Source: NORTHWEST FISHERIES SCIENCE CENTER submitted to
GENOME SEQUENCING OF THE VERTICALLY-TRANSMITTED FISH PATHOGEN RENIBACTERIUM SALMONINARUM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0198623
Grant No.
2004-35600-14173
Project No.
WNR-2004-00585
Proposal No.
2004-00585
Multistate No.
(N/A)
Program Code
23.2
Project Start Date
Dec 1, 2003
Project End Date
Nov 30, 2005
Grant Year
2004
Project Director
Strom, M. S.
Recipient Organization
NORTHWEST FISHERIES SCIENCE CENTER
2725 MONTLAKE BLVD. EAST
SEATTLE,WA 98112-2097
Performing Department
(N/A)
Non Technical Summary
Renibacterium salmoninarum, the causative agent of bacterial kidney disease, is a significant threat to the healthy and sustainable production of salmonid fish for consumption and species conservation efforts in the United States. Control of bacterial kidney disease is exceptionally difficult as there are no specific vaccines or approved therapeutics. The development of treatments are further complicated as R. salmoninarum is vertically transmitted from infected females to their eggs, is difficult to culture in vitro, and is a facultative intracellular pathogen. An understanding of the virulence mechanisms utilized by R. salmoninarum to cause disease in salmon, as well as the development of new and more effective vaccines or other therapeutics to control bacterial kidney disease, will be greatly aided by completing the sequencing of the R. salmoninarum genome. The goals of this project are to determine the complete nucleotide sequence of the R. salmoninarum ATCC strain 33209 and to make the sequence information and annotation freely and publicly available to the research community.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31140101160100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1160 - Pathology;
Goals / Objectives
Carry out the complete genome sequencing of the salmon bacterial pathogen Renibacterium salmoninarum. Identify open reading frames and carry out annotation of the genes based upon homologies to known bacterial genes in microbial genome databases. Upon completion of the project, deposit the sequence with annotated genes in the Genbank microbial genome database. Establish an undergraduate class in bioinformatics using the information garnered from the project. Host a workshop to present the findings of the project to interested parties in both the fish disease and broader scientific community.
Project Methods
A random, small-insert shotgun library will be constructed from R. salmoninarum DNA by standard genome sequencing methods and subjected to high throughput paired-end sequencing. Approximately 62,000 inserts will be sequenced to complete an 8x coverage of the 3.4 Megabase genome. Simultaneously, the sequences will be assembled into larger contiguous sequences (contigs) via a combination of Consed, PHRED, and PHRAP software to perform base calling and sequence assembly in an automated fashion. A large insert fosmid library will also be constructed to scaffold or order the contigs to correctly orient them. Fosmid clones that bridge gaps will then be sequenced to fill in the gaps using a primer-walking strategy. The finished sequence will be subjected to automated open reading frame (ORF) calling using CRITICA, followed by a preliminary assignment of gene names using the ERGO software suite. ERGO is a powerful annotation tool that performs a cross-genome comparative analysis to a large database of microbial genomes, and assigns functional names to the ORFs. After this stage, the computer-generated annotation of the genome will be subjected to manual annotation by examination of all potential open reading frames to correct any mistakes or to add information based on other genetic analyses. The annotated genome will then be reformatted and transferred to the Genbank Microbial Genomes database.

