|Wiens, Gregory - Greg|
|Welch, Timothy - Tim|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/3/2007
Publication Date: 5/2/2007
Citation: Wiens, G.D., Welch, T.J., Rexroad III, C.E., Lapatra, S., Call, D.R., Hunnicutt, D., Bhattacharyya, A., Campbell, J., Walunas, T. 2007. Genome Sequence of Flavobacterium psychrophilum Strain CSF 259-93 and Characterization of a Large Cluster of Genes Containing Short Repeats. Meeting Abstract Pg 12.
Technical Abstract: At the National Center for Cool and Cold Water Aquaculture, we have initiated an integrated approach to understand the pathogenesis and host response of rainbow trout to F. psychrophilum infection. As part of this program, and in cooperation with Clear Springs Foods Inc, we initiated a project to completely sequence the genome of F. psychrophilum strain CSF 259-93 in order to identify virulence factors and protective antigens. This strain was chosen as it is representative of lineage II strains causing significant disease in U.S. trout aquaculture, it is freely available, and a challenge protocol had been established. At the outset of this project, a single colony was isolated, virulence verified, and a large stock of DNA and viable bacteria were archived for future reference and challenge work. Genome sequencing and assembly was carried out by Integrated Genomics and a total of 40,527 shotgun sequence reads and 2966 fosmid sequences were assembled (8-fold sequence coverage). The completed genome consists of a single circular chromosome of 2,900,735 bp with a G+C content of 32.49%. A total of 87.96% of the genome is composed of open reading frames (ORFs) and 2634 ORFs were identified by Integrated Genomics ORF-calling software. The genome contains many transposase ORFs and they are associated with four regions of genome variability. A total of 1674 ORFs (63%) were assigned function and 1,086 ORFs are in asserted pathways. A particularly interesting set of 19 highly similar ORFs were identified that contain variable numbers (3 to 11 copies) of a 23 aa repeated motif. These ORFs were all in the same orientation and formed a large (21.5 kb) contiguous gene cluster. These repeats have homology to leucine-rich motifs while ends of these proteins are highly conserved with each other. A PCR assay was developed to distinguish between genes containing various numbers of repeats and this locus is highly polymorphic between strains. One of these ORFs, designated F. psychrophilum repeat protein N (frpN) was cloned and recombinant protein purified. This protein was weakly immunogenic in rainbow trout and did not induce a protective immune response against challenge. In summary, the genome of F. psychrophilum CSF 259-93 is relatively small compared to other sequenced bacteria from the Cytophaga-Flavobacterium-Bacteroides group and it contains abundant insertion elements that are associated with several regions of genome variability. The complete genome sequence is the first step toward a better understanding of metabolic and pathogenic processes, and the identification of potential vaccine candidates.