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

Abstract

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.

Molecular characterization of a locus required for hyaluronic acid capsule production in group A streptococci.

“…The 4.5-kb XbaI fragment hybridized with the pGAC106 probe and was therefore cloned into the pACYC184 plasmid using SalI and XbaI. The pACYC184 vector was chosen to bypass potential stability problems of this group A streptococcal insert in E. coli, as was reported by Kehoe and Timmis (34). Antibiotic sensitivity and DNA hybridization indicated that one recombinant plasmid, pGAC112, contained the proper insert.…”

The IgA‐binding β antigen of the c protein complex of Group B streptococci: sequence determination of its gene and detection of two binding regions

“…The reason for the deletions observed is not clear, but may be due to some instability of streptococcal DNA in E co//(e.g. see Kehoe and Timmis, 1984). High-level expression of the p antigen was only observed after thermo-induction with clone pPGJI (see Fig.…”

Biological Impact of a Large Scale Genomic Inversion that Grossly Disrupts the Relative Positions of the Origin and Terminus Loci of the Streptococcus pyogenes Chromosome

“…At that time, the chromosomal sequence of NZ131 had not been determined so gene Spy49_ 1180 was temporarily labeled as the Ex sequence, and gene Spy49_ 0145 was combined into the whole slo upstream sequence (17). The promoter-less ermB gene is silent because it is preceded by the chimerical Spy49_ 0145 -Spy49_ 1180 sequence in which a weak slo promoter found inside the Spy49_ 0145 gene (19, 20) is inactive due to the proximity of the Spy49_ 1180 sequence (17, 20). The insertion, selected by resistance to tetracycline, occurred via recombination between the plasmid and chromosomal Spy49_ 1180 sequences resulting in the strain OK173 (Fig.…”

“…1c). Since all experiments were performed in vitro (THY medium), the possibility remains that some unknown physiological factor might activate the transposed promoter or the previously identified weak slo promoter (19, 20) when synthesis of slo gene but not the nga gene is required.…”