Cloning and Characterization of the dagB Gene from Treponema denticola

Objectives: Treponema denticola is one of the major pathogens associated with adult periodontitis. The surface protease, dentilisin, is a potential pathogenic factor of these microorganisms. Previous results indicated that dentilisin co-purified with both 43kDa (DagA) and 38kDa proteins. In addition, dagA and the dentilisin gene, prtP, are part of the same operon. To examine the relationship of the 38 kDa protein gene with this operon, further analysis of the dentilisin operon was carried out. Methods: Northern blot analysis was performed with the prtP probe labeled with digoxigenin. To identify transcription start sites, RT-PCR using the SMART system (Clontech) was performed. The DNA sequence of the amplified sequence was determined using an ABI PRISM 310-2 DNA sequencer. Results: Northern blot analysis indicated that the mRNA coding for dentilisin is approximately 5 kb. This size is 1.5 kb larger than that of dagA and prtP. The products amplified by RT-PCR with primers designed from the 5'-ends of prtP and dagA were approximately 2.1kb and 1kb. This difference in size was consistent with the positions of the primers and indicated that the operon transcriptional start site was located 1kb upstream of the dagA gene. Sequence analysis of the amplified sequence revealed that an open reading frame (designated dagB) existed immediately upstream of dagA and coded for a 206 amino acid protein with a molecular mass of 22,674.85. Conclusions: The present study suggests that the dagB, dagA and prtP genes are translated from a polycistronic mRNA and dagB may play an important role in dentilisin activity. The relationship between dagB and the 38 kDa protein is currently under investigation. Supported by NIH grant DE09821.