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Applied and Environmental Microbiology, November 2005, p. 6910-6917, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6910-6917.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Efficient Procedure for Purification of Obligate Intracellular Wolbachia pipientis and Representative Amplification of Its Genome by Multiple-Displacement Amplification

Écologie Microbienne UMR CNRS 5557-USC INRA 1193,¹ Biométrie et Biologie Évolutive UMR CNRS 5558, Université Claude Bernard Lyon 1, Villeurbanne, France²

Received 24 December 2004/ Accepted 27 June 2005

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 x 10⁷ 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.

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