Synthesis of Glucan on the Cell Surface of Streptococcus mutans: Chemical and Scanning Electron Microscopic Studies

The appearance and continuing growth of extracellular material on Streptococcus mutans HS6 cells in sucrose-containing Merthiolated buffer was observed in a scanning electron microscope and was found to be related to the glucan synthesis on the cell and to adherence of the cell to a smooth surface. Cells grown in broth completely deprived of sucrose by invertase (HS6-IV) had a characteristic, slightly rugged surface structure. On incubation of HS6-IV in the sucrose-containing buffer, a few small globular particles appeared on the surface and grew to an irregular shape (globular to fibrilar) after several hours. The increase in the total glucan content of the cells paralleled the growth of the globular material, to which ferritin-conjugated anti-dextran globulin was found to bind. On the cell surface of cells harvested from conventional broth, both small globular and irregular structures, which possibly formed from sucrose in the broth, existed originally and continued to grow during incubation, along with the material newly appearing on the surface. The accumulation of glucan on the cells resulted in their adherence to a glass surface. The inhibition of growth of the extracellular material on the cells by trypsin, dextranase or anti-glucosyltransferase corresponded to the decrease in glucan synthesis and the loss of adhering ability. These results indicated that the material growing on the cell surface was glucan synthesized by glucosyltransferases.     

Autolytic Enzyme System of Streptococcus faecalis IV. Electron Microscopic Observations of Autolysin and Lysozyme Action

Cell walls (LOG walls) were isolated from cultures of Streptococcus faecalis ATCC 9790 in the exponential phase of growth. These walls were either allowed to undergo autolytic dissolution (in the presence or absence of trypsin) or wall autolysis was inactivated with sodium dodecylsulfate (SDS walls). Inactivated walls were treated either with lysozyme or with isolated, partially purified S. faecalis autolysin. During wall lysis, samples were removed, negatively stained with phosphotungstate, and examined in the electron microscope. Both lysozyme and isolated autolysin appeared to act over the entire surface of SDS walls. After partial dissolution, a fibrous network over the surface was revealed. Lysozyme digestion revealed the presence of prominent, highly-contrasted equatorial and subequatorial bands around the walls. After trichloroacetic acid extraction, the bands were seen less frequently and less distinctly in the partially lysozyme digested walls, suggesting that the bands contained nonpeptidoglycan polymers. In the absence of trypsin (which activates a latent form of the autolysin), autolysis of LOG walls appeared to start at the equatorial bands and to proceed back towards the apex of the coccus. Ribbons of wall material coming off the wide edge of the nearly hemispherical wall fragments were observed. Activation of latent autolysis resulted in lytic action over the entire wall surface. The results are consistent with the previously postulated location of active autolysin at the areas of new wall synthesis and the random location of latent autolysin in LOG walls.     

Effects of penicillin on synthesis and excretion of lipid and lipoteichoic acid from Streptococcus mutans BHT.

Cultures of Streptococcus mutans BHT grown for at least eight generations in a chemically defined medium containing [1(3)-14C]glycerol, when treated with growth-inhibitory concentrations (0.2 micrograms/ml) of benzylpenicillin (Pen G), produced and excreted increased amounts of lipid and lipoteichoic acid per unit of cells. Cellular lysis was not observed. Compared with untreated controls, lipid excretion increased 15-fold, and lipoteichoic acid excretion increased 6-fold, 4 h after the addition of Pen G. All lipid species showed increased synthesis and excretion after exposure to Pen G. Although the same lipid types were found in both the Pen G-treated and the untreated cultures, the percent composition was altered after treatment with Pen G. The most dramatic example of this was the percentage of intracellular diphosphatidylglycerol found in the Pen G-treated cultures, 22.6%, in contrast to 5.3% found in the untreated cultures.     

