Submitted to: American Society for Microbiology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/17/2009
Publication Date: 5/12/2009
Citation: Wiens, G.D., Lapatra, S.E., Call, D.R., Welch, T.J. 2009. Comparative genomics of the Flavobacterium psychrophilum Frp locus. American Society for Microbiology Meeting.
Technical Abstract: Flavobacterium psychrophilum is a Gram-negative, bacterium responsible for cold water disease and rainbow trout fry syndrome. These diseases are a source of significant economic loss in cool water aquaculture. Recently, the genome sequences of two strains, JIP02/86 and CSF259-93, have been determined. Both genomes contain a large cluster of putative cell surface proteins containing multiple identical or near identical repeats within each protein. We have designated this locus in strain CSF259-93 the Flavobacterium repeat protein (Frp) locus. The 23 aa repeats are similar in sequence to repeats found in the Tannerella forsythia leucine-rich repeat protein, BspA. Methods: The complete CSF259-93 genome sequence was determined and the Frp locus compared to the published JIP02/86 sequence. A degenerate PCR assay was developed to distinguish between ORFs containing different numbers of repeats and to also examine ORF transcription. Rabbit polyclonal antibody was generated against one protein, FrpN, and western blotting was used to probe culture supernatants and lysates for protein expression. Results: In strain CSF 259-93, the Frp locus formed a large (~21.5 kb) contiguous gene cluster comprised of 19 ORFs all in the same transcriptional orientation and each gene contained between 3 and 11 tandem repeats. The CSF259-93 locus differed in gene number, sequence and repeat structure from the JIP02/86 strain. Twenty-five additional strains were examined using degenerate PCR primers confirming that this locus is highly polymorphic between strains. Gene expression studies revealed simultaneous expression of multiple Frp ORFs in both broth and plate culture. Rabbit polyclonal antibody raised against FrpN bound to both secreted and bacterial-cell associated protein. Vaccination-challenge experiments employing purified recombinant FrpN demonstrated that FrpN is weakly immunogenetic in rainbow trout and failed to induce a protective immune response against intraperitoneal challenge. Conclusion: The Frp locus is highly polymorphic containing variation in gene number, organization and repeat copies.