Progress 12/01/03 to 11/30/07
Outputs
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. 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. 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. PROJECT MODIFICATIONS: We used 454 Life Sciences, which is a next generation sequencing technology, to assist in closure of the genome.
Impacts
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.
Publications
- Williams, M. W., G. C. Waldbieser, D. W. Dyer, A. F. Gillaspy, and M. L. Lawrence. 2007 In press. Characterization of the rrn operons in the channel catfish pathogen Edwardsiella ictaluri. J. Applied Microbiol.
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Progress 01/01/06 to 12/31/06
Outputs
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 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). As of November 8, 2006, 41,306 sequencing reads have been generated with a 75.8% success rate, yielding 31,304 high quality reads.
Average read lengths have been 684 bp. The assembly currently contains 32 contigs >2 Kb in length; the genome size is 3.84 Mb with a G+C content of 57.4%. The genome was annotated with the TIGR Annotation Engine, identifying a total of 3945 ORFs.
Impacts
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. Another surprising finding was that E. ictaluri has fewer genes classified as unknown function or hypothetical than E. coli O157:H7.
Publications
- Williams, M. L., D. W. Dyer, A. F. Gillaspy, R. L. Thune, C. A. Landry, G. C. Waldbieser, S. C. Schuster, and M. L. Lawrence. 2006. The Edwardsiella ictaluri genome sequencing project. International Symposium on Aquatic Animal Health, San Francisco, California.
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Progress 01/01/05 to 12/31/05
Outputs
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 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. Closure and validation are in progress. As of January 31, 2006, 40,100 sequencing reads have been generated with a 75.2% success rate, yielding 30,167 high quality reads. Average read lengths have been 588 bp. E. ictaluri contains a large number of
insertion sequences and prophages, which suggests the potential acquisition of a number of genes through horizontal transfer.
Impacts
E. ictaluri is composed of a single serotype, and the species is also relatively consistent in biochemical reactions. The finding of a large number of insertion sequences and prophages may indicate that the E. ictaluri genome has undergone/is undergoing more changes than was realized. Upon completion of the annotation, we anticipate the identification of genes similar to known virulence genes from other gram-negative enterics, and we anticipate the discovery of novel virulence genes specific to a poikilothermic host.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
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 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 6X coverage was achieved on November 8, 2004. As of January 31, 2005, we reached 8.03X coverage with addition of fosmid sequences. Closure and validation are in progress. Overall, 27,819 sequencing reads have been generated with a 81.2% success rate, yielding 22,577 high quality reads. Average read
lengths have been 555 bp. Preliminary analysis of the assembled contigs indicates that E. ictaluri contains a large number of insertion sequences and prophages. Biologically, this is interesting because it suggests the potential acquisition of a number of genes through horizontal transfer.
Impacts
E. ictaluri is composed of a single serotype, and the species is also relatively consistent in biochemical reactions. The finding of a large number of insertion sequences and prophages may indicate that the E. ictaluri genome has undergone/is undergoing more changes than was realized. Upon completion of the annotation, we anticipate the identification of genes similar to known virulence genes from other gram-negative enterics, and we anticipate the discovery of novel virulence genes specific to a poikilothermic host.
Publications
- No publications reported this period
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