Electron Microscopic Observations on the Effects of Penicillin on the Morphology of Chlamydia psittaci

L cells were infected at high multiplicity with meningopneumonitis organisms and incubated in medium containing 200 units per ml of penicillin. At intervals up to 48 hr after infection, cells were removed and thin sections were prepared for electron microscopic studies on the morphology of the developing organism. Penicillin had no effect on the initial reorganization of the infecting elementary body to form the developmental reticulate body (RB), and, up to 12 hr after infection, the treated and untreated cultures were identical. After that time, however, penicillin-treated organisms showed striking differences in that binary fission was prevented, large abnormal RB forms were seen in great numbers, masses of RB cytoplasmic membranes and envelopes were formed within and outside the RB itself, and large numbers of empty or partially filled small vesicles were pinched off the RB. After 36 hr immature nucleoids were formed within the RB. Throughout all of this period, both the outer cell envelope and the cytoplasmic membrane of these RB were recognized. When infected cells were transferred into penicillin-free medium, the abnormal RB showed recovery to form normal RB both by a budding-like process and by internal fragmentation or subdivision rather like endosporulation. We have concluded that penicillin inhibits binary fission and prevents the synthesis of certain components essential for the formation of the elementary body envelope.     

Electron Microscopic Examination of Corynebacterium ovis

Corynebacterium ovis (C. pseudotuberculosis) was examined by electron microscopy after being subjected to various methods of fixation. The organism exhibited a fine structure similar to other corynebacterial species in the appearance of its cell wall, plasma membrane, nuclear apparatus, cytoplasmic matrix, wealth and complexity of intracytoplasmic membrane systems, and polyphosphate granules. An outstanding structural feature was the existence of an electron-dense, floccular layer external to the cell wall which both ligroin and acetone-methanol extractions demonstrated to be the previously postulated surface lipid of this organism. The only variations in structure evident between virulent and attenuated strains was a quantitative difference in the thickness and appearance of the surface lipid. The observation of this layer provided a basis for explaining the surface properties of C. ovis, with particular respect to its clumping capacity in suspension, the waxiness of its growth on solid media, and its ability to grow as a pellicle on suitable liquid media. The variation in the visible amount of surface lipid between the virulent and avirulent strains adequately explained the divergence of these three surface properties between the strains.     

Electron microscopy of antibody-labelled cells of Streptococcus mutans.

Examination of immune complexes between cells of Streptococcus mutans and homologous antiserum by the techniques of thin-sectioning and freeze-etching revealed that the cells were embedded within and extensive matrix 80-90 nm thick with defined boundaries.     

Properties of mutacin b, an antibacterial substance produced by Streptococcus mutans strain BHT

An antibacterial substance produced by strain BHT of Streptococcus mutans (mutacin b) was found to be a small molecule (MW 3,500-6,000) with remarkable resistance to temperature, alkali and various solvents. Enzyme sensitivity tests of partially purified preparations indicated that mutacin b is a peptide. It is sensitive to several proteolytic enzymes and its lethal effects on sensitive cells can be prevented by adding trypsin to cells exposed to mutacin b. High concentrations of mutacin b inhibited the growth of producer cells, indicating that strain BHT is only partially immune to this substance.     

Effect of inoculation sequence and nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing on human teeth in an artificial mouth

Human teeth in an artificial mouth were inoculated with Streptococcus mutans BHT, Streptococcus mitior LPA-1, or sequentially with both organisms. Incubation was continued for 90 h. Mixed populations were largest when a nutrient supplement containing 5.0% (w/v) sucrose was supplied. Fewer organisms were recovered from experiments with synthetic saliva only, or when a supplement containing 0.05% (w/v) glucose was available. The inoculation sequence determined the total viable count and a larger population resulted when Strep. mutans was the initial colonizer (P less than 0.01). Strep. mutans was always able to become established even when super-infected on to a 24 h plaque of Strep. mitior. The final proportion of Strep mutans was lower when it was the superinfecting organism and the sucrose (P less than 0.01) or glucose (P less than 0.05) nutrient supplement was provided. This work confirms the importance of inoculation sequence and presence of sugars in plaque accumulation and demonstrates the fundamental role of microbial interactions in this process.     