Progress 12/01/03 to 11/30/05

Outputs
During the 12/1/04 to 11/30/05 reporting period, the following progress was made. In January 2005, PIs Strom and Wiens attended and participated in the NSF/USDA-CSREES Microbial Genome Sequencing Program Awardees Workshop. Annotation of the first draft sequence continued using the Integrated Genomics ERGO bioinformatics suite. The genome was closed in October 2005 by the University of Washington Genome Center. 9 X sequence coverage was obtained from small-insert library sequencing and the genome assembled using PHRED/PHRAP/CONSED software tools. The genome was closed by four rounds of AUTOFINISH followed by manual assembly. A total of 52,686 sequence reads were incorporated in the final assembly and the gross assembly was validated by pulsed-field gel electrophoresis. The assembly was also validated to a ~1 kb resolution using end sequences and fingerprint data from an independently derived fosmid library and comparison to the virtual fingerprint pattern derived from the assembled sequence using the SEQTILE software tool. At this time, the genome was re-analyzed through ERGO. The genome of R. salmoninarum is a single, circular chromosome of 3,155,250 base pairs with 56.27% G+C content that encodes 3,667 open reading frames (ORFs). ERGO assigned functions to 2,333 of these ORFs (63.62%), and manual annotation has increased the number of ORFs with a functional designation to ~88%. The genome contains 69 IS994 and 9 novel ISRsa2 insertional sequence elements. R. salmoninarum possesses genes for glycolytic, pentose phosphate, TCA, and pyruvate cycle metabolic pathways and thus can utilize many sugars and polyols; however, only a limited number of transporters have been identified. The bacterium appears to be able to synthesize most amino acids including cysteine and serine indicating that the high cysteine requirement in the medium is for a purpose other than protein biosynthesis. The bacterium is able to utilize L-kynurine and also contains the genes encoding superoxide dismutase, catalase and thioredoxin peroxidase suggesting adaptation to intracellular survival within eukaryotic cells. The genome sequence has also revealed the presence of several antibiotic resistance factors, including many specifically involved in macrolide resistance. The latter finding has implications for current BKD treatment regimens and suggests possible reasons for the inability of macrolides to completely eliminate the bacterium in infected fish. A workshop titled "Bacterial Kidney Disease: Challenges for the 21st Century," sponsored in part by this grant and organized by the PIs, was held in Seattle WA November 15-17, 2005. At this international workshop (http://www.nwfsc.noaa.gov/bkd-conference), one day was devoted to the announcement of first findings obtained through the R. salmoninarum Genome Project. Outreach has also included an undergraduate class taught at Oregon State University that utilized the genome sequence as a primary training tool. The UW Genome Center has recently completed final reorienting of the genome. Following one more ERGO analysis, final annotation and GenBank submission is anticipated to be completed by early summer of 2006.

Impacts
Knowledge of the R. salmoninarum genome will serve as a foundation for identification of virulence factors, DNA vaccine gene candidates and improved diagnostic tests. In addition, the completed R. salmoninarum genome sequence will be a template for comparison with clinical and environmental isolates. Sophisticated differentiation techniques for R. salmoninarum isolates are needed and will aid management decisions to either treat infected fish populations with antibiotics or to destroy infected gametes. The complete genome sequencing of R. salmoninarum will represent the first genome sequence from the Micrococcaceae family that includes seven described genera. This will add to the expanding diversity of genome sequences available to all scientists.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
The grant period was initiated 12/1/03. During the 12/1/03 to 9/30/04 reporting period, the following progress was made. Disbursement of funds to collaborators and contractors identified in the budget were carried out: cooperative agreements with the University of Washington Genome Center (for genomic sequencing and assembly) and Oregon State University (co-PI Rockey for bioinformatics and education); interagency transfer agreement to the USDA-ARS National Center for Cool and Coldwater Aquaculture (co-PI Wiens, for project direction and bioinformatics); subcontract to Integrated Genomics, Inc. (for annotation and bioinformatics). In January 2004, PIs Strom and Wiens attended and participated in the NSF/USDA-CSREES Microbial Genome Sequencing Program (MGSP) Awardees Workshop, and presented a poster describing the project. A public project web site was launched in March, 2004 (http://micro.nwfsc.noaa.gov/rs-genome). Genome sequencing by the University of Washington Genome Center commenced in March 2004. A first draft sequence (7.5 x coverage) was received in August 2004 and submitted to Integrated Genomics for the first computerized annotation using the ERGO bioinformatics suite. This draft sequence was also posted on the project web site. PIs Strom, Wiens, and Rockey attended a training workshop at Integrated Genomics for the ERGO bioinformatics platform and began manual annotation of the draft genome sequence. PIs Strom, Wiens, and Rockey also presented summaries of current research interests and goals related to this project at the workshop. PI Rockey has initiated plans for a genomics and bioinformatics course for undergraduates to be held in 2005 at Oregon State University.

Impacts
Knowledge of the R. salmoninarum genome will serve as a foundation for identification of virulence factors, DNA vaccine gene candidates and improved diagnostic tests. In addition, the completed R. salmoninarum genome sequence will be a template for comparison with clinical and environmental isolates. Sophisticated differentiation techniques for R. salmoninarum isolates are needed and will aid management decisions to either treat infected fish populations with antibiotics or to destroy infected gametes. The complete genome sequencing of R. salmoninarum will represent the first genome sequence from the Micrococcaceae family that includes seven described genera. This will add to the expanding diversity of genome sequences available to all scientists.

Publications

  • No publications reported this period