Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products.
Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.
Yamamoto
K
K
Department of Bacteriology and Serology, Osaka University, Japan.
Ichinose
Y
Y
Shinagawa
H
H
Makino
K
K
Nakata
A
A
Iwanaga
M
M
Honda
T
T
Miwatani
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000378:metabolism; D014158:Transcription, Genetic; D014734:Vibrio cholerae / Q000235:genetics*
D003603:Cytotoxins; D004251:DNA Transposable Elements; D006460:Hemolysin Proteins; D021382:Protein Sorting Signals