Effect of inoculation sequence and nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing on human teeth in an artificial mouth

Human teeth in an artificial mouth were inoculated with Streptococcus mutans BHT, Streptococcus mitior LPA-1, or sequentially with both organisms. Incubation was continued for 90 h. Mixed populations were largest when a nutrient supplement containing 5–0% (w/v) sucrose was supplied. Fewer organisms were recovered from experiments with synthetic saliva only, or when a supplement containing 0–05% (w/v) glucose was available. The inoculation sequence determined the total viable count and a larger population resulted when Strep, mutans was the initial colonizer ( P < 0–01). Strep, mutans was always able to become established even when super-infected on to a 24 h plaque of Strep, mitior. The final proportion of Strep mutans was lower when it was the superinfecting organism and the sucrose ( P < 0–01) or glucose ( P < 0–05) nutrient supplement was provided. This work confirms the importance of inoculation sequence and presence of sugars in plaque accumulation and demonstrates the fundamental role of microbial interactions in this process.     

Solute Size Effects on the Diffusion in Biofilms of Streptococcus mutans

The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.     

Electron Microscopic Observations on the Three-Dimensional Morphology of Apatite Crystallites of Human Dentine and Bone'

In sections of human dentine (carious and sound) and bone examined with the electron microscope, apatite crystallites were seen to present long thin profiles somewhat suggestive of a cylindrical shape, broad profiles indicative of a plate-like shape, and profiles intermediate between these two extremes. With a special stereoscopic specimen holder allowing the specimen to be tilted through an angle of 30° it was possible to record images of two profiles of the same crystallite from different angles and thus gain information concerning the 3-dimensional morphology of crystallites showing a thin profile. In all fields so examined, the thin-profile crystallites that were properly oriented with respect to the axis of tilt exhibited a different width dimension in each of the two micrographs. From this it is concluded that the thin profiles actually represented edge views of plate-like crystallites.     

Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans

The Streptococcus mutans atlA gene encodes an autolysin required for biofilm maturation and biogenesis of a normal cell surface. We found that the capacity to form biofilms by S. mutans, one of the principal causative agents of dental caries, was dramatically impaired by growth of the organism in an aerated environment and that cells exposed to oxygen displayed marked changes in surface protein profiles. Inactivation of the atlA gene alleviated repression of biofilm formation in the presence of oxygen. Also, the formation of long chains, a characteristic of AtlA-deficient strains, was less evident in cells grown with aeration. The SMu0629 gene is immediately upstream of atlA and encodes a product that contains a C-X-X-C motif, a characteristic of thiol-disulfide oxidoreductases. Inactivation of SMu0629 significantly reduced the levels of AtlA protein and led to resistance to autolysis. The SMu0629 mutant also displayed an enhanced capacity to form biofilms in the presence of oxygen compared to that of the parental strain. The expression of SMu0629 was shown to be under the control of the VicRK two-component system, which influences oxidative stress tolerance in S. mutans. Disruption of vicK also led to inhibition of processing of AtlA, and the mutant was hyperresistant to autolysis. When grown under aerobic conditions, the vicK mutant also showed significantly increased biofilm formation compared to strain UA159. This study illustrates the central role of AtlA and VicK in orchestrating growth on surfaces and envelope biogenesis in response to redox conditions.     

Effect of Nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 Growing in Pure or Mixed Culture on Human Teeth in an Artificial Mouth

Streptococcus mutans and Streptococcus mitior were both capable of colonizing the surfaces of human teeth in an artificial mouth. Although fermentable carbohydrate was not essential for colonization, highest numbers were recovered after 5 d if 5–0°_***0 (w/v) sucrose had been available intermittently. When grown together in mixed culture the interaction of the two species was affected profoundly by the available nutrients. In the presence of synthetic saliva alone, Strep. mitior was strongly antagonistic to Strep. mutans. When a nutrient broth containing sucrose was also provided intermittently there was slight inhibition of Strep. mutans accompanied by an increase in the proportion of Strep. mitior in the plaque, although the former was the dominant organism under these conditions. When 0–5% (w/v) glucose replaced the sucrose, mutual antagonism occurred and fewer organisms were recovered than if only synthetic saliva had been available. One reason why a high-sucrose diet encourages colonization by Strep. mutans may be that insoluble extracellular polysaccharide confers a competitive advantage upon it in the face of antagonistic agents such as the hydrogen peroxide produced by Strep. mitior.     

