A genomic library constructed from isolate MT444 DNA in the plasmid vector pBR328 was screened using host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 °C and found to contain a 31 kb dlll fragment of inserted DNA. This fragment was present in seven isolates of which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of and failed to identify any additional proteins compared to control containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for . The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 557. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designated Neither protease nor lecithinase activities were detectable in recombinants expressing gene Haemolytic activity was observed from 6 °C to 37 °C for erythrocytes from a number of mammalian species and also from fish. Gene was expressed in as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of cultured under conditions of iron-sufficiency or iron-restriction. The results indicate that the availability of iron modulates the expression of the gene.


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