In:
Infection and Immunity, American Society for Microbiology, Vol. 69, No. 2 ( 2001-02), p. 706-711
Abstract:
Brachyspira ( Serpulina ) hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs. The production of a beta-hemolysin has been considered a major virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein 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 the 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, tlyA to tlyC . The lack of homology between tlyA to tlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here. A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen a B. hyodysenteriae 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 been designated hlyA . A hemolysis-negative Escherichia coli 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-hemolysin. Based on sequence analysis, the translated protein had a pI of 4.3, an α-helical structure, and a phosphopantetheine binding motif. Hybridization 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 location of hlyA on the chromosomal map was distinct from the locations of tlyA , tlyB , and tlyC .
Type of Medium:
Online Resource
ISSN:
0019-9567
,
1098-5522
DOI:
10.1128/IAI.69.2.706-711.2001
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2001
detail.hit.zdb_id:
1483247-1
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