L'assemblaggio e l'annotazione del genoma di un endobatterio di un fungo come punto di partenza per le analisi filogenetiche e l'identificazione dei profili metabolici.

Chronic bacterial infections that inflict no evident harm on their hosts are widespread among eukaryotes. In the past years, studies on insect endobacteria revealed that these microbes can influence several aspects of the host biology, from nutrition to defence and reproduction, are often inherited and can pass through host generations. They show varying degrees of genome reduction, biased nucleotide base composition and gene degradation as pseudogenes, and often are obligate mutualists required to the host development. The associations between fungi and bacteria also show high complexity but are characterized only in a small number, and bacteria associated to mycorrhizal fungi are currently the best studied. Arbuscular mycorrhizal fungi (AMF, phylum Glomeromycota) are soil fungi that form a symbiotic association with most land plants. AMF are known to possess endobacteria in their cytoplasm, which were first recognized on the basis of electron microscopy studies. One population of these endobacteria is restricted to the Gigasporaceae AMF family, is rod-shaped, related to Burkholderia, and is vertically transmitted. These endobacteria were named Candidatus Glomeribacter gigasporarum (CaGg). Studies on a fungal line from which these endobacteria had been removed demonstrated that CaGg contributes to the fitness of its host by improving the expansion and branching of pre-symbiotic hyphae. The characterisation of fungal endobacteria is very limited, due to their uncultivability in cell-free medium, and their biological significance is still unknown. To understand the metabolic processes underpinning the interaction of CaGg with its fungal host, it has been sequenced the genome of a homogeneous bacterial population thriving in the BEG 34 strain of Gigaspora margarita, an AMF which in turn is an obligate plant biotroph. Two complementary strategies were used: Sanger sequencing and 454 pyrosequencing. Together, these high-throughput strategies produced a large amount of sequences that need to be assembled to obtain the complete genome. The assembly process involved several steps of sequence assembly and mapping (Phred/Phrap and MIRA programs) and contig editing (Consed software). Contigs were experimentally validated and gap-closure was performed in silico and with PCR, while the annotation of all CDSs, phylogenetic inference and metabolic reconstruction were performed using several bioinformatic tools. The final assembly revealed a genome of ~ 1.72 Mb. Analysis of genomic features placed CaGg in the Burkholderiaceae, while metabolic network analyses clustered it with insect endobacteria. This positioning of CaCg among different bacterial classes reveals that it has undergone convergent evolution to adapt it to intracellular life-style. The genome annotation of this mycorrhizal-fungal endobacterium has revealed an unexpected genetic mosaic where typical determinants of symbiotic, pathogenic and free-living bacteria are integrated in a reduced genome. CaGg is an aerobic microbe, which has an extreme dependence on its host for carbon, phosphorus and nitrogen supply; it possesses secretion systems, and synthesizes vitamin B12, antibiotics- and toxin-resistance molecules, which may contribute to the fungal host's ecological fitness. This genome sequencing project promotes Candidatus Glomeribacter gigasporarum as a model in the study of mycorrhizal fungi/bacteria symbiosis.