ABSTRACT

1. LA LA LB LB LIC1 LIC1 LIC2 LIC2 OO OO Unique Unique Amount Amount Putative Gene Function Putative Gene Function Large chromosome sequence comparison between Pomona and Lai Table 1 Comparison of the large Pomona chromosome sequence with that of Lai and Copenhageni Small chromosome sequence comparison between Pomona and Lai Small chromosome sequence comparison between Pomona and Copenhageni Large chromosome sequence comparison between Pomona and Copenhageni 292 genes in the small chromosome of Pomona 3356 genes in the large chromosome of Pomona X X 7 123 unknown unknown Libraries Construction X X 2 46 19 hypothetical proteins 9 transposase and the other functional proteins 2 hypothetical proteins X 10 7 hypothetical proteins 1 putative sugar transport protein 1 elongation factor P 1 orotidine-5'-monophosphate decarbo -xylase X 64 47 hypothetical proteins 2 transposases and the other functional proteins Cloning into pUC18 Small-insert library (initial shotgun) Hydroshear (2-4Kb) X 52 40 hypothetical proteins 11 transposases 1 N-acetylglutamate synthase X 4 3 hepothetical proteins 1 S-adenosylmethionine decarboxylase X X 51 51 transposases X X 4 1 transposase 3 hypothetical proteins Genomic DNA X X 6 1 DNA binding protein 1 FimH-like protein 1 lipoprotein 1 3-oxoacyl-(acyl-carrier protein) reductase 2 glutamate-1-semialdehyde aminotransferase Cloning into pCC1FOS X X 6 1 spermidine synthase 1 transcriptional regulator 2 transposase 2 hypothetical protein Pipette (40Kb) Fosmid library Note: LA-the large chromosome of Lai LB-the small chromosome of Lai LIC1-the large chromosome of Copenhageni LIC2-the small chromosome of Copenhageni OO-the other organisms neither Lai nor Copenhageni Unique-unique to Pomona X X X X 259 3014 Genes in both Lai and Copenhageni Genes in both Lai and Copenhageni Note: LA-the large chromosome of Lai LB-the small chromosome of Lai LIC1-the large chromosome of Copenhageni LIC2-the small chromosome of Copenhageni OO-the other organisms neither Lai nor Copenhageni Unique-unique to Pomona Exgap representation of the Pomona sequencing to date. 75,457 reads have been collected and assembled into 28 contigs (>2kb) with a Phrap coverage of 8.1. Preliminary analysis of Leptospira interrogans serovar Pomona genome sequence Jiaxin Qu1, Fares Z. Najar1, Yung-Fu Chang2, and Bruce A. Roe1 1. Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019 2. College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 ABSTRACT In an effort to accelerate our understanding of the pathogenic mechanism of leptospires and facilitate the identification of novel vaccine candidates, we are sequencing the genome of Leptospira interrogans serovar Pomona. To date, ~76,000 sequencing reads have been collected, yielding ~8-fold shotgun coverage in 28 contigs, and an estimated genome size of ~ 5 Mb. About 5,000 ORFs can be annotated by comparison to Leptospira interrogans serovar Pomona and serovar Copenhageni. The central metabolic pathways of glycolysis / gluconeogenesis, pentose phosphate, citric acid cycle, fatty acid metabolism, oxidative phosphorylation, and ATP synthesis, as well as the biosynthetic pathways for 20 amino acids now have been reconstructed using KEGG and are identically conserved in Lai and Copenhageni. Preliminary analysis reveals that there are 292, 367, and 276 predicted genes in the small chromosome of Pomona, Lai, and Copenhageni, respectively. However, of the 292 Pomona small chromosome genes, only 7 are unique to Pomona. There are 3356, 4360, and 3454 predicted genes in the large chromosome of Pomona, Lai, and Copenhageni, respectively. However, of the 3356 Pomona large chromosome genes, only 123 are unique to Pomona. Therefore, although there is extensive connection of genomic organization among these 3 serovars, the observed difference likely account for their altered infection profiles. Table 2 Comparison of the small Pomona chromosome with that of Lai and Copenhageni Sequencing Strategy Bacterial genome Physical shearing(Nebulization) Subcloning in pUC18 and electro- transformation into E. coli XL1blue-MRF’ DNA sequencing using ABI 3700 Computer-generated contig alignment using Phred-Phrap Primer-walking using large-insert clones, and MPCR. Primers generated using MerMade Gap Closure Strategies Multiplex-PCR using split genes MPCR- using multiple pairs of primers are selected based on split genes (genes with one part in one contig and another part in an adjacent contig) between the ends of contigs to generate PCR products. • Uniplex-PCR using paired reads • Pairs of primers are picked using the PrimOU program based on the read pairs Fos-End Sequencing Sequences of the Fosmid ends are overlaid on the WGS assemble to order and orient the contigs. The gap-spanning fosmids then are picked for primer walking to close the gap. Current Results CONCLUSION Comparative genomic analysis reveals that Pomona, Lai and Copenhageni have the overall genetic similarity and their central metabolic pathways are conserved. However, Pomona has 130 unique genes (7 in the small chromosome, 123 in the large chromosome). These unique genes may play a role in maintaining the Pomona host specificity and its disease phenotype.