The overall objective of this research project is to obtain a complete sequence of the genome from the channel catfish pathogen Edwardsiella ictaluri. Specifically, we are proposing to: <OL> <LI> Construct random E. ictaluri strain 93-146 genomic libraries, conduct random shotgun sequencing to 8-fold coverage, and assemble the draft sequence<LI> Close sequence gaps and resolve ambiguities<LI> Annotate and release the complete E. ictaluri strain 93-146 chromosome sequence. </ol> Secondary objectives of this proposal are to 1) educate and train personnel at Mississippi State University and Louisiana State University in genome sequencing techniques and annotation through hands-on participation, and 2) disseminate knowledge from this project to the aquatic animal health research community and formulate plans and collaborative teams for future functional genomics research based on the E. ictaluri genome sequence.
NON-TECHNICAL SUMMARY: The disease caused by Edwardsiella ictaluri, enteric septicemia of catfish (ESC), is the most economically important disease of farm-raised channel catfish, which is the largest aquaculture industry in the United States. Genetic systems and challenge models are well established for E. ictaluri, but current investigations on how E. ictaluri causes disease are slowed by the need to clone and sequence individual genes. The purpose of this study is to obtain a finished, complete sequence of the Edwardsiella ictaluri genome using a collaborative effort between investigators that will provide sequencing expertise and investigators that will provide the biological expertise for this particular pathogen. Whole-genome sequencing of E. ictaluri will accelerate research on the pathogenesis of ESC, possibly allowing the development of an effective treatment or preventive method.
APPROACH: To obtain a finished, complete sequence of the 2.15 Mb genome of E. ictaluri strain 93-146, at least two plasmid libraries (2-4kb and 1-2kb average insert sizes) will be constructed and shotgun sequenced to approximately 8-fold coverage. Simultaneously, a cosmid-sized (ca. 40kb) large-insert library will be constructed, and approximately 1000 clones will be end-sequenced. This data will be assembled with the shotgun data from the plasmid libraries and will form a backbone upon which the shotgun data can be organized for final closure and annotation. The data from the shotgun-sequencing phase will be assembled using Phred/Phrap. Final closure will rely on PCR and primer walking to close gaps, with primer-directed finishing employed to raise sequence quality in low-coverage regions of the genome. Annotation will utilize the TIGR Annotation Engine followed by manual editing. E. ictaluri sequence data will be made available on the Laboratory for Microbial Genomics web site within one month after 3X coverage of the genome is achieved, with subsequent release of updated sequences on a monthly basis. A scientific workshop will be held in conjunction with the annual meeting of the Fish Health Section of the American Fisheries Society at the conclusion of the project for dissemination of knowledge through the aquatic animal health community and for team building for future functional genomics research.
PROGRESS: 2003/12 TO 2007/11<BR>
OUTPUTS: To obtain a finished, complete sequence of the 3.5 Mb genome of E. ictaluri strain 93-146, three plasmid libraries were constructed and used to complete shotgun sequencing to approximately 8-fold coverage. A small insert library was constructed in pSMART (Lucigen, Middleton, WI) (1-2kb average insert size), medium insert libraries were constructed in pBK-CMV (3-5kb average insert sizes), and a large insert library (ca. 40kb) was also constructed in pCC1FOS (Epicenter, Madison,WI). The data from the shotgun-sequencing phase was assembled using Phred/Phrap. To assist in closure, 19X coverage of 454 sequencing was conducted (70,616,471 bp with an average read length of 99 bp). To provide improved scaffolding, paired-end 454 reads were added to the assembly. Final closure utilized PCR and primer walking to close gaps, with primer-directed finishing employed to raise sequence quality in low-coverage regions of the genome. The genome was annotated with the TIGR Annotation Engine. Completion of manual editing, submission to GenBank, and publication will occur in 2008.<BR> PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine (MSU-CVM) was the project director. Dr. David W. Dyer at the Oklahoma University Health Sciences Center (OUHSC), Dr. Allison Gillaspy at OUHSC, and Dr. Ron Thune at the Louisiana State University School of Veterinary Medicine (LSU-SVM) were the project co-directors. Dr. Michele Williams conducted closure and annotation. Dr. Geoff Waldbieser, a molecular biologist with the Catfish Genetics Research unit of the USDA-ARS, ran closure reactions. Jenny Gipson, Jeremy Zaitshik, and Edgar Scott from OUHSC conducted shotgun sequencing, assembly, and database management, respectively. Christie Landry and Denise Fernandez at LSU-CVM assisted in conducting closure reactions. <BR> TARGET AUDIENCES: The genome sequence of Edwardsiella icatluri will benefit the catfish health research community. The genome sequence should stimulate new research on pathogenesis of enteric septicemia of catfish (ESC) and accelerate research on vaccine development for this disease. This research ultimately targets catfish producers to provide new solutions to help control ESC, which is their most important disease problem. <BR> PROJECT MODIFICATIONS: We used 454 Life Sciences, which is a next generation sequencing technology, to assist in closure of the genome.
IMPACT: 2003/12 TO 2007/11<BR>
The E. ictaluri sequence data was released in September 2004 and is being provided as assembled contigs on the Laboratory for Genomics and Bioinformatics website (http://micro-gen.ouhsc.edu/e ictal/e ictal home.htm). Three-fold coverage of the genome was reached on October 8, 2004, and 8X coverage was achieved on January 31, 2005. To assist in closure, 19X coverage of 454 sequencing was conducted (70,616,471 bp with an average read length of 99 bp). The genome was annotated with the TIGR Annotation Engine followed by manual editing, identifying a total of 3945 ORFs. Functional predictions for the ORFs indicate that the physiology of E. ictaluri is similar in many respects to other members of the Enterobacteriaceae, including Escherichia coli O157:H7, Salmonella enterica, Yersinia pestis, and Erwinia carotovora. However, E. ictaluri does have some unique features; for example, its genome contains a higher number of genes involved in DNA recombination, replication, and repair; protein stabilization; and two-component regulatory systems. This may indicate that E. ictaluri is adapted to survival in more stressful or diverse environments than the other Enterobacteriaceae species we compared it to. Interestingly, the genome had 203 transposable elements, which is considerably higher than the other four species. This is surprising because E. ictaluri only has one serovar and is generally considered to be phenotypically homogenous. By contrast, E. coli and S. enterica are each composed of more than 100 serovars and are relatively diverse species, yet E. ictaluri has more transposable elements than both species combined. Pathogenesis-related genes were identified, including type III secretion system genes, O polysaccharide biosynthesis genes, heme transport genes, genes encoding at least two types of fimbriae, afimbrial adhesins, and two chondroitinase genes. Multiple tandem flagellin genes are present, but no siderophore or capsular polysaccharide biosynthesis genes were found. Functional genomics studies utilizing the genome sequence are underway, and the genome sequence is being utilized to accelerate pathogenesis research at multiple institutions.