Titre de la dissertation: « The microbial communities in calaminiferous soils ».

Promoteurs de thèse: Monsieur David Gillan et Monsieur Ruddy Wattiez

Résumé de la dissertation: Calaminiferous soils, or calaminarian grasslands, are open plant formations which colonize soils rich in metals (mainly zinc, cadmium and lead). They take their name from the town of La Calamine, in the east of Belgium, where various ores rich in zinc and lead were extracted. Metals have a significant influence on the composition and diversity of the flora and fauna of these areas. Unique and often entirely new biological processes with organisms resistant to high concentrations of these metals developed there. Surprisingly, very few studies on the microbiology of these soils have been performed.

The aims of the present work were (1) to investigate the structure of the microbial communities in the calaminiferous soil of Parc Péru, in Auby (Northern France), (2) to determine their main metal-resistance systems, and (3) to test the hypothesis that metal-sensible bacteria are present in the soil and may benefit from the protection of metal-resistant bacteria (bacterial cooperation).

For the first point, and to avoid as much biases as possible, an efficient molecular approach, metaproteogenomics was selected. The results showed that 7 bacterial genera were identified which represented the important members of the soil community. These genera were Mesorhizobium, Bradyrhizobium, Azospirillum, Variovorax, Nocardioides, Gaiella and Kouleothrix. These bacteria would be able to maintain important soil functions despite the presence of high metal levels. For the second point, metaproteomics indicated that the two main systems of metal resistance presented at the community level were HME-RND efflux systems homologous to CusA/CzcA, and P-type ATPases. Surprisingly resistance genes to tellurite were also expressed in the soils. Cultivation method was then used to isolate sensible and metal-resistant bacterial strains. Couples of strains obtained in Auby (1 metal-resistant, 1 metal-sensible) were then combined and studied in a drip-flow bioreactor in the presence of copper. The presence of the two strains was determined in the formed biofilm after various incubation times using methods such as PCR targeting the ITS spacer, cultivation, and fluorescent in situ hybridization. Results clearly indicate that metal-sensible bacteria are present in the soil and may benefit from the protection of metal-resistant bacteria in a dual-species biofilm. However, the most interesting dual-species biofilm formation could not be reproduced. Finally, a novel Cupriavidus campinensis strain was isolated in Auby. The genome of the strain (C19) was completely sequenced, assembled with SPAdes and annotated. Comparative genomic analyses were then performed with the other two sequenced campinensis strains (S14E4C and the type strain LMG19282). These comparisons indicate that strain C19 has a complete set of metal-resistance genes and important differences between the three strains were highlighted, such as the absence of mercury resistance operons (transposons) in strain C19, or its very limited set of accessory genes used for copper resistance (copTGJ).

Together these results suggest that calaminiferous soils feature very specific microbial communities and that metal-resistance might also be the result of cooperation between different bacterial species.