7/8/2014 ARS Project: GENOME SEQUENCING AND IDENTIFICATION OF VIRULENCE FACTORS IN FLAVOBACTERIUM PSYCHROPHILUM (408300) A...

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Research Project: GENOME SEQUENCING AND

IDENTIFICATION OF VIRULENCE FACTORS IN Project Team
FLAVOBACTERIUM PSYCHROPHILUM © Wiens, Gregory - Greg
Scott LaPatra - Principal

Location: Leetown, West Virginia * Investigator

2007 Annual Report Project Annual Reports
o FY 2008

1a.Objectives (from AD-416) o FY 2007

The objective of this cooperative research project is to identify °

virulence factors in the genome of Flavobacterium psychrophilum by o FY 2006

use of DNA sequencing. Flavobacterium psychrophilum is the o FY 2005

etiological agent of bacterial cold water disease. The disease causes °

significant production losses of rainbow trout and novel methods are o FY 2004

urgently needed for control of this disease. Knowedge of the F.
psychrophilum genome should serve as a foundation for future .
development of new vaccines, improved diagnostic tests, and novel Related National Programs

therapeutics. » Aquaculture (106)

1b.Approach (from AD-416)
Genomic DNA will be isolated from a virulent clone of Flavobacterium
psychrophilum strain CSF-259-93 and random, large-insert and small-
insert DNA libraries will be created. The DNA libraries will be used to
generate clones for 10,000 sequencing reactions for low-coverage
sequence analysis of the genome. Sequence data will be organized
into contigs and preliminary annotation will be performed using the
ERGO bioinformatics system developed by Integrated Genomics.
Preliminary annotation will allow assessment of the metabolic and
genetic organization of Flavobacterium psychrophilum. The partially
assembled genome sequence will be available on the NCCCWA web
site and a reference clone bank established.

3.Progress Report
This report serves to document research conducted under a Non-
Funded Cooperative Agreement between ARS and Clear Springs Foods
Inc. Bacterial cold water disease, caused by Flavobacterium
psychrophilum, is a serious problem for rainbow trout aquaculture.
Conventional approaches to vaccine development have been
ineffective and antimicrobial resistance is of concem. This year we
reported the completed genome of Flavobacterium psychrophilum.
The genome consists of a single circular chromosome of 2,900,735 bp
with a G+C content of 32.49%, A set of 19 highly similar genes were
identified that contain variable numbers of a short repeat. These
repeats have homology to leucine-rich motifs. A PCR assay was
developed to distinguish between genes containing various numbers
of repeats and this assay was used to show that this locus is highly
polymorphic between strains. One of these genes, 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.
The complete genome sequence is the first step toward a better
understanding of metabolic and pathogenic processes, and the
identification of potential vaccine candidates. The identified genes are
the basis of ongoing vaccine studies, The completion of the genome
sequence was the stimulus for a workshop titled Flavobacterium 2007
held in Shepherdstown WV on May 2-4th that was attended by over 70
scientists from eight countries. This work also addresses research
outlined in the National Program 106 Action Plan. Integrated Aquatic
Animal Health. Goal 1. Microbial Genomics. The ADODR is in frequent
contact with the cooperator through phone calls, email, and annual
meetings in addition to receipt of written reports,

Last Modifie

2 05/14/2009

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