The complete methylome of an entomopathogenic bacterium reveals the existence of loci with unmethylated Adenines

www.nature.com/scientificreports OPEN Received: 14 March 2018 Accepted: 3 August 2018 Published: xx xx xxxx The complete methylome of an entomopathogenic bacterium reveals the existence of loci with unmethylated Adenines Amaury Payelleville1, Ludovic Legrand 2, Jean-Claude Ogier1, Céline Roques3, Alain Roulet3, Olivier Bouchez3, Annabelle Mouammine1,4, Alain Givaudan1 & Julien Brillard 1 DNA methylation can serve to control diverse phenomena in eukaryotes and prokaryotes, including gene regulation leading to cell differentiation. In bacteria, DNA methylomes (i.e., methylation state of each base of the whole genome) have been described for several species, but methylome profile variation during the lifecycle has rarely been studied, and only in a few model organisms. Moreover, major phenotypic changes have been reported in several bacterial strains with a deregulated methyltransferase, but the corresponding methylome has rarely been described. Here we report the first methylome description of an entomopathogenic bacterium, Photorhabdus luminescens. Eight motifs displaying a high rate of methylation (>94%) were identified. The methylome was strikingly stable over course of growth, but also in a subpopulation responsible for a critical step in the bacterium’s lifecycle: successful survival and proliferation in insects. The rare unmethylated GATC motifs were preferentially located in putative promoter regions, and most of them were methylated after Dam methyltransferase overexpression, suggesting that DNA methylation is involved in gene regulation. Our findings bring key insight into bacterial methylomes and encourage further research to decipher the role of loci protected from DNA methylation in gene regulation. DNA methyltransferases (MTases) are enzymes that catalyze the transfer of a methyl group from the universal methyl donor S-Adenosylmethionine (SAM) to a nucleotide1. MTases are widespread from eukaryotes to prokaryotes: 5-methylcytosine (m5C) and N6-methyladenine (m6A) methylation marks have been described in eukaryotes2,3 whereas additional N4-methylcytosine (m4C) marks can be detected in bacteria and archaea4,5. For years, DNA methylation studies mostly used whole genome bisulfite sequencing (WGBS), which only detects m5C6, until the advent of Single Molecule Real Time (SMRT) sequencing made it possible to also detect m4C and m6A modifications7. DNA methylation in bacteria is involved in many cellular processes. It provides a defense against foreign DNA in restriction-modification systems (RM), where a restriction endonuclease (REase) acts in coordination with a DNA MTase. The MTase methylates self-DNA whereas exogenous DNA is cleaved by the cognate REase due to different methylation patterns. RM-MTases are classified into 4 different groups based on biochemical properties (e.g. their subunit organization, their recognition of palindromic vs asymmetric target DNA sequences…)8. The REBASE database is designed to identify REases and MTases and currently lists thousands of putative RM-MTases9. Many studies, particularly in Escherichia coli, have described the role of DNA methylation in discriminating between the parental and newly-synthesized strand using the DNA mismatch repair (MMR) system10. During replication, errors may occur, and the MMR system excises the wrong base on the newly-synthesized unmethylated DNA strand10. The MMR system requires Dam (DNA adenine MTase), an MTase that is not coupled with a restriction enzyme and so called “orphan” or “solitary” MTase5. In Gammaproteobacteria, Dam methylates 1 DGIMI, INRA, Univ. Montpellier, Montpellier, France. 2LIPM, Université de Toulouse, INRA, CNRS, CastanetTolosan, France. 3GeT-PlaGe, INRA, US 1426, Genotoul, Castanet-Tolosan, France. 4Present address: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Quartier UNIL/Sorge, Lausanne, CH1015, Switzerland. Amaury Payelleville and Ludovic Legrand contributed equally to this work. Correspondence and requests for materials should be addressed to J.B. (email: ) SCIeNTIFIC RePOrTs | (2018) 8:12091 | DOI:10.1038/s41598-018-30620-5 1 www.nature.com/scientificreports/ 5′-GATC-3′ motifs. Dam is also involved in epigenetic mechanisms by strongly contributing to the regulation of several genes expression5. In E. coli for example, the Lrp regulator can bind sites containing GATC sequences in the pap operon promoter region11. Lrp has greater affinity to unmethylated sites than fully-methylated (methylation on both strands) sites, and the competition between Dam and Lrp binding gives rise to two sub-populations, one expressing the Pap pilus and the other not12. Deregulation by mutation or overexpression of Dam in several bacterial species can lead to drastic phenotypic changes. For example, Dam overexpression leads to a decrease in virulence for Yersinia pseudotuberculosis, Vibrio cholerae, Salmonella enterica, Pasteurella multocida, Aeromonas hydrophila and Photorhabdus luminescens13–18. Dam mutants have also been described as showing impaired virulence in S. enterica, Haemophilus influenzae, Klebsiella pneumoniae, A. actinomycetemcomitans and Y. pestis19–23. Dam mutants are not viable in Y. pseudotuberculosis and V. cholera16 or suspected to be unviable in other bacterial species18. DNA cytosine MTase (Dcm) is another solitary MTase that is well conserved in Gammaproteobacteria24. The dcm gene is associated to vsr which encodes the very-short-patch repair system involved in T/G mismatches correction24. Dcm adds a methyl group to the second cytosine of CCWGG motifs. This solitary MTase has been shown to be involved in drug resistance, translation25 and stationary phase gene expression26. In addition to Dam, the best known example of solitary MTase involved in epigenetic mechanisms in bacteria27 is the CcrM (Cell cycle regulated MTase) found in many Alphaproteobacteria. Its main role in Caulobacter crescensus is regulation of the cell cycle and cell division28,29. It is essential for cell viability in fast-growing conditions30 but not in slow-growing conditions such as minimal media31. It has not been described as involved in MMR31. Many other solitary MTases are present in many bacterial genomes, but their role has not yet been described32. SMRT sequencing can now detect all DNA methylation marks in genomes, opening opportunities to detect new methylated motifs7. This new-generation sequencing technology has been used to describe the methylome of several microorganisms. In bacteria, there has been a strong focus on animal pathogens, but the methylomes of some plant pathogens have also been reported32–41. P. luminescens TT01 is an entomopathogenic bacterium member of the Enterobacteriaceae. It is found in symbiosis with a soil nematode from the genus Heterorhabditis. This nematobacterial complex is able to kill many crop-pest insects and can be used in biocontrol42. During its lifecycle, this bacterium has to switch betwe (...truncated)