In contrast to group A streptococci or Streptococcus pneumoniae, cells of Streptococcus sanguis (group H) do not exhibit the irreversible effects of penicillin treatment, such as loss of viability or lysis. On the other hand, the same bacteria show typical effects of penicillin, such as morphological alterations, reduction in the rate of cell wall synthesis, and secretion of murein and lipoteichoic acid polymers into the medium. A novel effect of cell wall inhibitors was also noted: treatment with beta-lactams or with fosfomycin, D-cycloserine, or beta-halogeno-D-alanine caused the release of substantial amounts of glycerol lipids into the growth medium. The antibiotic "tolerance" of S. sanguis is interpreted in terms of the hypothesis that the activity of bacterial murein hydrolases is essential for the irreversible effects of cell wall inhibitors.

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilusMR-1C and Lactobacillus delbrueckii subsp.bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799–805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 andLactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, theepsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticusLH100 plus the non-EPS-producing mutant S. thermophilusDM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of d-galactose, l-rhamnose, andl-fucose in a ratio of 5:2:1.

Streptococcus pneumoniae has an auxotrophic requirement for choline, and choline residues that incorporate into the wall and membrane teichoic acids are intimately involved with the control of autolytic phenomena of this bacterium. We report here the re-examination of the role of choline in autolytic cell wall degradation using the choline-independent S. pneumoniae strain R6Cho- recovered from a heterologous cross with DNA from Streptococcus oralis. S pneumoniae Cho- cultured in choline-free medium grew with normal generation time but formed long chains, failed to undergo stationary-phase autolysis, and was also resistant to lysis induced by deoxycholate or penicillin. Cell walls produced under these conditions had reduced phosphorus content, contained no choline residues detectable by nuclear magnetic resonance, and had reduced binding capacity for the pneumococcal autolytic amidase, and complete hydrolysis of such walls by the amidase required prolonged incubation with high concentrations of the enzyme. Addition of choline to the growth medium reversed at these phenomena. High-performance liquid chromatography analysis of amidase digests of cell walls prepared from strain R6Cho- grown with or without choline produced identical stem peptide profiles, which were also similar to that of the parental S. pneumoniae strain R6. Peptidoglycans prepared by hydrofluoric extraction of cell walls from Cho- growth with or without choline or from the parental strain R6 were uniformly susceptible to the autolytic amidase and were fully degraded to the normal family of stem peptides, indicating that, in sharp contrast to the case of cell walls, the amidase degradation of teichoic acid-free peptidoglycan did not require the presence of choline residues in the substrate.

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Streptococcus oralis ATCC 35037 took up radioactively labeled choline from growth medium. Most of the choline (80 to 90%o) was incorporated into the cell wall teichoic acid, and about 10%Y was localized in the plasma membrane. While cells grew in choline-free medium, they did so at slow rates and produced cell walls with greatly reduced amounts of phosphate and no detectable choline. Cells grown in choline-free medium had grossly abnormal shape and size. Both biochemical and morphological abnormalities were reversible by addition of choline to the medium.Teichoic acids, complex anionic wall polymers ubiquitous in gram-positive bacteria (30,31), are known to have important ecological roles such as receptors for bacteriophage (1), as type-or group-specific antigens (7), and as reservoirs of surface-bound divalent cations (2, 31). Structurally similar so-called membrane teichoic acid polymers linked to glycolipids are also present in smaller quantities in the plasma membrane of all gram-positive bacteria. The precise physiological role(s) of these wall-and membrane-linked polymers in bacterial growth remains to be determined. Cell wall teichoic acids play a role in cellular morphogenesis in Bacillus subtilis and Bacillus licheniformis (3, 9, 10,22), and genetic studies with teichoic acid mutants ofB. subtilis imply that these polymers are essential for normal cell growth (5,16,19). Amino alcohols (e.g., choline), which are unique components of both the wall and membrane teichoic acids of Streptococcus pneumoniae, are essential nutrients for this bacterium (2,27). Choline is also present in the wall teichoic acid of several viridans group streptococci and clostridia (20,24,26). Streptococcus oralis is frequently identified among viridans group streptococci that colonize human upper respiratory tracts (4,20). While closely related to S. pneumoniae, this bacterium is classified as a distinct species on the basis of nucleic acid hybridization and cell wall composition data (20,25,26).In preliminary studies with S. oralis ATCC 35037, it was noticed that omission of choline from a synthetic growth medium (28) caused striking abnormalities in the growth and morphology of these bacteria. In this communication, we describe an analysis of these phenomena. MATERIALS AND METHODSBacterial strains and culture conditions. S. oralis ATCC 35037 was cultivated in a chemically defined (CDen) medium, a synthetic medium (21) modified by replacing the Casamino Acid hydrolysate with a mixture of pure individual amino acids. The medium was modified as indicated in several experiments by omission of choline. Cultures of bacteria grown in choline-free medium were produced in the following manner. Bacteria grown in full (choline-contain-* Corresponding author. ing) medium were diluted 100-fold into choline-free medium and grown at 370C overnight, after which the culture was further diluted 10-to 20-fold into the same amino alcoholfree medium. The doubling time of cell mass was 1 h in the presence and about 2 to 2.5 h in the absence of cholin...