ANALYSIS OF A 2ND BACTERIOPHAGE HYALURONIDASE GENE FROM STREPTOCOCCUS-PYOGENES - EVIDENCE FOR A 3RD HYALURONIDASE INVOLVED IN EXTRACELLULARENZYMATIC-ACTIVITY

The hyaluronidase gene (hylP2) from a second group A streptococcal bac teriophage was isolated from ATCC T-type-22 hyaluronidase-producing st rain 10403, a strain known to produce increased amounts of extracellul ar hyaluronidase. Sequence analysis of hylP2 and alignment with the pr eviously described bacteriophage hyaluronidase gene (hylP) showed a hi gh degree of similarity; however, hylP2 had deletions of regions speci fying 34 amino acids. Twenty-eight of the deleted amino acids were in a region of HylP containing a series of collagen-like Gly-X-Y repeatin g units. By employing primers for both hylP and hylP2, PCR amplificati on resulted in fragments of appropriate sizes in 97% of the strains te sted, with some strains producing two fragments, indicating the presen ce of at least two phages. When the hylP2 gene was introduced via a pl asmid vector into a non-hyaluronidase-producing Streptococcus pyogenes strain, this strain was still unable to produce extracellular hyaluro nidase, although intracellular hyaluronidase was present. These result s, along with the absence of a typical N-terminal signal peptide, indi cate that HylP2 is unable to be secreted into the extracellular milieu . Examination of more than 100 strains for production of hyaluronidase showed that only 23% of the strains produced extracellular hyaluronid ase. One of these strains (strain 10403) contains a single bacteriopha ge hyaluronidase gene (hylP2) which, when inactivated by allelic repla cement, still produces large amounts of extracellular hyaluronidase. T hese results suggest the presence of a different hyaluronidase gene en coding a protein that is actively secreted into the extracellular mili eu.