Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme

Section of Periodontology 1 , Section of Molecular and Cellular Oncology and Microbiology 2 and Dental Hospital, Section of Periodontology 3 , Tokyo Medical and Dental University, Graduate School, 1-5-45, Yushima, Bunkyo-Ku, Tokyo 113-8549, Japan Correspondence Shinichi Arakawa shinichi.peri{at}tmd.ac.jp Received June 11, 2003 Accepted September 2, 2003 Tannerella forsythensis (previously named Bacteroides forsythus ) is a Gram-negative, anaerobic, fusiform bacterium that is a primary or secondary aetiological agent in periodontal disease in humans. T. forsythensis expresses several putative virulence factors, including a sialidase; however, there has been no molecular genetic characterization of this enzyme. A sialidase clone (pHI-1) was screened from a total of 455 recombinant clones of a genomic DNA library using the 2'- (4-methylumbelliferyl)- - D - N -acetylneuraminic acid (MUNeuAc) filter-paper spot assay. The sialidase gene ORF ( siaHI ) consists of a 1395 bp coding sequence and encodes a protein with 465 amino acids with an overall molecular mass of 52 kDa. The sialidase does not have sequence similarity to any other bacterial sialidase. The entire sialidase ORF was expressed in Escherichia coli . Furthermore, the sialidase was purified from the type strain of T. forsythensis and from a recombinant clone, pHI-1 : 1, and was analysed using a non-denaturing gel, revealing that the enzyme preparations were respectively separated as two major bands and as a single band. Southern blot hybridization analysis revealed similar patterns of siaHI -hybridizing bands among clinical isolates of T. forsythensis from periodontitis patients. This is the first study on the cloning and expression of a T. forsythensis sialidase gene and the purification of the SiaHI enzyme from T. forsythensis ATCC 43037 T and recombinant E. coli . Abbreviation: MUNeuNAc, 2'-(4-methylumbelliferyl)- - D - N -acetylneuraminic acid. The GenBank/EMBL/DDBJ accession number for the siaHI gene sequence of T. forsythensis ATCC 43037 T is AY069941.