Characterization of glucosyltransferase-deficient, plasmid-containing mutants of Streptococcus mutans LM-7

Abstract

The possibility that glucosyltransferase (GT)-mediated insoluble-glucan synthesis from sucrose is controlled by the 3-megadalton plasmid pAM7 in Streptococcus mutans LM-7 has been examined. A low-sucrose agar medium was developed to readily detect and quantitate presumptive GT-negative mutants. Such mutants were isolated from Todd-Hewitt broth cultures grown either with or without sodium dodecyl sulfate (10 [Lg/ml) or acriflavine (0.5 ig/ml) at frequencies ranging from about 0.01 to 1%. Independently isolated mutants had the following characteristics: (i) cells were virtually devoid of cell-associated GT and did not aggregate upon addition of sucrose; (ii) cell-free culture fluids synthesized 1OX less insoluble glucan than those of the parent; and (iii) cultures grown with sucrose did not form adherent deposits on the wall of the culture tube, as is typical of S. mutans. Both parent and mutants formed relatively little soluble glucan in 1-h assays. Three independently isolated mutants and the parent were found to contain similar amounts of plasmid DNA. Analysis by sucrose density gradient centrifugation and agarose gel electrophoresis did not reveal a size difference between the plasmids from parent and mutants. These results show that (i) S. mutans LM-7 generates GT-deficient mutants at relatively high frequency that still contain a 3-megadalton plasmid; (ii) both cell-associated and extracellular GT levels are depressed in the mutants, which suggests that these activities are directly or indirectly controlled by the same gene or by genes that segregate as a unit.About 10 years ago, several investigators demonstrated that the smooth-surface cariogenicity of Streptococcus mutans in animal model systems is associated with the formation of an adherent and water-insoluble glucan containing primarily a-1-6-and a-1, 3-linkages (16, 19). This concept has since been amply confirmed. The glucosyltransferase (GT) activity responsible for insoluble-glucan synthesis from sucrose has been relatively refractile to biochemical and genetic characterization. This appears to be at least partly due to its tendency to form complexes with itself and other sucrose-and dextran-metabolizing enzymes with apparent molecular weights greater than 106 (3,14,26,31). However, at least three major and several minor forms of the enzyme have been isolated from strain OMZ-176 (18), and electrophoretically distinct activities with molecular weights ranging from about 160,000 to 225,000 have since been reported from the genetically and biochemically related strain 6715 (3). It is not known whether the observed enzyme multiplicity is due to multiple GT genes or to posttranscriptional modification of a single GT gene product. The location of the gene(s) controlling insoluble-glucan synthesis is also open to question. Whereas it was first proposed that this trait is associated with an inducible prophage (17, 24), later evidence suggested that GT synthesis is controlled by a plasmid because plasmid-curing agents induced mutants at high frequency that s...

Ability of Streptococcus mutans and a Glucosyltransferase-Defective Mutant to Colonize Rodents and Attach to Hydroxyapatite Surfaces

“…Unstandardized whole cultures of strain A17 contained 31% as much total glucosyltransferase activity as cultures of strain LM7; however, standardized, washed A17 cells possessed less than 3.0% of the activity of parental cells (Table 1). This observation confirms the findings of Donkersloot et al (4). When strain A17 was propagated in 2% sucrose broth, it formed little macroscopically evident adherent growth on the walls of culture vessels.…”

More sensitive test agar for detection of dextranase-producing oral streptococci and identification of two glucan synthesis-defective dextranase mutants of Streptococcus mutans 6715

“…To examine the possibility that certain glucan synthesis-defective mutants might, in fact, be dextranase mutants, several mutants with phenotypically altered glucan synthesis were compared with their respective parents on dex-10 and SGS agar. Figure 2 exemplifies that (i) the S. mutans LM-7 mutants that we described earlier (6) were indistinguishable from their parent on the test agars, and (ii) dextranase activity was more readily detected on dex-10 than on SGS agar. Identical results were obtained with the other LM-7 mutants ( Table 2).…”

“…The glucan synthesis-defective mutants were kindly provided by the investigators listed in Table 2. All cultures were maintained and checked for purity as described previously (6). Downloaded from which will be designated as SGS agar, and the newly developed medium, which will be designated as dex-10 agar, were used for the detection of endo-dextranase activity.…”

Biology, immunology, and cariogenicity of Streptococcus mutans.

“…The few occurrences of plasmids in S. mutans argue against earlier claims of plasmid-mediated polysaccharide synthesis by this bacterium. Related to this, Donkersloot et al (112) isolated mutants of S. mutans LM7 that had essentially no GTase activity, but still retained plasmid DNA. Therefore, these mutants are different from the plasmidless, GTase-deficient mutants of strains PK1 and JC2 (253).…”