1. Testing Bacterial Proteins for Evidence of Horizontal Gene Transfer James Godde, John Iverson, Kabi Neupane, and Sara Penhale

2. Repetitive DNA Found in abundance in Eukaryotes Only 1% of the human genome encodes protein, while more than half of the genome consists of repetitive DNA. Relatively rare in Prokaryotes Nearly 89% of the E. coli genome encodes protein, while less than 1% consists of repetitive DNA

3. Different Classes of Repetitive DNA

4. CRISPRs What is a CRISPR? –Clustered Regularly Interspersed Short Palindromic Repeats –Class of repeats found exclusively in prokaryotes How widespread are they? Frequency of Occurrence Unknown What is their function? Function Unknown How did they get there in the first place? Mode of Transmission Unknown

5. What are Cas genes? In addition to the CRISPR sequences themselves, there are a number of genes usually found in close association with the regions of repetitive DNA These genes were termed Cas (CRISPR associated) genes There are 4 Cas genes which have been characterized to date. The function of each gene can be guessed at due to similarities they share with known genes: Cas 1 is homologous to a DNA repair gene Cas 2 is homologous to a transposase Cas 3 is homologous to a helicase Cas 4 is homologous to RecB exonuclease

6. Finding Cas genes Cas genes were found by using NCBI BLAST to search for homologs to previously characterized Cas genes (Jansen et al., 2002), as well as to any newly characterized ones In addition to homology with other genes, Cas genes had to be located near CRISPR sequences themselves

7. Cas 1Cas 2Cas 3 Cas 4

8. Formation of a total evidence tree Cas genes have been found in 115 different species of prokaryotes Analysis was limited to the 58 species for which sequence data were available for all four Cas genes Protein sequences for all Cas genes were concatenated and aligned using Clustal W Combined dataset was used to draw a neighbor-joining tree with MacVector

9. Classical rRNA-based Phylogeny Archaea Eukarya Bacteria Yang et al., 2005

10. Classical rRNA-based Phylogeny Archaea Bacteria Yang et al., 2005

11. Method:Neighbor Joining; Best Tree; tie breaking = Random Distance:Absolute (# differences) Gaps distributed proportionally Nanoarchaem Pyrococcus hor 1 Archaeoglobus 2 Methanobacterium Thermotoga Rubrobacter Clostridium ther Desulfobacterium 2 Thermoanaerobacter Fusobacterium Moorella 2 Porphyromonas Bacteroides Methanosarcina bar Methanosarcina acet Methanococcus Pyrococcus hor 2 Pyrococcus fur Chloroflexus Corynebacterium Chlorobium 2 Desulfovibrio desul Rhodospillium 1 Salmonella typhi CT18 Salmonella typhimurium E. coli K12 E. coli 0157 Geobacter sulf Photobacterium (mega) Sulfolobus tok Sulfolobus sol Archaeoglobus 1 Methanosarcina maz Leptospira (lai) Streptococcus pyo 1 Streptococcus aga 2603 Streptococcus aga NEM316 Streptococcus pyo 2 Streptococcus mut Moorella 1 Geobacter meta Methylococcus Magnetococcus Chlorobium 1 Desulfovibrio vul (mega) Shewanella (Sargasso Sea) Rhodospillium 2 Xanthomonas Chromobacterium Azotobacter Bacillus halo Desulfobacterium 1 Pyrobaculum aero Thermus HB8 (mega) Synechocystis (mega) Nostoc pun Nostoc 7120 200.0 492.327 531.322 431.986 525.293 462.82 400.18 503.823 461.177 396.724 443.276 420.201 444.953 397.047 466.496 491.409 397.048 356.952 261.984 364.747 229.253 590.433 549.823 407.176 461.936 230.257 240.755 233.245 344.971 367.033 364.967 350.083 410.917 361.245 511.755 310.026 443.945 384.555 324.992 276.008 356.965 340.257 327.743 410.048 300.729 329.271 370.48 328.255 243.306 215.778 228.222 410.47 414.53395.173 Archaea

12. Method:Neighbor Joining; Best Tree; tie breaking = Random Distance:Absolute (# differences) Gaps distributed proportionally Nanoarchaem Pyrococcus hor 1 Archaeoglobus 2 Methanobacterium Thermotoga Rubrobacter Clostridium ther Desulfobacterium 2 Thermoanaerobacter Fusobacterium Moorella 2 Porphyromonas Bacteroides Methanosarcina bar Methanosarcina acet Methanococcus Pyrococcus hor 2 Pyrococcus fur Chloroflexus Corynebacterium Chlorobium 2 Desulfovibrio desul Rhodospillium 1 Salmonella typhi CT18 Salmonella typhimurium E. coli K12 E. coli 0157 Geobacter sulf Photobacterium (mega) Sulfolobus tok Sulfolobus sol Archaeoglobus 1 Methanosarcina maz Leptospira (lai) Streptococcus pyo 1 Streptococcus aga 2603 Streptococcus aga NEM316 Streptococcus pyo 2 Streptococcus mut Moorella 1 Geobacter meta Methylococcus Magnetococcus Chlorobium 1 Desulfovibrio vul (mega) Shewanella (Sargasso Sea) Rhodospillium 2 Xanthomonas Chromobacterium Azotobacter Bacillus halo Desulfobacterium 1 Pyrobaculum aero Thermus HB8 (mega) Synechocystis (mega) Nostoc pun Nostoc 7120 200.0 492.327 531.322 431.986 525.293 462.82 400.18 503.823 461.177 396.724 443.276 420.201 444.953 397.047 466.496 491.409 397.048 356.952 261.984 364.747 229.253 590.433 549.823 407.176 461.936 230.257 240.755 233.245 344.971 367.033 364.967 350.083 410.917 361.245 511.755 310.026 443.945 384.555 324.992 276.008 356.965 340.257 327.743 410.048 300.729 329.271 370.48 328.255 243.306 215.778 228.222 410.47 414.53395.173 Proteobacteria

13. Conclusions The total evidence tree is a good representation of the individual Cas gene trees, and can be used to draw the same conclusions The trees support the hypothesis that Cas genes have been passed via horizontal gene transfer More work is required to eliminate the alternate hypothesis that the trees reflect convergent evolution in response to similar environments

14. Yang, S. Doolittle, R. F., and Bourne, P. E. 2005. Phylogeny determined by protein domain content. PNAS 102:373-378. Jansen, R., van Embden, J. D., Gaastra, W., and Schouls, L. M. 2002. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43:1565- 1575. References