INACTIVATION OF THE STREPTOCOCCAL ERYTHROGENIC TOXIN-B GENE (SPEB) INSTREPTOCOCCUS-PYOGENES

Streptococcal proteinase precursor (SPP) is a zymogen secreted by Stre ptococcus pyogenes that becomes activated to a cysteine proteinase. SP P has been shown to be immunologically identical to streptococcal eryt hrogenic toxin B (SPE B), and sequence comparison has shown a high deg ree of homology between the two proteins. In this study, we have const ructed a speB mutant strain of S. pyogenes by insertional inactivation . An internal fragment of the cloned speB gene in plasmid pCR1000 was replaced with an erythromycin resistance determinant, and the recombin ant plasmid was introduced into strain NZ131 by electrotransformation. Following the selection of erythromycin-resistant clones, Southern hy bridization experiments confirmed the presence of the recombinant plas mid containing the erm gene in the chromosome of the resistant strains . Analysis of extracellular proteins produced by the wild-type and spe B mutant strains by Ouchterlony immunodiffusion and isoelectric focusi ng revealed the presence of SPE B in the wild-type strain but not the speB mutant. Additionally, SPP, which has an isoelectric focusing patt ern similar to that of SPE B and reacts with SPE B antiserum, was not detected among the extracellular proteins of the speB mutant strain. P roteinase activity as assayed by two different methods was present in the extracellular proteins produced by the wild-type strain, but the s peB mutant strain had no extracellular proteinase activity. The mutant strain had a growth rate similar to that of the wild-type strain and produced normal levels of other extracellular products, suggesting tha t proteinase was not essential for viability as previously suggested. Our data are consistent with the view that a single gene (speB) produc es a single protein that has been identified and/or assayed as either SPE B or SPP.