Cloning and expression in Escherichia coli of the streptolysin O determinant from Streptococcus pyogenes: characterization of the cloned streptolysin O determinant and demonstration of the absence of substantial homology with determinants of other thiol-activated toxins.
A gene bank of Streptococcus pyogenes Richards was constructed in Escherichia coli by using the bacteriophage replacement vector lambda L47.1, and hybrid phage expressing streptolysin O (SLO) were identified among the recombinants. DNA sequences encoding SLO were subcloned from an slo+ hybrid phage into a low-copy-number vector plasmid to yield an slo+ hybrid plasmid, pMK157. This plasmid contains 5.6 kilobase pairs of cloned streptococcal DNA sequences, is stable, and expresses SLO at easily detectable levels in E. coli. Transposon gamma delta insertion mutants and in vitro-generated deletion mutants of pMK157 were isolated and analyzed. This analysis showed that a single gene is sufficient for production of SLO in E. coli and allowed this slo gene to be mapped to within +/- 100 base pairs. Two forms of the slo gene product, with molecular weights of 68,000 and 61,000, were detected in E. coli minicells harboring slo+ plasmids and by immunoblotting of E. coli whole cells harboring slo+ plasmids. Southern blotting hybridization experiments with the cloned SLO DNA sequences as probes failed to demonstrate homology between the cloned SLO determinant and DNA isolated from bacteria expressing thiol-activated cytolysins related to SLO.
D001426:Bacterial Proteins; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003429:Cross Reactions; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D010957:Plasmids; D013297:Streptococcus pyogenes / Q000235:genetics*; D013301:Streptolysins / Q000235:genetics* / Q000276:immunology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D013301:Streptolysins; C025964:streptolysin O; D004262:DNA Restriction Enzymes