THE 46-KILODALTON-HEMOLYSIN GENE FROM TREPONEMA-DENTICOLA ENCODES A NOVEL HEMOLYSIN HOMOLOGOUS TO AMINOTRANSFERASES

The 46-kDa hemolysin produced by Treponema denticola may be involved i n the etiology of periodontitis. In order to initiate a genetic analys is of the role of this protein in disease, its gene has been cloned. S ynthetic oligonucleotides, designed on the basis of the previously rep orted amino-terminal amino acid sequence of the 45-kDa hemolysin, were used as primers in a PCR to amplify part of the hemolysin (hly) gene. This PCR product was then used to clone the entire hly gene from libr aries of T. denticola genomic DNA, Constructs containing the entire cl oned region on plasmids in Escherichia coil produced both hemolysis an d hemoxidation activities either on sheep blood agar plates or in liqu id assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis a nd Western blot (immunoblot) analysis revealed that the constructs syn thesized a protein with molecular size of about 46 kDa which was react ive with anti-T. denticola hemolysin. Nucleotide sequence analysis ind icated that the largest open reading frame could encode a protein with a calculated molecular size of 46.2 kDa. The first 31 amino acids enc oded by this open reading frame were identical to the experimentally d etermined amino-terminal sequence of the 45-kDa hemolysin. These resul ts indicate that the entire hly gene has been cloned. The deduced amin o acid sequence of the T. denticola hly gene is homologous (23 to 37% identity) to those of proteins that are members of a family of pyridox al-phosphate-dependent aminotransferases. This suggests that the 46-kD a hemolysin may be related to an aminotransferase and have a novel mec hanism of hemolysis. However, the functional aspects of this relations hip remain to be investigated.