INTRODUCTION OF THE EMM6 GENE INTO AN EMM-DELETED STRAIN OF STREPTOCOCCUS-PYOGENES RESTORES ITS ABILITY TO RESIST PHAGOCYTOSIS

To complete the formal proof that the M protein functions to protect the group A streptococcus from phagocytosis and to lay the groundwork for the molecular genetic dissection of this major virulence factor, we have reintroduced a wild-type allele of the M protein gene (emm) into the chromosome of a strain from which it had previously been deleted. For this purpose, we used homologous recombination with an engineered plasmid that was introduced into the Streptococcus pyogenes chromosome by an electroporation technique that we developed. The plasmid pJRS120E was constructed in Escherichia coli and contains the structural gene for the antiphagocytic M6 protein (emm6) of S. pyogenes JRS4 and a selectable marker (ermAM) between the chromosomal regions that flank emm6 in S. pyogenes. This plasmid was introduced into S. pyogenes strain JRS75, a strain that is not resistant to phagocytosis as a result of the allelic replacement of the emm6 gene by a kanamycin resistance gene (aphA3). DNA hybridization analysis indicated that in erythromycin-resistant strain JRS115, homologous recombination resulted in the replacement of the aphA3 gene of JRS75 with the introduced emm6 allele. Strain JRS115 produces approximately the same amount of M6 as does the parental emm6+ strain JRS4, as assayed by Western blot analysis, and is resistant to phagocytosis.