Cloning of a beta-hemolysin gene of Brachyspira (Serpulina) hyodyseneteriae and its expression in Escherichia coli

Brachyspira (Serpulina) hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs, The production of a beta-hemolysin has been considered a m ajor virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic pr otein sequence. Thus, questions still remain concerning the structural gene sequence of the hemolysin, To answer this question unequivocally, the beta -hemolytic toxin was purified from extracts of log-phase spirochetes, and t he N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K ) and compared with the translated sequences of previously cloned genes, tl yA to tlyC. The lack of homology between tlyA to tlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that i s reported here. A degenerate probe was designed based on the N-terminal am ino acid sequence of the purified beta-hemolysin and used to screen a B. hy odysenteriae genomic library. Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8. 93-kDa polypeptide containing the N-terminal sequence of the purified beta- hemolysin, To distinguish this gene from the tlyA to tlyC genes, it has bee n designated hlyA. A hemolysis-negative Escherichia coil strains containing hlyA was beta-hemolytic on blood agar media. Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of native B. hyodysenteriae beta-hemo lysin, Based on sequence analysis, the translated protein had a pI of 4.3, an alpha -helical structure, and a phosphopantetheine binding motif, Hybrid ization analysis of genomic DNA indicated that the hlyA gene was present in B. hyodysenteriae and B, intermedia but was not detected in B. innocens, B . pilosicoli, or B. murdochii under high-stringency conditions. The locatio n of hlyA on the chromosomal map was distinct from the locations of tlyA, t lyB, and tlyC.