Deletions in the carboxyl-terminal region of Streptococcus gordonii glucosyltransferase affect cell-associated enzyme activity and sucrose-associated accumulation of growing cells

Vickerman, M. Margaret; Clewell, Don B.

doi:10.1128/aem.63.5.1667-1673.1997

Cited by 12 publications

(4 citation statements)

References 37 publications

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“…To confirm that the levels of GTF activity were related to levels of GTF protein, gels were immunoblotted, a process described in Materials and methods, and GTF antibody‐reactive protein was detected with antiserum directed against strain Challis GTF. These lower molecular weight forms of GTF are thought to be due to the degradation of the native enzyme by endogenous proteases, and occur even in the presence of protease inhibitors, as previously described (10,32). Results shown are representative of a minimum of four independent experiments.…”

Section: Resultsmentioning

confidence: 79%

“…The sensitivity of this assay is comparable to GTF activities determined by measuring [ 14 Cglucose] sucrose incorporation into glucan polymers (30,36). To confirm that GTF activity correlated to amounts of GTF protein, immunoblots were done as previously described (32). Briefly, after electrophoresis proteins were transferred to PVDF membranes and incubated overnight with polyclonal antiserum directed against strain Challis GTF (a gift from H. F. Jenkinson), and antibody‐reactive protein was detected with goat antirabbit IgG‐alkaline phosphatase.…”

Section: Methodsmentioning

confidence: 99%

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Initial characterization of the Streptococcus gordonii htpX gene

Vickerman

Mather²,

Minick³

et al. 2002

Oral Microbiology and Immunology

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Examination of the Streptococcus gordonii chromosomal region, which lies immediately upstream of the glucosyltransferase positive regulatory determinant rgg, revealed two open reading frames. Based on nucleotide sequences, these genes were similar to the Listeria monocytogenes lemA gene, which is involved in antigen presentation, and the Escherichia coli htpX heat shock gene, which has an unknown function. Northern hybridization analysis indicated that S. gordonii lemA and htpX genes were associated with a ca. 1.7-kb polycistronic transcript. Although levels of the lemA/htpX transcript did not increase in response to heat to levels seen with dnaK controls, insertional inactivation of htpX resulted in changes in adhesiveness, cellular morphology and detergent-extractable surface antigens in cells grown at 41 degrees C, implying that htpX may be involved in surface protein expression. Insertional inactivation of lemA and htpX indicated that, despite their proximity to rgg and the structural gene, gtfG, these upstream genes do not affect S. gordonii glucosyltransferase activity.

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Section: Resultsmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

Initial characterization of the Streptococcus gordonii htpX gene

Vickerman

Mather²,

Minick³

et al. 2002

Oral Microbiology and Immunology

View full text Add to dashboard Cite

show abstract

“…The glucansucrases of oral streptococci and Leuconostoc are generally regarded as extracellular enzymes but nevertheless they can also be detected in a cell‐associated form. C‐terminal deletions result in an increase in the secretion of GTF [29,30]. The observations described here suggest that further exploration is required into the contribution of N‐ and C‐terminal repeats to surface localisation as well as to the complex process of glucan synthesis and binding.…”

Section: Resultsmentioning

confidence: 82%

Location of repeat elements in glucansucrases of Leuconostoc and Streptococcus species

Janeček

Svensson

Russell

2000

FEMS Microbiology Letters

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Glucosyltransferases of oral streptococci, dextransucrases and alternansucrase of Leuconostoc mesenteroides, collectively referred to as glucansucrases, are large extracellular enzymes that synthesise glucans with a variety of structures and properties. A characteristic of all these glucansucrases is the possession of a C-terminal domain consisting of a series of tandem amino acid repeats. These repeat units are thought to interact with glucan but closely resemble the cell wall binding domain motif found in choline binding proteins in Streptococcus pneumoniae and surface-located proteins in a range of other bacteria. Analysis of dextransucrase and alternansucrase sequences has now shown that they also contain these repeat motifs in the N-terminal region, raising questions about their evolutionary origin and functional importance. ß

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“…With regard to the role of GBD repeats, a number of studies have been performed to understand the importance of some specific amino acid residues in such repeats for glucan binding as well as the minimum number of repeats essential for GTF functions (8,(10)(11)(12)(13). Dissociation con- stants have recently been estimated for the binding of the GBDs of GTFs and GBPs to dextran (8,14).…”

mentioning

confidence: 99%

Thermodynamics of the Binding of the C-Terminal Repeat Domain ofStreptococcus sobrinusGlucosyltransferase-I to Dextran

et al. 2007

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Glucosyltransferases (GTFs) secreted by mutans streptococci and some other lactic acid bacteria catalyze glucan synthesis from sucrose, and possess a C-terminal glucan-binding domain (GBD) containing homologous, directly repeating units. We prepared a series of C-terminal truncated forms of the GBD of Streptococcus sobrinus GTF-I and studied their binding to dextran by isothermal titration calorimetry. The binding of all truncates was strongly exothermic. Their titration curves were analyzed assuming that the GBD recognizes and binds to a stretch of dextran chain, not to a whole dextran molecule. Both the number of glucose units constituting the dextran stretch (n) and the accompanying enthalpy change (DeltaH degrees ) are proportional to the molecular mass of the GBD truncate, with which the Gibbs energy change calculated by the relation DeltaG degrees = -RT ln K (R, the gas constant; T, the absolute temperature; K, the binding constant of a truncate for a dextran stretch of n glucose units) also increases linearly. For the full-length GBD (508 amino acid residues), n = 33.9, K = 4.88 x 10(7) M-1, and DeltaH degrees = -289 kJ mol-1 at 25 degrees C. These results suggest that identical, independent glucose-binding subsites, each comprising 14 amino acid residues on average, are arranged consecutively from the GBD N-terminus. Thus, the GBD binds tightly to a stretch of dextran chain through the adding up of individually weak subsite/glucose interactions. Furthermore, the entropy change accompanying the GBD/dextran interaction as given by the relation DeltaS degrees = (DeltaG degrees - DeltaH degrees)/T has a very large negative value, probably because of a loss of the conformational freedom of dextran and GBD after binding.

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Deletions in the carboxyl-terminal region of Streptococcus gordonii glucosyltransferase affect cell-associated enzyme activity and sucrose-associated accumulation of growing cells

Cited by 12 publications

References 37 publications

Initial characterization of the Streptococcus gordonii htpX gene

Initial characterization of the Streptococcus gordonii htpX gene

Location of repeat elements in glucansucrases of Leuconostoc and Streptococcus species

Thermodynamics of the Binding of the C-Terminal Repeat Domain ofStreptococcus sobrinusGlucosyltransferase-I to Dextran

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