Streptococcus mutans fructosyltransferase (ftf) and glucosyltransferase (gtfBC) operon fusion strains in continuous culture

Wexler, D. L.; Hudson, Michael; Burne, Robert A.

doi:10.1128/iai.61.4.1259-1267.1993

Cited by 65 publications

(34 citation statements)

References 50 publications

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“…In a previous study, the expression of the gtfBC and ftf genes was examined at steady state with limiting glucose at a dilution rate of 0.05 or 0.3 h À1 , and pH 7 and 6 [8]. The results presented here confirm and significantly extend those results.…”

Section: Resultssupporting

confidence: 85%

RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression inStreptococcus mutans

Browngardt¹,

Wen²,

Burne³

2004

FEMS Microbiology Letters

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Glucosyltransferases (Gtfs) and fructosyltransferase (Ftf), and the exopolysaccharides they produce, facilitate bacterial adherence and biofilm formation, and enhance the virulence of Streptococcus mutans. In this study, we used continuous chemostat cultures and reporter gene fusions to study the expression of ftf and gtfBC in response to carbohydrate availability and pH, and to asses the role of a protein similar to catabolite control protein A (CcpA), RegM, in regulation of these genes. Expression of ftf was efficient at pH 7.0 and 6.0, but was repressed at pH 5.0 under glucose-excess conditions. At pH 7.0, ftf expression was 5-fold lower under glucose-limiting conditions than in cells growing with an excess of glucose. Expression of gtfBC was also sensitive, albeit to a lesser extent, to pH and glucose availability. Inactivation of regM resulted in decreases of as much as 10-fold in both ftf and gtfBC expression, depending on growth conditions. These findings reinforce the importance of pH and carbohydrate availability for expression of two primary virulence attributes of S. mutans and reveal a critical role for RegM in regulation of expression of both gtfBC and ftf.

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Section: Resultssupporting

confidence: 85%

RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression inStreptococcus mutans

Browngardt¹,

Wen²,

Burne³

2004

FEMS Microbiology Letters

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“…Western blot analysis S. mutans cells grown in continuous culture were collected by centrifugation, washed and lysed using a homogenizer obtained from BioSpec Products, as described previously (Wexler et al, 1993). Protein lysates were collected by centrifugation and subjected to SDS-PAGE electrophoresis along with high molecular mass protein markers (LTI).…”

Section: Isolation Of Total Rnamentioning

confidence: 99%

“…Cells were collected by centrifugation, washed and resuspended in 1.0 ml of 10 mM Tris-HCl buffer, pH 7.4. Clarified whole-cell lysates were prepared by homogenizing the cell suspension in the presence of a one-third volume of glass beads (average diameter ¼ 0.1 mm) as detailed previously (Wexler et al, 1993), and chloramphenicol acetyltransferase enzyme (CAT) activity was measured. The determination of CAT specific activities was accomplished using the spectrophotometric assay of Shaw (1979).…”

Section: Northern Analysismentioning

confidence: 99%

Transcriptional analysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification

Jayaraman

Penders

Burne

1997

Molecular Microbiology

113

109

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SummaryThe dental pathogen Streptococcus mutans persists and causes diseases in highly dynamic environments and gains a selective ecological advantage in environmental conditions that frequently exceed the limits for growth of the organism, particularly with regard to environmental pH. The goal of this study was to begin a molecular genetic analysis of a major stress protein, DnaK/Hsp70, to begin to understand how stress responses are regulated in this lactic acid bacterium and to establish a relationship between dnaK gene expression and exposure to acidic environments. Cloning and nucleotide sequence analysis revealed that the dnaK gene is preceded by, and is in an operonlike arrangement with, the hrcA and grpE genes, although intergenic spacing was unlike that described in other bacteria. An inverted repeat (a CIRCE element) was identified by sequence analysis and, using primer extensions, a heat shock-responsive, A -type promoter, P1, 5Ј to the hrcA gene, and a B -type promoter, 5Ј to the grpE translational start site, were identified. No promoters were detected between grpE and dnaK. A strain carrying a strongly polar insertion in the hrcA gene had markedly diminished levels of dnaK mRNA, indicating that dnaK was transcribed as part of an operon from P1, and to a lesser extent from P2. Results from physiological manipulation of S. mutans in continuous chemostat culture demonstrated that steadystate levels of S. mutans dnaK mRNA and DnaK protein were (i) increased in response to acid shock; (ii) elevated in acid 'adapted' cells; and (iii) induced in response to alkali shock of acid 'adapted' cells. In all cases, increased amounts of dnaK mRNA could be correlated with enhanced transcription from P1. This study provides the first detailed analysis of the expression of a heat shock gene from an oral isolate, and the evidence provided suggests that B -like promoters may also be involved in class I heat shock gene expression in some Gram-positive organisms.

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“…S. mutans cells that had been heat shocked at 40°C or 42°C for 1 h were collected by centrifugation, washed and lysed as previously described [26]. The protein lysates were collected by centrifugation and subjected to SDS-PAGE electrophoresis along with high molecular mass protein markers (Life Technologies Gibco BRL).…”

Section: Western Blot Analysismentioning

confidence: 99%

DnaK expression in response to heat shock of Streptococcus mutans

Jayaraman

1995

FEMS Microbiology Letters

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The oral pathogen, Streptococcus mutans, persistently colonizes human hosts and initiates oral disease despite extreme variations in environmental conditions. To begin to investigate the role of the stress protein, DnaK (Hsp70), in environmental stress responses by S. mutans, pulse-chase experiments were initially used to establish that a functional heat shock response existed in this organism. A C-terminal fragment of the S. mutans dnaK gene was cloned and engineered to be expressed with a histidine tag. Using the recombinant DnaK protein that had been purified by nickel affinity chromatography, an antibody specific for the S. mutans DnaK protein was generated to analyse DnaK expression under homeostatic and heat shock conditions. Western blot analysis indicated that the anti-recombinant DnaK antibody specifically recognized a protein (molecular mass approx. 68 kDa) which was induced in response to thermal stress. Elucidating the role of DnaK in responses by S. mutans to various environmental stressors will provide a better understanding of how DnaK is involved in survival of extreme environments and the contribution of the DnaK protein to the virulence of S. mutans.

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Streptococcus mutans fructosyltransferase (ftf) and glucosyltransferase (gtfBC) operon fusion strains in continuous culture

Cited by 65 publications

References 50 publications

RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression inStreptococcus mutans

RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression inStreptococcus mutans

Transcriptional analysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification

DnaK expression in response to heat shock of Streptococcus mutans

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