Create 3 different phylogenetic trees from the alignments Evolutionary Divergence of the Origin of Replication of Various Group II Clostridium Vidal, Kevin§, Hunicke-Smith, S.¥, Blinkova A.¥, Walker J.R.¥, León A.J.§, and Ginés-Candelaria E.§ §Miami Dade College-Wolfson Campus, Department of Natural Sciences, Health & Wellness, Miami, FL 33132 ¥University of Texas at Austin, School of Biological Sciences, Section of Molecular Genetics and Microbiology, Austin TX 78712 Discussion Based on results gathered from the phylogenetic trees constructed from multi-wise alignments from full replication origin regions, we observed a relative high homology between Clostridium taeniosporum and the Clostridium botulinum strains E3 Alaska and B strain Eklund, namely 85.5% and 89.5%, respectively. This result contrasts with the relative relatedness at the 70% level with other Group II strains (see Table 1). Also interesting is the homology score for strain BKT015925, when the orientation of the origin was reversed to correspond to the orientation of the remaining origins. Figure 1 illustrates the actual phylogenetic tree produced by Geneious Pro v6.1.5 for the full replication origin regions the displays the substitutions per site of each genome. We could preliminary infer that the lesser the score value the lesser the substitutions detected. As revealed from the phylogenetic tree, Clostridium taeniosporum had only 0.0405 substitutions per site with regards to the node connecting it to the strains Clostridium botulinum E3 Alaska and Clostridium botulinum B strain Eklund. This means that the origin of replication of Clostridium taeniosporum has only diverged from those strains by .0405 substitutions per site. This result, along with percent identity values shown in table 1, seem to indicate that Clostridium taeniosporum is most closely related to Group II Clostridium botulinum strains E3 Alaska and B strain Eklund. The latter result seems to be supported when we observed the percent identities between the short-sequence dnaA box region 3 detected by OriFinder of the various Group II C. botulinum strains with that same region 1 of Clostridium taeniosporum. A similar outcome results when we analyzed the long-sequence dnaA box regions 2. Region 3 was also run in each alignment to verify our hypothesis that it may not contribute to the activation of the origin of replication in Clostridium taeniosporum. Thus suspicion arose when looking at the annotated genome of Clostridium taeniosporum and seeing that while region 1 and 2 are located in the vicinity of the dnaA gene operon that includes dnaN, recF and gyrB, region 3 is located about 20kbp upstream near the annotation for the gene encoding the ammonium transporter. Whether this represents a factual possibility will require further testing. As seen from table 2 the strains with the highest percent homology to region 1 is Clostridium botulinum E3 Alaska and Clostridium botulinum B strain Eklund, with a percent homology of 71.4 and 70.6, respectively, while the homology between these same two strains with region 3 is only 14.9 and 16.1, respectively. A similar result was obtained when exploring the percent homology for the long-sequence dnaA box region 2, which exhibits higher homology with the long regions than region 1. The latter result, along with the others observed by this study supports our hypothesis that region 1 perhaps may not contribute to the function of the origin in addition of confirming results obtained by rDNA reported by Iver, et al., 2008, which is also confirmed with the reduction in substitutions per site (0.0443) for the branch connecting C. taeniosporum origin to that of the node that links C. botulinum E3 str. Alaska and C. botulinum B str. Eklund 17B. References /Acknowledgements Feng Gao and Chun-Ting Zhang (2008) Ori-Finder: a web-based system for finding oriCs in unannotated bacterial genomes. BMC Bioinformatics.9:79. Geneious v6.5.1 created by Biomatters. Available from http://www.geneious.com/ Iyer A.V. et al. 2008. Clostridium taeniosporum is a close relative of the Clostridium botulinum Group II. Anaerobe 14:318-324. We would like to thank the National Science Foundation Advanced Technological Education Program NSF ATE DUE 0802508 “The Biotechnbology Research Learning Collaborative” (BRLC) and the US Department of Education, HIS-STEM Program P031C110190 STEM-TRACK for their support of this research project. We would like to thank Dr. Edwin Ginés-Candelaria and Professor Alfredo León for their guidance during this project. Introduction Clostridium taeniosporum is an anaerobic, Gram positive bacterium that is nonpathogenic, which has been found to be very closely related to Clostrodium botulinum Group II strains, some of which produce very potent neurotoxins. Despite this close relationship, C. taeniosporum produces endospores with distinctive such as the spores that are formed by Clostridium taeniosporum large, flat, ribbon-like appendages (Iyer, A.V. et al., 2009). Another key distinction is the absence of the toxin produced by some members of their group. The purpose of this study is to determine the evolutionary divergence of clostridial replication origins from the various strains in Group II to reveal if there were any changes in replication strategy from those observed in the Firmicutes. The strategy employed required the localization of the origins of replication for various clostridial strains in Group II directly from their annotated genomes. These sequences were extracted from the National Center for Biotechnology Information (NCBI) depository GenBank. These origins were then aligned compared to the replication origin of Clostridium taeniosporum (Thompson, J., Bode, A., Shrenker, N., et al., personal communication) using CLUSTALW Multi-wise nucleotide alignment software. The resulting alignment was to generate a phylogenetic tree for all the origins. We employed Escherichia coli as the Enterobacteriaceae out-group control. From this preliminary phylogenetic tree, we could observe that Clostridium taeniosporum is most closely related to the Clostridium botulinum group II strains E3 Alaska and B Eklund, thus confirming results obtained by Iyer, et al., using rDNA analyses. Using this information may provide us greater insight into the evolutionary divergence of replication strategies in Clostridium taeniosporum and ultimately, the Firmicutes. Results Figure 1. Phylogenetic tree obtained from CLUSTALW alignments of the complete origins of replication for various Group II clostridia. Materials & Methods The project began by extracting the sequences of the dnaA box regions for the various Clostridium strains along with the Escherichia coli out-group control using the Bioinformatics software OriFinder. The extracted long (ranging from 378-1074 nucleotides in length) and short (260-432 nucleotide range) dnaA box regions from selected Group II clostridia and E. coli were then entered into Bioinformatics software Geneious Pro v6.1.5 to perform a CLUSTAL W multi-wise sequence alignment. The alignment was used to generate a phylogenetic tree for the dnaA box region of origin activation. A similar approach was employed for the complete replication origin where each region was extracted by using the region of approximately 10-15 Kbps encompassing two indicator genes: From gidA (encoding the glucose-inhibited division protein A) to the gyrA (encoding the gyrase subunit A), with the exception of C. taeniosporum where this region consisted of approximately more than 35 Kbps. Extract long and short dnaA box regions on OriFinder for Group II Clostridium botulinum, Clostridium taeniosporum, and Escheria coli Find complete origin of replication for each group of strains using origin indicator genes Run a multiple sequence alignment for the complete origin, and dnaA box long and short regions Create 3 different phylogenetic trees from the alignments Examine the distances and the percent homology of each strain to determine preliminary evolutionary relatedness in Group II clostridia