Three autolytic-defective mutants of Streptococcus faecium (S. faecalis ATCC 9790) were isolated. All three autolytic-defective mutants exhibited the following properties relative to the parental strain: (i) slower growth rates, especially in chemically defined medium; (ii) decreased rates of cellular autolysis and increased survival after exposure to antibiotics which block cell wall biosynthesis; (iii) decreased rates of cellular autolysis when treated with detergents, suspended in autolysis buffers, or grown in medium lacking essential cell wall precursors; (iv) a reduction in the total level of cellular autolytic enzyme (active plus latent forms of the enzyme); (v) an increased ratio of latent to active forms of autolysin; and (vi) increased levels of both cellular lipoteichoic acid and lipids.

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Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattem of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme. 'o.2 S.A.

Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed.

SPOl bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVBl. The injected DNA is partially degraded to small trichloroacetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPOl DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVBI. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPOl-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in heterogeneous in size. MATERIALS AND METHODS Bacteria and bacteriophage. B. subtilis CU403 divIVB1 thyA, thyB, metB (24) was used throughout this study as a source of minicells and bacteriophage host. SPOl bacteriophage was obtained from C. Yehle. SPOl phage stocks were produced by infecting host bacteria at an adsorbance at 660 nm of (A ...) 0.5 in TY medium (2) containing 5 x 10-3 M CaCl2 at 37 C with multiplicity of infection of 10. After cell lysis the lysate was cleared by centrifugation at 5,000 x g for 10 min. The supernatant was centrifuged at 27,000 x g for 3 h at 4 C and the phage pellet was allowed to resuspend overnight at 4 C in TBT buffer (2). The phage suspension was layered on top of a three-step CsCl gradient containing CsCl solutions in TBT of densities 1.