WHOLE GENOME SHOTGUN SEQUENCING OF STREPTOCOCCUS MITIS

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DESCRIPTION (Adapted from the Investigator?s Abstract): The goal is to determine the complete genome sequence of the type strain of Streptococcus mitis, the predominant pioneer colonizer of tooth surfaces, and thus an initiator of dental plaque formation relevant to caries and periodontal disease. These pioneer streptococci facilitate or inhibit the establishment of late colonizers and oral pathogens. The predominance of IgA1 protease producing S. mitis variants in the pharynx is associated with allergic disease. S. mitis is a causative agent of endocarditits. It is an emerging source of septicemia in cancer patients, and the first vancomycin-resistant S. mitis isolate was reported in one such case. Phylogenetically, S. mitis and the overt pathogen S. pneumoniae, the causative agent of otitis, pneumoniae, septicemia and meningitis, are nearest neighbors. There is clear demonstration that the S. mitis type strain constitutes a reservoir of genetic determinants that contribute to penicillin resistance in pathogenic S. pneumoniae. The fear is that resistance to vancomycin will be transferred in a similar manner. The approach is a whole genome random sequencing strategy used successfully at TIGR to sequence twelve prokaryotic genomes. The yearlong project will consists of four phases: 1) Construction of small, medium and large insert size genomic libraries from S. mitis type strain, NCTC 12261. 2) Sequencing both ends of a sufficient number of small insert clones to provide 8-fold sequence coverage of the genome. Medium and large insert clones will be sequenced to 6-fold and 10-fold clone coverage, respectively. This will provide a sequence scaffold that will aid in verifying genome architecture. Further, 10-fold clone coverage from the large (50 kb) insert library will ensure a predicted sequence hit every 5,000 bp of the 2.2 Mb S. mitis genome. 3) Assembling the genome sequence from the collection of sequence reads, and closing remaining gaps. 4) Annotation of the genome sequence to identify all open reading frames, assignment of gene names and functional roles based on database similarity searches. Accomplishing this goal will provide a unique opportunity to compare the repertoire of genes in the commensal S. mitis to that of other streptococci, such as the highly virulent, type-4 S. pneumoniae strain, nearing completion at TIGR, allowing the identification of virulence-associated genes.