Synthesis of M Protein by Group A Hemolytic Streptococci in Completely Synthetic Media During Steady-State Growth

Abstract

Strains of type 6 (S 43) and type 14 group A streptococci were grown with Mprotein production in the presence of chemically defined synthetic media slightly modified from that previously employed for the growth of a nonproducer of M protein (type 4). The M protein, which is associated with virulence in group A streptococcus, was previously produced in growing cultures only with complex media. The bacterial growth with the biosynthesis of M protein in synthetic medium was obtained by successive adaptation in steady-state culture with decreasing amounts of Todd-Hewitt broth. The synthesis continued for at least 480 generations at pH 7.3 and with a generation time of 84 min. Glucose was the limiting nutrilite and the concentration of reducing agents in the medium was critical. The M protein was identified by gel diffusion against type-specific antisera from the Communicable Disease Center and from R. Lancefield. The yield of M protein obtained from organisms grown in the continuous-culture device was comparable to that from standard broth stationary cultures. In a previous paper, Davies, Karush, and Rudd (5) investigated the amino acid utilization of a type 4-derived group A streptococcus growing under steady-state conditions in a continuousculture device with a completely synthetic medium. The type 4 streptococcus was a nonproducer of M protein. Since the M protein is the antigen which confers type specificity upon the group A streptococci and is associated with virulence, it was of interest to establish the growth conditions in a chemically defined medium which would allow its synthesis. There have been many attempts to grow group A streptococci that produce M protein in such a medium. The most recent one was reported by Mickelson and Slade (20) and Mickelson (19), who found that, after repeated subculturing in a proteinor peptidefree medium, the streptococci lacked M protein. The same strains, Richards (type 3), N19 (type 19), and S43 (type 6), when grown on a medium that contained reduced ovalbumin, showed no 1 This work was presented in part before the American Society of Biological Chemists at a meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.

M proteins of group A streptococci.

“…Over 80% of all cultures were typable by the T agglutination pattern, but only about 30 to 50% of the strains were typable by M precipitation (depending on the individual laboratories and their supply of anti-M sera). By both techniques, over [5,11,12,27,44], and [8,25,Imp. 19].…”

Growth characteristics of group A streptococci in a new chemically defined medium

“…In addition to the above-mentioned CDM, Davies et al (3,4) have been able to grow group A streptococci in CDM under steady-state growth conditions. They reported production of M protein (4), but must add Todd-Hewitt broth to their CDM and slowly dilute it out to get the bacteria to adapt to the CDM.…”

Affinity and temporal variation of isoelectric fractions of rabbit antilactose antibody

“…The sterility of the preparation was checked by plating on nutrient agar. The dry weight concentration of the cells was determined with a Klett-Summerson colorimeter using a value of 100 Klett units for a 0.288-mg/ml cell suspension (Davies et al, 1968).…”