Although catalase is thought to be a major defense against hydrogen peroxide (H202), the (17) were provided by P. C. Loewen (University ofManitoba). E. coli were grown statically in brain heart infusion broth or M9 minimal salts medium supplemented with 10 mM glucose (M9/glucose) (25) at 370C in room air overnight (18-20 hr) to early stationary-phase (OD600 = 2.2-2.4). Thiamin was not added to the growth medium because these strains of E. coli were phenotypically Thi+, probably from a spontaneous Thi--Thi+ reversion. Bacteria in midexponential growth were obtained by diluting these stationary-phase cultures 1:100 in prewarmed fresh brain heart infusion broth and incubated further until the OD6(o of the culture reached 0.5-1.0. Cells were harvested from the culture by centrifugation (10,000 x g for 10 min at 20C), washed thrice in cold phosphate-buffered saline (140 mM NaCI/10 mM Na2HPO4/10 mM KH2PO4, pH 7.2) or in M9/glucose before being resuspended in the indicated buffers, and the absorbance at 600 nm was measured. The correlation of OD6(, with bacterial concentration was affirmed by direct microscopic counts and by colonyforming units obtained by growth on LB agar. Bacterial stocks were maintained on LB agar plates, stored at 2-4C. The catalase status of colonies was constantly monitored by direct catalase assay and by H202 drop tests.Streptococcal strains were obtained from the American Type Culture Collection (Streptococcus sobrinus; 33478), G. Germaine (University of Minnesota) (Streptococcus mitis, 9811), and E. L. Thomas (University of Tennessee) (Streptococcus mutans OMZ176 and S. mutans GS5). Organisms were cultured in Todd-Hewitt broth statically at 370C either in room air (S. mutans) or in a candle extinction jar (S. sobrinus and S. mitis) and washed as described for E. coli.H202 King of E. cofi. Suspensions of E. coli were diluted to the concentration of 5 x 107 cells per ml (concentrated) or 500 cells per ml (dilute) in M9/glucose and prewarmed to 370C before being exposed to H202 at the indicated concentrations. Bacterial viability, or, more accurately, ability ofthe bacteria to replicate, was assayed by pour-plating diluted aliquots (in triplicate) of the H202/bacteria mixture in bovine catalase-supplemented (100 units per ml) LB agar at different times during incubation. Killing experiments were performed not only in pure cat(+) and pure cat(-) E. coli suspensions but also were carried out in mixtures of cat(+) and cat(-) E. coli as indicated. To differentiate cat(+) from cat(-) E. coli after H202 exposure of such mixtures, bacteria ofboth strains were made resistant to ampicillin by pUC118 (a derivation of Abbreviations: LB agar, Lennox L agar; M9, minimal salts medium; M9/glucose, M9/10 mM glucose; cat(-), catalase-deficient mutant; cat(+), wild-type strain CSH7. *To whom reprint requests should be addressed. 7924The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S...

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