Inactivation of the streptococcal erythrogenic toxin B gene (speB) in Streptococcus pyogenes.
Streptococcal proteinase precursor (SPP) is a zymogen secreted by Streptococcus pyogenes that becomes activated to a cysteine proteinase. SPP has been shown to be immunologically identical to streptococcal erythrogenic toxin B (SPE B), and sequence comparison has shown a high degree of homology between the two proteins. In this study, we have constructed 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 recombinant plasmid was introduced into strain NZ131 by electrotransformation. Following the selection of erythromycin-resistant clones, Southern hybridization experiments confirmed the presence of the recombinant plasmid containing the erm gene in the chromosome of the resistant strains. Analysis of extracellular proteins produced by the wild-type and speB mutant strains by Ouchterlony immunodiffusion and isoelectric focusing revealed the presence of SPE B in the wild-type strain but not the speB mutant. Additionally, SPP, which has an isoelectric focusing pattern similar to that of SPE B and reacts with SPE B antiserum, was not detected among the extracellular proteins of the speB mutant strain. Proteinase activity as assayed by two different methods was present in the extracellular proteins produced by the wild-type strain, but the speB 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 that proteinase was not essential for viability as previously suggested. Our data are consistent with the view that a single gene (speB) produces a single protein that has been identified and/or assayed as either SPE B or SPP.
Chaussee
M S
MS
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Gerlach
D
D
Yu
C E
CE
Ferretti
J J
JJ
D001426:Bacterial Proteins; D001483:Base Sequence; D005098:Exotoxins / Q000032:analysis / Q000235:genetics*; D005798:Genes, Bacterial; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D013297:Streptococcus pyogenes / Q000235:genetics* / Q000472:pathogenicity
D001426:Bacterial Proteins; D005098:Exotoxins; D008565:Membrane Proteins; C117980:SpeA protein, Streptococcus pyogenes; C027951:erythrogenic toxin