Bacterial genomics, metagenomics and nematode microbiota

Evolutionary history and host adaptation factors in bacteria of the genera Photorhabdus and Xenorhabdus

Bacteria of the genera Photorhabdus and Xenorhabdus are symbiotic of nematodes and pathogenic of insects. Through various contracts (Genoscope 2007-2008; Nosopharm start-up, Nîmes 2010-2011) and international collaborations (American Xenorhabdus consortium, University of Wisconsin and University of Arizona), we have assembled genomes covering a large part of the phylogenetic diversity of these two bacterial genera in the MicroScope sequence databases (https://mage.genoscope.cns.fr/microscope/home/index.php). Comparative and functional genomics approaches were conducted and led to the following main results:

• The genera Photorhabdus and Xenorhabdus are genomically divergent despite a convergence in lifestyle (Chaston et al, 2011).
• Within these two bacterial genera, regions of genomic plasticity (genomic islands, prophages, and hypervariable regions) are rich in genes encoding bacterial adaptation factors to the biotic and abiotic environment and consist of a mosaic of functional and mobile modules (Ogier et al, 2010) (Figure 1).
• In some Xenorhabdus species, genomic reduction has occurred by deletion of genomic modules. The streamlining hypothesis following significant functional complementarity between symbionts and their nematode host is proposed (Ogier et al, 2014).
• In other Xenorhabdus species, comparative genomics between a virulent and a non-virulent strain suggests the existence of a trade-off between optimal cadaver exploitation and the ability to inhibit microorganisms in the insect cadaver (Bisch et al, 2016).
• Some factors of adaptation to the parasitic cycle could be identified: type 5 (Ogier et al, 2016), type 6 (McMullenII et al, 2017) secretion system, secondary metabolites involved in insecticidal activity (Kim et al, 2017) or in antimicrobial activities (Pantel et al, 2018).

Figure 1. Schematic diagram of the modular structure of a RGP (region of genomic plasticty) in the genome of Xenorhabdus nematophila ATCC19061 (Xn) (panel A) and the corresponding modules in the genome of Photorhabdus luminescens TT01 (Pl), Photorhabdus asymbiotica ATCC43949 (Pa), Xenorhabdus bovienii SS-2004 (Xb) (panel B).

Metagenomics and nematode microbiota

The explosion of knowledge on the microbiota, i.e. the community of microbes associated with a host, and the consequences of its variations (e.g. chronic intestinal diseases following alterations of the human intestinal microbiota) are currently leading the microbiology pathology community to revise its vision based on the equation "a disease = a pathogen" and to broaden it to the notion of pathobiome, i.e. "a disease = a pathogen + a community of microbes that can influence the course and outcome of the disease".

We were interested in describing the bacterial community associated with entomopathogenic nematodes (EPNs) in addition to their symbionts Xenorhabdus or Photorhabdus. To do so, we designed tools adapted to describe the bacterial species associated with the infesting forms of EPNs, the IJs (Figure 2), by molecular metabarcoding approaches (Ogier et al, 2019) or culturomic approaches (Pagès et al, 2020).

Figure 2. Detail of an infesting form of entomopathogenic nematode or IJ (magnification x 400) with inter-cuticular bacteria that are sometimes observed, which could be members of the AFM.

We have identified a bacterial community of about ten cultivable species (mainly Proteobacteria) frequently associated with IJs (FAM for Frequently associated microbiota) (Figure 3). Within this community, the species Pseudomonas protegens is as entomopathogenic as the symbionts (Ogier et al, 2020). We will continue these studies to assess the potential role of each member of the IJs microbiota in the parasite cycle in the insect. This associated microflora is likely to be of importance for optimal efficacy of entomotoxic activities of nematobacterial complexes as biocontrol agents against crop pests. Our results have therefore operational implications for the production of these agents with a minimum maintenance of their microflora (in vitro production in fermenters or on insects).

Figure 3. Occurrence (x-axis) and high abundance frequency (y-axis) of OTUs (operational taxonomic units) associated with infesting forms of Steinernema carpocapsae. OTUs are described by metabarcoding with the 16S rRNA gene marker rpoB or V3V4.

Bibliography

Bisch, G., Ogier, J.-C., Médigue, C., Rouy, Z., Vincent, S., Tailliez, P., et al., 2016. Comparative genomics between two Xenorhabdus bovienii strains highlights differential evolutionary scenarios within an entomopathogenic bacterial species. Genome Biol Evol 8, 148-160. DOI : 10.1093/gbe/evv248.

Chaston, J.M., Suen, G., Tucker, S.L., Andersen, A.W., Bhasin, A., Bode, E., et al., 2011. The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes. PLoS One 6. DOI : 10.1371/journal.pone.0027909.

Kim, I.-H., Aryal, S.K., Aghai, D.T., Casanova-Torres, Á.M., Hillman, K., Kozuch, M.P., et al., 2017. The insect pathogenic bacterium Xenorhabdus innexi has attenuated virulence in multiple insect model hosts yet encodes a potent mosquitocidal toxin. BMC Genomics 18, 25 p. DOI : 10.1186/s12864-017-4311-4.

McMullen II, J.G., McQuade, R., Ogier, J.-C., Pagès, S., Gaudriault, S., Stock, P.S., 2017. Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts. Microbiology 163, 510-522. DOI : 10.1099/mic.0.000449.

Ogier, J.C., Calteau, A., Forst, S., Goodrich-Blair, H., Roche, D., Rouy, Z., et al., 2010. Units of plasticity in bacterial genomes: new insight from the comparative genomics of two bacteria interacting with invertebrates, Photorhabdus and Xenorhabdus. BMC Genomics 11, 568. DOI : 10.1186/1471-2164-11-568.

Ogier, J.-C., Pagès, S., Bisch, G., Chiapello, H., Médigue, C., Rouy, Z., et al., 2014. Attenuated virulence and genomic reductive evolution in the entomopathogenic bacterial symbiont species, Xenorhabdus poinarii. Genome Biol Evol 6, 1495-1513. DOI : 10.1093/gbe/evu119.

Ogier, J.-C., Duvic, B., Lanois, A., Givaudan, A., Gaudriault, S., 2016. A new member of the growing family of contact-dependent gowth inhibition systems in Xenorhabdus doucetiae. PLoS One 11, 19 p. DOI : 10.1371/journal.pone.0167443.

Ogier, J.-C., Pagès, S., Galan, M., Barret, M., Gaudriault, S., 2019. rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing. BMC Microbiol 19. DOI : 10.1186/s12866-019-1546-z.

Ogier, J.C., Pagès, S., Frayssinet, M., Gaudriault, S., 2020. Entomopathogenic nematode-associated microbiota: from monoxenic paradigm to pathobiome. Microbiome 8, 25. DOI : 10.1186/s40168-020-00800-5.

Pagès, S., Ogier, J.-C., Gaudriault, S., 2020. A novel semi-selective medium for Pseudomonas protegens isolation from soil samples. J Microbiol Methods, 105911. DOI : 10.1016/j.mimet.2020.105911.

Pantel, L., Florin, T., Dobosz-Bartoszek, M., Racine, E., Sarciaux, M., Serri, M., et al., 2018. Odilorhabdins, antibacterial agents that cause miscoding by binding at a new ribosomal site. Mol Cell, 70, 83-94.e7. DOI : 10.1016/j.molcel.2018.03.001.