Purification and partial genome characterization of the bacterial endosymbiont Blattabacterium cuenoti from the fat bodies of cockroaches.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
The present study showed successful purification and characterization of 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.
Tokuda
Gaku
G
Center of Molecular Biosciences (COMB), University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan. tokuda@comb.u-ryukyu.ac.jp.
Lo
Nathan
N
Takase
Aya
A
Yamada
Akinori
A
Hayashi
Yoshinobu
Y
Watanabe
Hirofumi
H