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R G Quivey

Publications and source records attributed to R G Quivey.

18 recordsLinked to original sources
PubMed2001

Genetics of acid adaptation in oral streptococci.

A growing body of information has provided insights into the mechanisms by which the oral streptococci maintain their niches in the human mouth. In at least one case, Streptococcus mutans, the organism apparently uses a panel of proteins to survive in acidic conditions while it promotes the formation of dental caries. Oral streptococci, which are not as inherently resistant to acidification, use protective schemes to ameliorate acidic plaque pH values. Existing information clearly shows that while the streptococci are highly related, very different strategies have evolved for them to take advantage of their particular location in the oral cavity. The picture that emerges is that the acid-adaptive regulatory mechanisms of the oral streptococci differ markedly from those used by Gram-negative bacteria. What future research must determine is the extent and complexity of the acid-adaptive systems in these organisms and how they permit the organisms to maintain themselves in the face of a low-pH environment and the microbial competition present in their respective niches.

Acids↗
PubMed2000

Shifts in membrane fatty acid profiles associated with acid adaptation of Streptococcus mutans.

Cells of Streptococcus mutans UA159 physiologically adapted to acidification during growth at pH 5 in glucose-limited chemostat cultures were enriched in mono-unsaturated and longer chain fatty acids compared with unadapted cells grown under the same conditions but at pH 7. Ratios of unsaturated to saturated fatty acids in the cells were, respectively, 1.2 and 0.3. Cyclopropane fatty acids were not detected. Streptococcus sobrinus 6715, which is known to have minimal acid-adaptive capacity, showed only minimal change in membrane fatty acids.

Adaptation, Physiological↗
PubMed2000

Adaptation of oral streptococci to low pH.

The strategies employed by oral streptococci to resist the inimical influences of acidification reflect the diverse and dynamic niches of the human mouth. All of the oral streptococci are capable of rapid degradation of sugar to acidic end-products. As a result, the pH value of their immediate environment can plummet to levels where glycolysis and growth cease. At this point, the approaches for survival in acid separate the organisms. Streptococcus mutans, for example, relies on its F-ATPase, to protect itself from acidification by pumping protons out of the cells. S. salivarius responds by degrading urea to ammonia and S. sanguis produces ammonia by arginolysis. The mechanisms by which these organisms regulate their particular escape route are now being explored experimentally. The picture that emerges is that the acid-adaptive regulatory mechanisms of the oral streptococci differ markedly from those employed by Gram-negative bacteria. What remains to be elucidated are the breadth of the acid-response systems in these organisms and how they permit the microbes to sustain themselves in the face of low pH and the bacterial competition present in their respective niches. In this article, we summarize reports concerning the means by which oral streptococci either utilize acidification to subdue their competitors or protect themselves until pH values return to a more favorable level.

Acids↗
PubMed1999

Physiologic homeostasis and stress responses in oral biofilms.

Studies performed since the early, 1970s have yielded tremendous amounts of information about the physiology, genetics, and interactions of oral bacteria. This pioneering work has provided a solid foundation to begin to apply the knowledge and technologies developed using suspended populations for studying oral bacteria under conditions that more closely mimic conditions in the oral cavity, in biofilms. Our current understanding of phenotypic capabilities of individual and complex mixtures of adherent oral bacteria is in its infancy. There is ample evidence that oral streptococci have different patterns of gene expression than planktonic cells, but we have little understanding of the basis for these observations. Even in biofilmforming bacteria with very well-developed genetic systems it is only very recently that genetic loci involved in biofilm formation and responses to surface growth have been identified. A comprehensive study of the physiology and gene expression characteristics of adherent oral bacteria not only will enhance our abilities to control oral diseases, but it will provide critical information that can be applied to a variety of other pathogenic microorganisms.

Adaptation, Physiological↗
PubMed1999

Induction of an AP endonuclease activity in Streptococcus mutans during growth at low pH.

The oral microbe Streptococcus mutans uses adaptive mechanisms to withstand the fluctuating pH levels in its natural environment. The regulation of protein synthesis is part of the mechanism of acid adaptation and tolerance in S. mutans. Here, we demonstrate that the organism's acid-inducible protein repertoire includes an AP endonuclease activity. This abasic site-specific endonuclease activity is present at greater levels in cells grown at low pH than in cells grown at pH 7, and is apparently independent of the RecA protein. Experiments using tetrahydrofuran or alpha-deoxyadenosine-containing substrates indicate that the activity induced at low pH may be similar to the activity of exonuclease III from E. coli. Acid-adapted S. mutans also shows an increased survival rate after exposure to near-UV radiation in both the wild type and a recA strain. Far-UV radiation resistance is observed in the wild type only. The endonuclease activity was purified approximately 500-fold from an S. mutans recA mutant strain grown at pH 5. Initial characterization revealed a 3' to 5' exonuclease activity, and showed additional functional similarities to DNA repair enzymes from other organisms.

Calcium Chloride↗
PubMed1996

Cloning and nucleotide sequence analysis of the Streptococcus mutans membrane-bound, proton-translocating ATPase operon.

The function of the membrane-bound ATPase in S. mutans is to regulate cytoplasmic pH values for the purpose of maintaining delta pH. Previous studies have shown that as part of its acid-adaptive ability, S. mutans is able to increase H(+)-ATPase levels in response to acidification. As part of the study of ATPase regulation in S. mutans, we have cloned the ATPase operon and determined its genetic organization. The structural genes from S. mutans were found to be in the order: c, a, b, delta, alpha, gamma, beta, and epsilon; where c and a were reversed from the more typical bacterial organization. The operon contained no I gene homologue but was preceded by a 239-bp intergenic space. Deduced aa sequences from open reading frames indicated that genes encoding homologues of glycogen phosphorylase and nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase flank the H(+)-ATPase operon, 5' and 3' respectively. Sequence analysis indicated the presence of three inverted-repeat nt sequences in the glgP-uncE intergenic space. Primer extension analysis of mRNAs prepared from batch-grown or steady-state cultures demonstrated that the transcriptional start site did not change as a function of culture pH value. The data suggest that potential stem-and-loop structures in the promoter region of the operon do not function to alter the starting position of ATPase-specific mRNA transcription.

Amino Acid Sequence↗
PubMed1996

Interactions of streptococcal glucosyltransferases with alpha-amylase and starch on the surface of saliva-coated hydroxyapatite.

The salivary pellicle consists of various proteins and glycoproteins which may interact with one another. Experiments were performed to elucidate the interactions of streptococcal glucosyltransferase (Gtf) enzymes with human salivary alpha-amylase in solution and on the surface of saliva-coated hydroxyapatite (SHA) beads. The Gtf enzymes -B, -C and -D, when immobilized on to SHA beads, reduced the activity of adsorbed amylase; GtfD showed the highest inhibition of salivary amylase activity. The presence of glucan produced by immobilized GtfD did not further reduce amylase activity. The amount of amylase adsorbed on to hydroxyapatite beads was reduced when salivary amylase was added simultaneously with any of the Gtf enzymes, suggesting that amylase and Gtfs may compete with each other for binding sites on hydroxyapatite. Starch hydrolysates produced by SHA-surface-bound salivary amylase were tested for their effect on glucan production from sucrose by Gtf enzymes in solution and on SHA beads; glucan production by SHA-immobilized GtfB was stimulated in the presence of starch hydrolysates. Glucan synthesized by SHA-immobilized GtfB in the presence of starch hydrolysates was less susceptible to hydrolysis by the fungal enzyme mutanase than was glucan made by SHA-immobilized GtfB in the absence of starch hydrolysates. Glucan production by GtfB associated with streptococci immobilized on to SHA was also enhanced in the presence of starch hydrolysates. The adhesion of oral micro-organisms to SHA coated with glucan made in the presence and absence of starch hydrolysates was investigated, and some bacteria displayed higher adhesion activities for the glucan made in the presence of the hydrolysates. Therefore, the interaction of amylase and Gtf enzymes on a SHA surface may modulate the formation of glucan and the adherence of oral micro-organisms.

