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Cloning and characterization of the genes encoding the haemolysin of Haemophilus ducreyi
Author(s) -
Palmer Katherine L.,
Munson Robert S.
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.18050821.x
Subject(s) - hemolysin , biology , haemophilus ducreyi , proteus mirabilis , microbiology and biotechnology , mutant , nucleic acid sequence , gene , molecular cloning , haemophilus influenzae , escherichia coli , genetics , peptide sequence , virulence , pasteurellaceae , antibiotics
We previously identified a heat‐ and protease‐labile haemolytic activity expressed by Haemophilus ducreyi . In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram‐negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore‐forming family of haemolysins. Tn 916 mutagenesis was employed to isolate haemolysin‐deficient mutants. Approximately 5000 Tn 916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35 000‐1, was further characterized. Sequences flanking the Tn 916 element in strain 35 000‐1 were employed to identify clones from a λDASHII library of H. ducreyi strain 35 000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35 000‐1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB , were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens .