Umi Watanabe* – [email protected] * Corresponding authorsPublished: 25 November 2008 BMC Research Notes 2008, 1:118 doi:10.1186/1756-0500-1-Received: 14 October 2008 Accepted: 25 NovemberThis article is available from: http://www.biomedcentral.com/1756-0500/1/118 ?2008 Tokuda et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any VER-52296 site medium, provided the original work is properly cited.AbstractBackground: Symbiotic relationships between intracellular bacteria and eukaryotes are widespread in nature. Genome sequencing of the bacterial partner has provided a number of key insights into the basis of these symbioses. A challenging aspect of sequencing symbiont genomes is separating the bacteria from the host tissues. In the present study, we describe a simple method of endosymbiont purification from complex environment, using Blattabacterium cuenoti inhabiting in cockroaches as a model system. Findings: B. cuenoti cells were successfully purified from the fat bodies of the cockroach Panesthia angustipennis by a combination of slow- and fast-speed centrifugal fractionations, nylon-membrane filtration, and centrifugation with Percoll solutions. We performed pulse-field electrophoresis, diagnostic PCR and random sequencing of the shoutgun library. These experiments confirmed minimal contamination of host and mitochondrial DNA. The genome size and the G+C content of B. cuenoti were inferred to be 650 kb and 32.1 ?7.6 , respectively. Conclusion: The present study showed successful purification and characterization of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27607577 the genome of B. cuenoti. Our methodology should be applicable for future symbiont genome sequencing projects. An advantage of the present purification method is that each step is easily performed with ordinary microtubes and a microcentrifuge, and without DNase treatment.BackgroundMany eukaryotes have developed symbiotic relationships with bacterial endocytosymbionts [1,2]. These symbioses typically involve provision of nutrients by the bacterial partner to its host, and/or manipulation by the bacterial partner of the hosts’ reproductive system [3]. Prior to therecent genomic revolution, details of their metabolism and physiology of many of these bacteria remained unknown, in part due to the difficulty of culturing these bacteria. Genome analyses have provided a wealth of information that will be crucial for elucidating the biology of these endosymbionts. The focus has been on symbiontsPage 1 of(page number not for citation purposes)BMC Research Notes 2008, 1:http://www.biomedcentral.com/1756-0500/1/in the phylum proteobacteria [4-13], although a few representatives of the other phyla have recently been sequenced [14,15]. A challenging step in sequencing symbiont genomes is purification of the bacterial cells. In some cases, bacterial DNA that was directly extracted from accumulated bacteriocytes was pure enough for sequencing analyses [5,9]. However, in other cases, it was necessary to remove contaminated DNA derived from host nuclei and/or mitochondria. For example, purification of Blochmannia and Wolbachia required the precise adjustment of treatment conditions of DNase I in order to remove DNA of contaminated organelles [6,16]; failure of this treatment caused complete digestion of the bacterial genome [16]. This problem was avoided d.