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Infection and Immunity, July 2000, p. 4245-4254, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Genetic Locus for Streptolysin S Production by Group A Streptococcus

Victor Nizet,1 Bernard Beall,2 Darrin J. Bast,3 Vivekananda Datta,1 Laurie Kilburn,3 Donald E. Low,3 and Joyce C. S. De Azavedo3,*

Department of Pediatrics, Division of Infectious Diseases, University of California, San Diego, La Jolla, California 920931; Centers for Disease Control and Prevention, National Center for Infectious Diseases, Respiratory Diseases Branch, Atlanta, Georgia 303332; and Department of Microbiology, Mount Sinai Hospital and University of Toronto, Toronto, Ontario, Canada M5G 1X53

Received 17 February 2000/Returned for modification 9 March 2000/Accepted 23 March 2000

Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis. Streptolysin S (SLS) is the cytolytic factor that creates the zone of beta-hemolysis surrounding GAS colonies grown on blood agar. We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S. D. Betschel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671-1679, 1998). Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project (www.genome.ou.edu/strep.html), we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production. The sag locus is conserved among GAS strains regardless of M protein type. Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype. Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon. A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant. Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis. We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins.

* Corresponding author. Mailing address: Department of Microbiology, Room 1483, Mount Sinai Hospital, 600 University Ave., Toronto, Canada M5G 1X5. Phone: (416) 586-8459. Fax: (416) 586-8746. E-mail: jdeazavedo{at}mtsinai.on.ca.

Infection and Immunity, July 2000, p. 4245-4254, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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