CLONING AND CHARACTERIZATION OF THE GENES ENCODING THE HEMOLYSIN OF HAEMOPHILUS-DUCREYI

We previously identified a heat- and protease-labile haemolytic activi ty expressed by Haemophilus ducreyi. In order to characterize the haem olysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram-negative organisms. The haemolys in genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia p ore-forming family of haemolysins. Tn916 mutagenesis was employed to i solate haemolysin-deficient mutants. Approximately 5000 Tn916 transpos on mutants were screened for the loss of haemolytic activity and two m utants were identified. One mutant, designated 35000-1, was further ch aracterized. Sequences flanking the Tn916 element in strain 35000-1 we re employed to identify clones from a lambda DASHII library of H. ducr eyi strain 35000 DNA. A 13 kb insert from one lambda clone was selecte d for further study. This 13 kb fragment was able to both confer haemo lytic activity to Escherichia coli and complement the haemolysin defic iency in strain 35000-1. The haemolysin gene cluster was cloned from t his 13 kb insert and two genes, designated hhdA and hhdB, were identif ied. The derived amino acid sequence of these genes demonstrated homol ogy to the haemolysin and activation/secretion proteins of P. mirabili s and Serratia marcescens.