Demonstration of shared antigenic determinants between Streptococcus mutans BHT cell membrane, human heart tissue and myosin using monoclonal antibodies to S. mutans

Monoclonal antibodies (MAb) raised to intact Streptococcus mutans P-4 cells (serotype e) were used to demonstrate the presence of shared antigenic determinant(s) between S. mutans BHT (serotype b) cell membranes and human heart tissue. MAb binding to both BHT membrane and human heart tissue was demonstrated by ELISA. Common antigens were identified by immunoblot analysis following separation of BHT membrane components and human heart antigens by SDS-PAGE. MAb 22C4 recognized three polypeptides from the BHT membrane preparation, having molecular masses of 42, 56 and 85 kDa. MAb 22C4 also recognized an 85 kDa component and a 200 kDa component from human heart tissue. MAb D159 was specific for a single 82 kDa polypeptide in BHT membrane, and also bound to two high molecular mass components in human heart (165 and 200 kDa). When both MAb D159 and 22C4 were first absorbed with S. mutans P-4 cells, subsequent reactivity to the aforementioned BHT membrane components was inhibited, indicating that these cross-reactive components are found in S. mutans P-4 as well as in S. mutans BHT micro-organisms. Competitive binding analysis showed that both MAb D159 and MAb 22C4 bound to myosin, indicating that S. mutans BHT membrane, human heart tissue and myosin share at least one immunodeterminant. This indicates that myosin could be the cross-reactive tissue component in human heart.     

Studies on the antibacterial activity of dodecylglycerol. Its limited metabolism and inhibition of glycerolipid and lipoteichoic acid biosynthesis in Streptococcus mutans BHT

Growth-inhibitory concentrations of racemic sn-1(3)-dodecylglycerol inhibit the incorporation of [14C] glycerol into lipids and lipoteichoic acid of Streptococcus mutans BHT and alter the per cent composition of the glycerolipids. Increases in phosphatidic acid and diphosphatidylglycerol (at the expense of phosphatidylglycerol) contribute the most to the change in lipid composition. No cellular lysis occurs under these conditions. Radioactive racemic sn-1(3)-dodecylglycerol is readily taken up by the cell and is metabolized primarily to lysophosphatidic acid and phosphatidic acid with smaller amounts converted to phosphatidylglycerol and diacylglycerol. The accumulation of phosphatidic acid and the loss of viability respond in parallel to different concentrations of dodecylglycerol. An increase in CTP is also observed which together with the increase in phosphatidic acid suggests a possible impairment in the synthesis of CDP-diacylglycerol.     

Effects of Panose on Glucan Synthesis and Cellular Adherence by Streptococcus mutans

The effects of panose on glucan synthesis and sucrose-dependent cellular adherence by Streptococcus mutans were investigated. Panose effectively inhibited glucan synthesis from sucrose by glucosyltransferases from S. mutans strain 6715, but increasing amounts of panose increased the release of fructose from sucrose by the enzymes. On the other hand, production of a series of oligosaccharides of increasing size by the enzymes was markedly enhanced in the presence of panose. These results indicate that panose activates the enzymes and that the inhibition of glucan synthesis by panose is due to the transfer of the glucosyl group of sucrose to panose. Sucrose-dependent adherence of cells of various S. mutans strains to a glass surface was also inhibited by panose.     

变异链球菌和血链球菌全代谢途径比对分析

为了比较变异链球菌和血链球菌全代谢途径,依据KEGG数据库(http://www.genome.ad.jp/kegg)对变异链球菌和血链球菌的全部代谢途径作逐项比对。结果显示,二者参与了85个代谢途径,包括多数以相同的酶参与的中央代谢途径,即糖酵解、三羧酸循环、磷酸戊糖途径等,和多数以不同的酶参与的双组分感应系统等。通过变异链球菌和血链球菌整体代谢网络对比,了解了变异链球菌和血链球菌理论上的全部代谢途径,为全面揭示二者代谢交流研究奠定了基础。