Efficient procedure for purification of obligate intracellular Wolbachia pipientis and representative amplification of its genome by multiple-displacement amplification.
Bacteria belonging to the genus Wolbachia are obligatory microendocytobionts 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 10(7) cells). We obtained sufficient amounts (8 to 10 microg) 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.
Mavingui
Patrick
P
Ecologie Microbienne UMR CNRS 5557-USC INRA 1193, Université Claude Bernard Lyon 1, Bāt. G. Mendel, 43 bd du 11 Novembre 1918, 69622 Villeurbanne, France. mavingui@biomserv.univ-lyon1.fr
Van
Van Tran
VT
Labeyrie
Estelle
E
Rancès
Edwige
E
Vavre
Fabrice
F
Simonet
Pascal
P
D000818:Animals; D001431:Bacteriological Techniques; D002460:Cell Line; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification*; D004330:Drosophila / Q000254:growth & development / Q000382:microbiology*; D016521:Electrophoresis, Gel, Pulsed-Field; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010063:Ovum / Q000382:microbiology; D017422:Sequence Analysis, DNA; D020577:Wolbachia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial