Role of the Photorhabdus Dam methyltransferase during interactions with its invertebrate hosts

Abstract

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.

Fig 1. Methylation-sensitive restriction enzyme (MSRE) PCR analysis.

(A), MboI restriction of a DNA region with a methylated (grey box with black circles) or unmethylated (grey box) GATC site, followed by PCR amplification. (B), PCR amplification of a locus harboring a previously found unmethylated GATC site (chromosomal position 10531) was performed on P. luminescens WT or Chr_dam strains DNA digested by MboI or Bsp143I (which digests GATC sites, whatever the methylation state). Detection of a 114 bp amplicon revealed that no digestion occurred.

Fig 2. Nemato-bacterial complex pathogenicity by infestation.

(A) Survival of G. mellonella larvae after infestation by 10 nematodes associated with Chr_gfp bacterial strain (green) or Chr_dam strain (blue). A significant difference of 2 hours was observed for the time needed to kill 50% of the larvae between the two strains (Wilcoxon, p-value<0.05). (B) Survival of S. littoralis larvae after infestation as described above. A significant difference was observed with an almost 6 hours delay for the Chr_dam strain (Wilcoxon, p-value<0.001).

Fig 3. Number of emerging IJs from each cadaver.

(A) Emerging IJs from each G. mellonella cadaver for each strain. The amount of IJs exiting from larvae cadaver were not significantly different between the two strains (Wilcoxon, p-value = 0.991). (B) Emerging IJs from S. littoralis larvae cadaver for each strain. The amount of IJs exiting from larvae cadaver were not significantly different (Wilcoxon, p-value = 0.31).

Fig 4. CFU in IJs nematodes for each strain.

After crushing of 10 IJs and plating of the resulting suspension, CFU were numerated. A significant difference was observed between the two strains (glmm, p-value<0.01).