INTRODUCTION

1. INTRODUCTION METHODOLOGY REFERENCE Walker, Jame R., Alexandra Blinkeva, and Philip Tucker. "CLOSTRIDIUM TAENIOSPORUM SPORES AND SPORE APPENDAGES AS SURFACE DISPLAY HOSTS, DRUG DELIVERY DEVICES, AND NANOBIOTECHNILIGICAL STRUCTURES." Patent Aplication Publication (2011): 1-40. Web. July 2012. Casjens, S., Palmer, N., Van Vugt, R., Mun Huang, W., Stevenson, B., Rosa, P.,et al. (2000) A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochaete Borrelia burgdorferi. Mol Microbiol 35: 490–516. Zhou, Y, Liang, Y., Lynch, K., Dennis, J.J., Wishart, D.S. 2011. “PHAST”: A Fast Phage Search Tool. Nucl. Acids Res. 39: (suppl 2):W347-352. Altschul S.F., Gish W., Miller W., Myers E.W. and Lipman D.J. (1990). Basic local alignment search tool. J. Mol. Biol. 215: 403-410.  Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2011) Geneious v5.4. Available from http://www.geneious.com • Table 1. Identified prophage sequences in PHAST from annotated sequences in C. taeniosporumscaffolds by Geneious Pro We have identified 3 distinct prophage sequences in the Clostridium taeniosporum genome assembly. Two of these sequences seem to be intact, one seems to have resulted from defective integration or excision events occurring during the prophage replication cycle (see Figure 1). Although the percent identity in the alignments are reduced; this could have resulted from lack of sequence information resulting from de novo genome sequencing as our scaffold assembly represents 97% of the potential genome (Hunicke-Smith, personal communication). The low identity might also be due to a gradual accumulation of genomic changes, and other integration events contributing to host-prophage coevolution. Additional support to our predicted prophage localizations include the ability to induce one of the prophages using mitomycin C, resulting in the identification of Clostridiumphage phi3626 (Blinkova, personal communication). Because the prophage’s identified sequence resulted larger than scaffold 13, we hypothesize that flanking sequences may be located in the 3% of the genome still unresolved by sequencing at the time of this study. Re-sequencing of some of these unresolved genomes segments have confirmed this prophage as an intact sequence (Cambridge, personal communication). Initial attempts to confirm positioning for Clostridium phage phiC2 resulted in an unconfirmed sequence in PHAST, however multiple staggered sections of scaffold 1 containing phage protein annotations resulted in the identification and confirmation of this defective phage integration event, upon their submission to PHAST (see Figure 2 B and C). ACKNOWLEDGMENTS RESULTS We would like to thank the National Science Foundation Advanced Technological Education ProgramNSF ATE DUE 0802508 “The Biotechnology Research Learning Collaborative (BRLC)”, the US Department of Education HIS-STEM Program P031C110190 STEM-TRAC Summer Research Program, and School of Science at Miami Dade for their support. A Identification of Prophages Sequences within theClostridium taeniosporumGenome Assembly DISCUSSION/CONCLUSION Moreno, Madayξ; Borrero, Katherineξ; Leon, Alfredoξ;Gines-Candelaria, Edwinξ; Walker, Jamesψ; S. Hunicke-Smithψ; Blinkova, Alexandraψ ξMiami Dade College Wolfson Campus, Miami, FL; ψUniversity of Texas at Austin, Austin, TX Misalignment of potential Clostridium phage phiC2 B Corrected alignment of potential Clostridium phage phiC2 C Bioinformatics tools such as Geneious Pro. 5.5.6, BLAST, and PHAST V 2 were utilized to predict and annotate possible coding sequences, phage proteins, and prophages. Geneious Pro. 5.5.6, a DNA and protein sequences assembly, analysis, and alignment software was used to predict the possible Open Reading Frames (ORFs) in the 18 scaffolds obtained from high-throughput 454 pyrosequencing of C. taeniosporum. The predicted ORFs were placed in the Basic Alignment Search Tool (BLAST) database from the National Center for Biotechnology Information (NCBI), which aligned the predicted ORFs to similar known sequences of other microbial genomes. Only phage proteins and other exogenous DNA sequences were annotated in Geneious Pro. 5.5.6. Regionscontaining high numbers of phage proteins and other exogenous DNA sequences were selected and assessedin PHAST (PHAge Search Tool), which aligns sequences to that of known phages topredict the phage identity and structure. The sequences of prophages identified by PHAST were accessed from the NCBI GenBank database in intact form and then re-aligned in Geneious Pro with their corresponding C. taeniosporum genome scaffold (see Table1).The realigned sequences were selected and reintroduced in PHAST. The latter allowed confirmation of prophage localization to the previously identified region of the scaffold and presumptive identification of the prophage(s). Clostridium taeniosporum is a Gram-positive, anaerobic, rod-shaped non-toxigenic bacterium. This bacterium is unique in forming endospores that display characteristic ribbon-like appendages that may be used as components for many products such as vaccines, fusion proteins, and drug delivery devices. (Walker et al., 2011). Different bioinformatics tools such as BLAST, PHAST V 2, and Geneious Pro 5.6.3 were used to scan for phage proteins, prophages, and their location within the genome respectively. Three prophages were predicted using PHAST but only Geobacillus virus E2 (40,863 bp) has been identified to be intact. If C. taeniosporum contains one or more prophages, using UV as a stressor will induce the prophages using Clostridium sporogenesand/or Bacillus cereus as indicator strains; which are close relatives of C. taeniosporum. Exogenous DNA sequences such as prophages, transposons, insertion sites (IS units), and plasmid insertions constitute a striking part of a bacterial genome. Prophages may constitute up to 20% of typical bacterial genomes (Casjenset al., 2000). These sequences might bring with them novel properties for the bacterial species such as antibiotic resistance or could also be mutagenic by interrupting protein gene coding sequences. Distinguishing which exogenous sequences are present and where they have been inserted will provide a framework to understand genomic function, variation, and evolution of C. taeniosporum. • Legend for consensus identity: • Dark green = 100% identity • Greenly-brown = Larger than 30% • smaller than100% identity • Red = smaller than 30% identity Figure 2. Realignments of prophage sequences identified in PHAST; accessed intact from the NCBI database and then realigned with corresponding C. taeniosporum genome scaffold by Geneious Pro v. 5.5.6. A. Realignment identified Clostridium phage phi3626 (accession # NC_003524) to a region in scaffold 13 with 53.3% identity and 7267 bp shorter than the prophage. B. Realignment identifiedGeobacillus virus E2 (accession # NC_009552) to a region in scaffold 1 with 50% identity. The prophageis located in coordinates from 1,082,060bp to 1,122,262bp in the forward DNA strand. C. Realignment identified Clostridium phage phi C2 (accession # NC_009231) to a region in scaffold 1 with 53.7% identity. The scaffold contains approximately 50% of the phage sequence integrated in coordinates 777,290bp to 805,322bp. The latter indicates defective integration event. Figure 1. The Bacteriophage Replication Cycle.