Ultrastructural Localization of Sucrases in Streptococcus mutans GS-5 and an Extracellular Polysaccharide Mutant: a Comparative Cytochemical and Immunocytochemical Study

Abstract

Electron microscopy and cytochemical and immunocytochemical procedures were used to study the ultrastructural distribution of sucrase enzymes in two strains of Streptococcus mutans. In a strongly adherent and virulent parent strain, GS-5, most of the invertase and fructosyltransferase activities were demonstrated extracellularly or bound to the cell surfaces. Intracellularly, enzymatic sites were detected near the plasma membrane on the periphery of the nucleoid and central mesosome. In GS-511, a mutant of diminished virulence and adherence, most of the enzymatic activity was not located on the cell surfaces, but was found away from the cell walls and associated with extracellular polysaccharides. Intracellularly, GS-511 manifested the same distribution of invertase and fructosyltransferase as did GS-5; however, the close association of these enzymes with the plasma membrane was not shown in GS-511. In both strains, extracellular areas near regions associated with cross wall formation appeared to show localized concentrations of these sucrases. Antibodies against partially purified glucosyltransferase (GTF) enzymes from GS-5 were used to localize GTF by immunocytochemical techniques. Indirect ferritin localization procedures showed that the extracellular and cell-bound GTF enzymes were distributed in similar locations as the fructosyltransferase and invertase enzymes. By absorption of the antiserum with whole GS-511 cells, the location of extracellular GTF and surface antigens unique to GS-5 was demonstrated. The dramatically reduced levels ofcell-bound sucrase activity in GS-511 indicates the significant role of these enzymes in adherence and cariogenicity. 447 on August 1, 2020 by guest http://iai.asm.org/ Downloaded from

Localization of Streptococcus mutans GS-5 glucosyltransferases and intracellular invertase

“…However, recent results (16) suggest that a major antibody fraction against this complex is directed against adherent glucan-synthesizing GTF activity. Furthermore, the labeling pattern with anti-GTF-A is similar to that observed in the previous investigation utilizing an anti-GTF antibody preparation preabsorbed with cells of a mutant strain of GS-5 which synthesized little insoluble glucan (1). Nevertheless, the unavailability of significant quantities of homogeneous GTF preparations capable of synthesizing insoluble glucans exclusively indicates that the labeling experiments with anti-GTF-A are not entirely unequivocal.…”

“…Controls were treated in exactly the same manner except that (i) normal rabbit globulins were reacted with the sections of S. mutans at step iv, and (ii) sections of a mixed culture of S. aureus and E. coli were reacted with the anti-GTF and invertase sera. Electron micrographs were prepared as previously described (1).…”

Effect of sucrose in culture media on the location of glucosyltransferase of Streptococcus mutans and cell adherence to glass surfaces

“…Synthesis of the glucans is mediated by the enzymatic action of cell-free (extracellular) and/or cell-bound glucosyltransferase(s) (GTase[s]). The location of S. mutans GTase appears to be complicated (1,3,11,16,20,22,23,28) and remains to be elucidated.…”

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

“…By the same procedure described here, Gram-negative bacteria such as Escherichia coli and Vibrio cholerae revealed the same characteristic wavy or gyrus-like surface structure (not shown) as appeared in a recent report (1), and some other streptococci likewise showed an individual characteristic surface appearance (smooth to rugged). Therefore, it would be possible to conclude that the characteristic still remained on the HS6 cell surface, although on the surface there were possibly left such artificial effects that the fuzzy coat and fibrillar material found with a transmission electron microscope (2,11,18,27) were partially smoothed by the metal particles to small slightly rugged and irregular-shaped structures, respectively (Figs. 1, 2, 5, and 6).…”

“…The extracellular enzymes of S. mutans HS6 (serotype a) (10, IS) are able to bind to heat-treated cells and subsequently synthesize water-insoluble glucan from sucrose, resulting in the adherence of cells (10,16). A transmission electron microscopic study of thin sections of S. mutans cells revealed the presence of a cell surface-associated fuzzy coat as the characteristic surface structure of S. mutans (2,II,18,27) and extracellular fibrillar glucan as the material responsible for the adherence and virulence (18). In a recent study the three-dimensional fine structure of the S. haemolyticus cell surface was examined with a high resolution scanning electron microscope (1).…”