molecular approaches in a mixed population. Bacterial variants frequently arise among Photorhabdus luminescens, a nematode-symbiotic and insect-pathogenic bacterium. We therefore studied genome plasticity within Photorhabdus variants. Results: We used a combination of macrorestriction and DNA microarray experiments to perform a comparative genomic study of different P. luminescens TT01 variants. Prolonged culturing of TT01 strain and a genomic variant, collected from the laboratory-maintained symbiotic nematode, generated bacterial lineages composed of primary and secondary phenotypic variants and colonial variants. The primary phenotypic variants exhibit several characteristics that are absent from the secondary forms. We identify substantial plasticity of the genome architecture of some variants, mediated mainly by deletions in the 'flexible' gene pool of the TT01 reference genome and also by genomic amplification. We show that the primary or secondary phenotypic variant status is independent from global genomic architecture and that the bacterial lineages are genomic lineages. We focused on two unusual genomic changes: a deletion at a new recombination hotspot composed of long approximate repeats; and a 275 kilobase single block duplication belonging to a new class of genomic duplications. Conclusion: Our findings demonstrate that major genomic variations occur in Photorhabdus clonal populations. The phenotypic consequences of these genomic changes are cryptic. This study provides insight into the field of bacterial genome architecture and further elucidates the role played by clonal genomic variation in bacterial genome evolutio

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

The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points

flhDC, the Flagellar Master Operon of Xenorhabdus nematophilus: Requirement for Motility, Lipolysis, Extracellular Hemolysis, and Full Virulence in Insects

Xenorhabdus is a major insect pathogen symbiotically associated with nematodes of the family Steinernematidae. This motile bacterium displays swarming behavior on suitable media, but a spontaneous loss of motility is observed as part of a phenomenon designated phase variation which involves the loss of stationary-phase products active as antibiotics and potential virulence factors. To investigate the role of one of the transcriptional activators of flagellar genes, FlhDC, in motility and virulence, the Xenorhabdus nematophilus flhDC locus was identified by functional complementation of an Escherichia coli flhD null mutant and DNA sequencing. Construction of X. nematophilus flhD null mutants confirmed that the flhDC operon controls flagellin expression but also revealed that lipolytic and extracellular hemolysin activity is flhDC dependent. We also showed that the flhD null mutant displayed a slightly attenuated virulence phenotype in Spodoptera littoralis compared to that of the wild-type strain. Thus, these data indicated that motility, lipase, hemolysin, or unknown functions controlled by the flhDC operon are involved in the infectious process in insects. Our investigation expands the view of the flagellar regulon as a checkpoint coupled to a major network involving bacterial physiological aspects as well as motility

A New Member of the Growing Family of Contact-Dependent Growth Inhibition Systems in Xenorhabdus doucetiae

Xenorhabdus is a bacterial symbiont of entomopathogenic Steinernema nematodes and is pathogenic for insects. Its life cycle involves a stage inside the insect cadaver, in which it competes for environmental resources with microorganisms from soil and the insect gut. Xenorhabdus is, thus, a useful model for identifying new interbacterial competition systems. For the first time, in an entomopathogenic bacterium, Xenorhabdus doucetiae strain FRM16, we identified a cdi-like locus. The cdi loci encode contact-dependent inhibition (CDI) systems composed of proteins from the two-partner secretion (TPS) family. CdiB is the outer membrane protein and CdiA is the toxic exoprotein. An immunity protein, CdiI, protects bacteria against inhibition. We describe here the growth inhibition effect of the toxic C-terminus of CdiA from X. doucetiae FRM16, CdiA-CTFRM16, following its production in closely and distantly related enterobacterial species. CdiA-CTFRM16 displayed Mg2+-dependent DNase activity, in vitro. CdiA-CT (FRM16)-mediated growth inhibition was specifically neutralized by CdiI(FRM16). Moreover, the cdi(FRM16) locus encodes an ortholog of toxin-activating proteins C that we named CdiC(FRM16). In addition to E. coli, the cdiBCAI-type locus was found to be widespread in environmental bacteria interacting with insects, plants, rhizospheres and soils. Phylogenetic tree comparisons for CdiB, CdiA and CdiC suggested that the genes encoding these proteins had co-evolved. By contrast, the considerable variability of CdiI protein sequences suggests that the cdiI gene is an independent evolutionary unit. These findings further characterize the sparsely described cdiBCAI-type locus

simple bacteriological tests for phenotypic characterization of Xenorhabdus and Photorhabdus phase variants

International audienc

Cloning and nucleotide sequence of a flagellin encoding genetic locus from Xenorhabdus nematophilus : phase variation leads to differential transcription of two flagellar genes (fliCD)

International audienc

- PM1072 - Bistability and antimicrobial peptide resista nce in the entomopathogenic bacteria Photorhabdus

- PM1072 - Bistability and antimicrobial peptide resista nce in the entomopathogenic bacteria Photorhabdus. International Union of Microbiological Societies Congresses - IUMS201