Changes in the carboxyl-terminal repeat region affect extracellular activity and glucan products of Streptococcus gordonii glucosyltransferase.
Glucans produced by the glucosyltransferase (GTF) of Streptococcus gordonii confer a hard, cohesive phenotype (Spp+) on colonies grown on sucrose agar plates. S. gordonii strains with specific mutations in the region of gtfG that encodes the GTF carboxyl terminus were characterized. In the parental strain Challis CH1, this region included a series of six direct repeats thought to function in glucan binding. The spontaneous mutant strain CH107 had a 585-bp deletion resulting in the loss of three internal direct repeats. Insertional mutagenesis was used to construct strain CH2RPE, which had the parental repeat region but was missing 14 carboxyl-terminal amino acids. The similarly constructed strain CH4RPE had an in-frame addition of 390 nucleotides encoding two additional direct repeats. Although strains CH1, CH2RPE, and CH4RPE all had similar levels of extracellular GTF activity, strain CH107 had less than 15% of the parental activity; however, Western blots (immunoblots) indicated that the amounts of extracellular GTF protein in all four strains were similar. 13C NMR analyses indicated that partially purified GTFs from the Spp+ strains CH1, CH2RPE, and CH4RPE all produced glucans with similar ratios of alpha1,6 and alpha1,3 glucosidic linkages, whereas the Spp- strain CH107 GTF produced primarily alpha1,6-linked glucans. Transformation of strain CH107 with pAMS57, which carries the gtfG positive regulatory determinant, rgg, increased the amount of GTF activity and GTF antibody-reactive protein ca. fivefold but did not confer a hard colony phenotype on sucrose agar plates, suggesting that the type of glucan product affects the sucrose-promoted colony phenotype.
Vickerman
M M
MM
Department of Microbiology and Immunology, School of Medicine, University of Michigan, Ann Arbor 48109, USA. vickerma@umich.edu
Sulavik
M C
MC
Minick
P E
PE
Clewell
D B
DB
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D005936:Glucans / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D013291:Streptococcus / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D005936:Glucans; D005964:Glucosyltransferases