A GENE FROM RENIBACTERIUM-SALMONINARUM ENCODING A PRODUCT WHICH SHOWSHOMOLOGY TO BACTERIAL ZINC-METALLOPROTEASES

A genomic library constructed from Renibacterium salmoninarum isolate MT444 DNA in the plasmid vector pBR328 was screened using Escherichia coli host strain DH1 for the expression of genes encoding putative vir ulence factors. A single haemolytic clone was isolated at 22 degrees C and found to contain a 3.1 kb HindIII fragment of inserted DNA. This fragment was present in seven Defence Establishment, isolates of R. sa lmoninarum which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised aga inst either cellular or extracellular components of R. salmoninarum an d failed to identify any additional proteins compared to control E. co li containing pBR328. However, minicell analysis revealed that a polyp eptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for R. sa lmoninarum. The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 54 8 amino acids with a predicted molecular mass of 66757 Da and a pl of 5.57. The deduced amino acid sequence of the gene possessed strong sim ilarities to those of a range of secreted bacterial zinc-metalloprotea ses and was tentatively designated hly. Neither protease nor lecithina se activities were detectable in E. coli recombinants expressing gene hly. Haemolytic activity was observed from 6 degrees C to 37 degrees C for erythrocytes from a number of mammalian species and also from fis h. Gene hly was expressed in E. coli 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 pur ified by affinity chromatography. Antiserum raised against the purifie d fusion protein was used to probe Western blots of cell lysates and e xtracellular products from seven isolates of R. salmoninarum cultured under conditions of iron-sufficiency or iron-restriction. The results indicate that the availability of iron modulates the expression of the hly gene.