The long-term objective of the proposed research is to develop a comprehensive molecular understanding of the organism Porphyromonas gingivalis, a species strongly associated with human periodontal diseases. P. gingivalis is a small genome representative of the little studied Cytophaga/Flavobacteria/Bacteroides phylum, a phylogenetically and medically important group of organisms. The complete genome sequence of virulent strain W83, about 2.2 Mb, will be determined (Aim 1). For sequencing, a 2.0 kb average insert size library will be prepared in the pUC18 vector and an 18. kb average insert size library in lambda DashII. Approximately 35,200 sequences from both ends of 17,600 pUC clones will yield approximately 8-fold coverage of the genome. The ends of 1,000 lambda DashII clones will also be sequenced to create a genome scaffold, and to verify the orientation and integrity of the contigs formed from the assembly of sequences from the plasmid library. The genome sequence will be assembled from the collection of randomly sequenced fragments; sequencing gaps will be closed by walking appropriate clones, and physical gaps by polymerase chain reaction. The sequence will be analyzed and annotated (Aim 2). Open reading frames (Orfs) will be identified related to proteins of known and unknown function. The sequence will be analyzed to identify a number of features, including metabolic pathways, virulence factors, and exported proteins. DNA structural motifs (such as promoters, replication origins and repeat elements) and genes for rRNA, and small RNAs, such as tRNA, 4.5S, 6S, 10S, and RNAse P, will be identified. The information from the annotated sequence will be integrated with experimental data from the P. gingivalis research community, including in vivo expression technology (Aim 3). The genome sequence and annotation data will be made available on the TIGR Microbial Database web site, as well as deposited in GenBank and other appropriate databases (Aim 4). The sequence information generated in this proposal will accelerate ongoing P. gingivalis research to identify and characterize virulence factors and to develop an effective P. gingivalis vaccine. Periodontal disease affects one in five Americans, and therefore, advances in prevention and therapy will have a significant effect on the health of the American people, and the cost of dental care.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE012082-02
Application #
2770267
Study Section
Genome Study Section (GNM)
Project Start
1997-09-01
Project End
2001-08-31
Budget Start
1998-09-01
Budget End
2001-08-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Institute for Genomic Research
Department
Type
DUNS #
City
Rockville
State
MD
Country
United States
Zip Code
20850
Hosogi, Yumiko; Duncan, Margaret J (2005) Gene expression in Porphyromonas gingivalis after contact with human epithelial cells. Infect Immun 73:2327-35
Chen, Tsute; Hosogi, Yumiko; Nishikawa, Kiyoshi et al. (2004) Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis. J Bacteriol 186:5473-9
Nishikawa, Kiyoshi; Yoshimura, Fuminobu; Duncan, Margaret J (2004) A regulation cascade controls expression of Porphyromonas gingivalis fimbriae via the FimR response regulator. Mol Microbiol 54:546-60
Duncan, Margaret J (2003) Genomics of oral bacteria. Crit Rev Oral Biol Med 14:175-87
Dashper, S G; Brownfield, L; Slakeski, N et al. (2001) Sodium ion-driven serine/threonine transport in Porphyromonas gingivalis. J Bacteriol 183:4142-8
Slakeski, N; Dashper, S G; Cook, P et al. (2000) A Porphyromonas gingivalis genetic locus encoding a heme transport system. Oral Microbiol Immunol 15:388-92
Califano, J V; Kitten, T; Lewis, J P et al. (2000) Characterization of Porphyromonas gingivalis insertion sequence-like element ISPg5. Infect Immun 68:5247-53
Rocha, E R; Owens Jr, G; Smith, C J (2000) The redox-sensitive transcriptional activator OxyR regulates the peroxide response regulon in the obligate anaerobe Bacteroides fragilis. J Bacteriol 182:5059-69
Chen, T; Dong, H; Yong, R et al. (2000) Pleiotropic pigmentation mutants of Porphyromonas gingivalis. Microb Pathog 28:235-47
Dashper, S G; Hendtlass, A; Slakeski, N et al. (2000) Characterization of a novel outer membrane hemin-binding protein of Porphyromonas gingivalis. J Bacteriol 182:6456-62

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