Copyright C 1992, American Society for Microbiology Functional Analysis of the Ca2+-Regulated Hemolysin I

The genetic determinant encoding the synthesis and secretion of hemolysin I (HlIy; gene designation, hiyl) by ActinobaciUilus pleuropneumoniae serotype 1 4074T was cloned in the lambda vector EMBL4. A 10.2-kb fragment that encoded hemolytic activity in the phage lysate was aligned by Southern blot hybridization to genes hlyC, hlyA, hlyB, and hblyD of the Escherichia coli hemolysin operon, and expression of the A. pleuropneumoniae genes in E. coli revealed that they have the same functions as their E. coli analogs: hlyIC encodes a protein that activates inactive 105-kDa prohemolysin I (encoded by hlyL4) to active hemolysin I, while hlyIB and hlyID are necessary for H1yIA secretion. Northern (RNA) hybridization of A. pleuropneumoniae RNA revealed that the gene cluster is transcribed as two RNA species, a major one of 3.5 kb, corresponding to hlyIC4, and a second, minor one of 7.5 kb, corresponding to the whole operon, hlyICABD. The level of hlyI mRNA was substantially higher inA. pleuropneumoniae 4074T cells grown in the presence of Ca2+, supporting the view that the expression ofthe hlyI determinant is Ca2+ regulated. Parallel RNA hybridization with random gene probes suggested that this Ca2+ regulation is specific for the hdyI determinant. Actinobacillus pleuropneumoniae, a gram-negative bacte-rium belonging to the family Pasteurellaceae, is the cause of porcine pleuropneumonia (38), and the strong hemolyti