Efficient Procedure for Purification of ObligateIntracellular Wolbachia pipientis and RepresentativeAmplification of Its Genome by Multiple-DisplacementAmplification

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Abstract
Bacteria belonging to the genus Wolbachia are obligatorymicroendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis , for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi , have been fully completed and six other genome sequencing projects are ongoing ( http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin ), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by multiple-displacement amplification from limited infected host tissue (0.2 g or 2 × 10 7 cells). We obtained sufficient amounts (8 to 10 μg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans , shares a similar overall architecture with its relative strain wMel.