Fish-pathogenic aeromonads produce several extracellular toxins of which hemolysin is thought to be one of the important virulence factors. In this study, the DNA structure and characterization of the hemolysin genes from Aeromonas hydrophila, A. salmonicida, and A. sobria have been examined.
Nine hemolysin genes have been cloned and their nucleotide sequences determined. Two(AHH1, AHH2) were from A. hydrophila strain ATCC7966, two(AHH3, AHH4) were from A. hydrophila strain 28SA, one(AHH5) was from A. hydrophila strain AH-1, one(ASH1) was from A. salmonicida strain ATCC 14174, two(ASH3, ASH4) were from A. salmonicida strain 17-2, and one(ASA1) was from A. sobria. The nucleotide sequences of the open reading frames and predicted molecular mass of the nines respectively were 1,731 base pair(bp) and 63,658 Daltons(Da), 978 be and 37,798 Da, 1,476 bp and 54,177 Da, 1476 bp and 54,159 Da, 1563 bp and 53,779 Da, 1716 bp and 64,780 Da, 1,467 bp and 54,188 Da, 1,734 bp and 63,400 Da, and 1,464
bp and 53,920 Da. The molecular mass predicted from the nucleotide sequences were similar to that of the molecular mass from maxicell analysis, in all except AHH2.
Hemolysin genes were classified into two groups depending on their nucleotide sequences. AHH1 and ASH4 belonged to one group which contained part of a homologous sequence of the hemolysin genes of Vibrio cholerae E1 Tor and V. vulnificus. This was designated group I and possessed the two highly conservative regions, and the location of cystein residue was conserved. regions may be important in the control of hemolytic activity. The other, group II is the previous reported aerolysin gene from A. hydrophila and A. trota, and AHH3, AHH4, AHH5, ASH3, and ASA1 were classified into this group. However AHH2 and ASA1 had no significant sequence homologies with those of the bacterial hemolytic proteins present in GENETYX-CD data bases.
AHH1 gene(group I) and the AHH5 and ASA1 genes(group II) were widely distributed in aeromonads.
Hemolysin was secreted by Escherichia coli containing each of the cloned genes except AHH2. Sensitivity to lysis by the cloned hemolysins was different for various species of erythrocytes as demonstrated by hemolysis on blood agar plates. Less