Abstract
The parasitic nematode,
Brugia malayi, causes lymphatic filariasis in humans, which in severe cases leads to the condition known as elephantiasis. The parasite contains an endosymbiotic α-proteobacterium of the genus
Wolbachia that is required for normal worm development and fecundity and is also implicated in the pathology associated with infections by these filarial nematodes. Bacterial artificial chromosome libraries were constructed from
B. malayi DNA and provide over 11-fold coverage of the nematode genome.
Wolbachia genomic fragments were simultaneously cloned into the libraries giving over 5-fold coverage of the 1.1
Mb bacterial genome. A physical framework for the
Wolbachia genome was developed by construction of a plasmid library enriched for
Wolbachia DNA as a source of sequences to hybridise to high-density bacterial artificial chromosome colony filters. Bacterial artificial chromosome end sequencing provided additional
Wolbachia probe sequences to facilitate assembly of a contig that spanned the entire genome. The
Wolbachia sequences provided a marker approximately every 10 kb. Four rare-cutting restriction endonucleases were used to restriction map the genome to a resolution of approximately 60 kb and demonstrate concordance between the bacterial artificial chromosome clones and native
Wolbachia genomic DNA. Comparison of
Wolbachia sequences to public databases using BLAST algorithms under stringent conditions allowed confident prediction of 69
Wolbachia peptide functions and two rRNA genes. Comparison to closely related complete genomes revealed that while most sequences had orthologs in the genome of the
Wolbachia endosymbiont from
Drosophila melanogaster, there was no evidence for long-range synteny. Rather, there were a few cases of short-range conservation of gene order extending over regions of less than 10 kb. The molecular scaffold produced for the genome of the
Wolbachia from
B. malayi forms the basis of a genomic sequencing effort for this bacterium, circumventing the difficult challenge of purifying sufficient endosymbiont DNA from a tropical parasite for a whole genome shotgun sequencing strategy.