DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis.

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.

Genotypic analysis of strains of mutans streptococci by pulsed-field gel electrophoresis.

The species and serotypes of various strains of S. mutans and S. sobrinus were characterized by pulsed-field gel electrophoresis after the genomic DNA from the various strains had been digested with five restriction enzymes (EcoR I, Xba I, Hind III, Sfi I and BssH II) separately. Among these restriction enzymes, BssH II was very useful for the characterization of species and serotypes and, in particular, digestion discriminated between serotypes d and g. The restriction patterns obtained from the genomic DNA of isolates isolated from children's saliva were essentially identical to those from the genomic DNA of the standard laboratory strains. Patterns of BssH II digests of the genomic DNA of 10 isolates identified as S. sobrinus were characteristic of serotype g of the standard laboratory strains. Our results indicate that digestion with BssH II and subsequence analysis by pulsed-field gel electrophoresis should be useful for the characterization of species and serotypes and for epidemiological studies of mutans streptococci.

Detection of antigenic surface proteins of Actinobacillus actinomycetemcomitans using the immunoblotting method.

Antigenic surface proteins of Actinobacillus actinomycetemcomitans (three strains), which can be recognized by antibodies in human serum, were examined using the Western blot method. By comparing the immunoblotting profiles between protease-treated cells and untreated cells, IgG-antigenic and IgM-antigenic surface proteins were found. The IgG-antigenic proteins revealed the following molecular weights: strain ATCC 29522, 52 and 49 kD; strain ATCC 29523, 45, 49, 52 and 70 kD; strain Y4, 36, 38, 44, 53 and 58 kD. Molecular weights of the IgM-antigenic proteins ranged from 50 to 92 kD: strain ATCC 29522, 68, 80, 90 and 92 kD; strain ATCC 29523, 62, 68 and 80 kD; strain Y4, 50, 64, 73, 81 and 86 kD. The IgG-antigenic proteins were very sensitive to trypsin and Bacillus licheniformis protease, but were resistant to V8 protease, while the IgM-antigenic proteins were sensitive to various proteases. These results suggested that IgG-antigenic and IgM-antigenic components were different from the serotype-specific antigen or species-specific antigen associated with polysaccharides or lipopolysaccharides with respect to molecular weights and that they were proteins.

Partial purification and some properties of pyroglutamyl peptidase from Enterococcus faecalis.

Pyroglutamyl peptidase was partially purified from Enterococcus faecalis ATCC 19433 by anion-exchange chromatography, gel filtration and salting out after lysis of cell walls with N-acetylmuramidase. Pyroglutamyl peptidase was purified 46-fold with a yield of about 2% based on the total activity of the crude extract. The molecular mass of the bacterial enzyme was estimated to be about 82 kD by gel filtration. The pl of the enzyme was 4.2 and the optimum pH and temperatures for the reaction were 7.2-7.5 and 35-45 degrees C, respectively. The enzyme was relatively stable below 45 degrees C, but almost all the activity was lost after heat-treatment at 55 degrees C for 15 min. The apparent K(m) value for pyroglutamyl-beta-naphthylamide was 0.55 mM. The bacterial enzyme specifically cleaved pyroglutamyl residues from the amino termini of pyroglutamyl compounds, such as Pyr-Asn-Gly, Pyr-His-Gly, Pyr-Ala-Glu, Pyr-Ala, neurotensin, thyrotropin-releasing hormone and bradykinin-potentiator B. However, human IgG and Bence Jones protein, which are high-molecular-mass proteins, were not hydrolysed. Neither derivatives of free amino acids, such as Ala-, Gly-, Pro- and Leu-p-nitroanilide, nor benzoyl-DL-Arg-p-nitroanilide were hydrolysed. The activity was strongly inhibited by thiol-blocking reagents (p-CMB, N-ethylmaleimide, monoiodoacetic acid). In addition, protease inhibitors, such as TLCK and PMSF, reduced the activity by 54 to 73%. These results suggest that the bacterial enzyme is a cysteine protease with sulphydryl residues in its active site and, possibly, histidine or serine residues near the active site.

Some properties of gamma-glutamyl transpeptidase from Fusobacterium nucleatum.

A series of chromatographic techniques was used to purify gamma-glutamyl transpeptidase from Fusobacterium nucleatum ATCC 23726. The purified enzyme was homogenous with a molecular weight (MW) of approximately 101 kD consisting of two different subunits with MW of 67 kD and 31 kD. Its distribution by treatment of lysozyme-EDTA suggested that the enzyme was a periplasmic protein. The pl of the enzyme was 5.9 to 6.2, and the optimum pH of the transpeptidation and the hydrolytic reaction was 8.0. Glycylglycine, glycine and methionine were good acceptors, and the enzyme reaction, in the presence of glycylglycine, was especially efficient. Glutamic acid, glutamine and serine were poor acceptors, and the enzyme activity was inhibited by these amino acids. The apparent Km value for the gamma-glutamyl donor (gamma-glutamyl-p-nitroanilide) was 4.9 x 10(-4)M and that for the acceptor (glycylglycine) was 0.19 M. Affinity-labelling reagents, such as DON, azaserine and AT-125 strongly inhibited the enzyme activity, and its activity was inhibited by L-serine plus borate, as is mammalian gamma-glutamyl transpeptidase. The antibodies against gamma-glutamyl transpeptidase from bovine kidney reduced the activity of the bacterial enzyme by 65%. These results indicate that the catalytic sites in F. nucleatum gamma-glutamyl transpeptidase were similar to those in mammalian gamma-glutamyl transpeptidase.

Partial purification and some properties of gamma-glutamyl peptide-hydrolysing enzyme from Actinobacillus actinomycetemcomitans.

A gamma-glutamyl peptide-hydrolysing enzyme was partially purified from Actinobacillus actinomycetemcomitans. The enzyme required metal ions, and 1 to 2 mM Mn2+ ions, especially, were essential for hydrolytic reaction. Its distribution by treatment of cells with lysozyme-EDTA suggested that the enzyme was a membrane-bound protein. The pI of the enzyme was in range of pH 5.1 to 5.6, and the apparent Km value for gamma-glutamyl-p-nitroanilide was 3.0 x 10(-4) M. The enzyme hydrolysed specifically gamma-glutamyl residues from the N-terminal of gamma-glutamyl compounds such as gamma-Glu-Met, gamma-Glu-Ala, gamma-Glu-Leu and gamma-Glu-Tyr, but did not catalyse the transpeptidation reaction. Neither free amino acids such as Ala-, Pro-, Gly- and Leu-p-nitroanilide nor alpha-glutamyl derivatives were hydrolysed. Its activity was strongly inactivated by metal chelators (EDTA or o-phenanthroline) and amino acids (Glu, Gln). In addition, the activity was specifically inactivated by gamma-glutamyl affinity-labelling reagents such as AT-125, 6-diazo-5-oxo-L-norleucine and azaserine, which are inhibitors of the gamma-glutamyl donor sites of mammalian gamma-glutamyl transpeptidase. Antibodies against bovine kidney gamma-glutamyl transpeptidase decreased the activity of the bacterial enzyme by 65%. These results suggested that the active sites in the bacterial enzyme were similar to those in mammalian gamma-glutamyl transpeptidase.

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.