Total DNA enriched in bacterial endosymbionts was extracted from viscera of 20–30 adult female insects in sterile conditions and mechanically homogenized. In order to reduce insect DNA contamination, the samples were subjected to consecutive centrifugations at 1150 g and 1300 g for 10 minutes, and genomic DNA was obtained from the supernatant following a CTAB (Cetyltrimethylammonium selleck screening library bromide) extraction method [48]. Genome sequencing and assembly The purified genomic DNA was shotgun sequenced using 454/Roche GS-FLX Titanium technology at the Genomics and Health area of the Public Health Research Center (CSISP, Generalitat Valenciana). One half-plate

single-ends, and one-fourth plate paired-ends (3 kb of fragment size) sequencing experiments were performed, yielding a total of 1.3 million reads. Sequences of ARS-1620 eukaryotic origin were eliminated after a taxonomic assignation process by Galaxy [49]. Filtered reads were automatically assembled by MIRA [50] and the resulting

contigs were manually edited with the Gap4 program from the Staden package software [51]. The remaining gaps in the genome of M. endobia str. PCVAL were closed by ABI sequencing of PCR products obtained with designed primers, at the sequencing facility of the Universitat de València. Potential oriC on both genomes were sought with the OriginX program [52]. Total DNA samples obtained from the P. citri populations from Murcia and Almassora were used to further analyze the rplQ region PX-478 from

the T. princeps genome. The region comprised between genes rpoA and aroK was amplified and sequenced using the primers rpoA-F (5′-TGCCAGGCCTAGTGCTAAACATCA-3′) and aroK-R (5′-TGTCGCCAGGACTGCTATCAATGT-3′). Gene annotation and functional analysis ARAGORN [53], tRNAscan Staurosporine [54], and Rfam [55] sowftware packages were used for RNA genes prediction. Coding genes were annotated by BASys (Bacterial Annotation System, [56], RAST [57] and refined by BLAST searches [58]. Finally, functional domain studies in Pfam database [59] were performed when coding-genes functionality assessment was required. Artemis [60] and MEGA5 [61] programs were used for genome statistics calculation and codon usage analysis. Metabolic capabilities were analyzed with Blast2Go [62] and KAAS [63] programs. Functional information from the BioCyc [64], KEEG [65] and BRENDA [66] databases were also used in this context. Genome alignments were performed using MAFFT [67]. Annotated ORFs were considered as functional genes following two non-exclusionary criteria: the conservation of at least 80% of the sequence length of the closest orthologs found by BLAST in non-redundant databases, and/or the maintenance of the essential functional domains detected by Pfam [59]. Accession numbers The genome sequence of M. endobia strain PCVAL has been deposited at the GenBank (accession number CP003881).