Adsorption↗
PubMed1995

Acid adaptation in Streptococcus mutans UA159 alleviates sensitization to environmental stress due to RecA deficiency.

A RecA-deficient stain of Streptococcus mutans, isolated previously, was found to be more susceptible than the prototroph organism to acid killing and also showed reduced colony-forming ability on sucrose-containing medium. The deficient strain was able to grow in chemostat culture at a low pH value of 5 and did not show reduced capacity to produce acid in standard pH-drop experiments with excess glucose. Moreover, it was able to undergo an adaptive response when grown at a low pH to become more resistant to acid killing and also to killing by ultraviolet radiation or hydrogen peroxide. In fact, after adaptation, it was nearly as resistant as the prototroph strain. These findings were interpreted, in part, in terms of an acid-inducible DNA repair system which functions independently of RecA.

Adaptation, Physiological↗
PubMed1993

Raffinose-induced mutanase production from Trichoderma harzianum.

The enzyme alpha(1-->3),3-glucanohydrolase (referred to as mutanase) from the filamentous fungus Trichoderma harzianum OMZ 779 is capable of degrading the water-insoluble glucan in dental plaque. Previously, it was necessary to produce the glucan (referred to as mutan) in vitro for use as the sole carbon source and inducer of mutanase synthesis in fungal cultures. We report here that raffinose also induces the production of mutanase. The metabolism of raffinose differed from that of other sugars in metabolic end products and secreted protein profile. In addition to mutanase, we observed an approximately 15,000 M(r) protein that was also regulated by carbon source and by illumination conditions.

Enzyme Induction↗
PubMed1992

In vivo inactivation of the Streptococcus mutans recA gene mediated by PCR amplification and cloning of a recA DNA fragment.

The inactivation of the RecA protein in pathogenic oral streptococci would facilitate genetic analysis of potential virulence factors in these strains. Comparison of recA nucleotide (nt) sequences from a number of bacteria has suggested that two regions of highly conserved RecA amino acid (aa) sequence could be used as a basis for synthesizing degenerate oligodeoxyribonucleotide primers with which to amplify recA homologues from the streptococci. Accordingly, primer mixtures were used to amplify a 693-bp fragment of the Streptococcus mutans chromosome by PCR. The amplified fragment was cloned and its identity confirmed via hybridization to an Escherichia coli recA gene probe and by nt sequence determination. The recA homologue fragment from S. mutans GS-5 was 63% and 75% homologous to the deduced aa sequences of the E. coli and Bacillus subtilis RecA enzymes, respectively. The S. mutans recA fragment was mutagenized in vitro via insertional inactivation and returned to the chromosome using allelic exchange. The resulting strains of S. mutans were shown to be substantially more sensitive to UV irradiation than the wild-type strain. Further, the ability to incorporate linear markers into the chromosome was abolished in putative S. mutans recA strains, thus indicating the functional inactivation of RecA in these microorganisms.

Amino Acid Sequence↗
PubMed1991

Polymerase chain reaction amplification, cloning, sequence determination and homologies of streptococcal ATPase-encoding DNAs.

The highly conserved portion of the catalytic subunit (beta-subunit) of the membrane-bound, proton-translocating ATPase from three strains of oral streptococci has been amplified via the polymerase chain reaction. Hybridization studies demonstrated the existence of homology between Escherichia coli and Bacillus megaterium beta-subunit probes at the streptococcal DNA level. Highly degenerate primers, based on the E. coli and B. megaterium amino acid (aa) sequences, were used to amplify the homologues in Streptococcus mutans, S. sanguis and S. sobrinus. The 600 bp fragment from S. sobrinus has been cloned and its nucleotide (nt) sequence determined. Comparison of its nt and deduced aa sequence to that of E. coli and B. megaterium reveals a high degree of homology at the aa level.

Amino Acid Sequence↗
PubMed1991

A computer program designed for the analysis of data from rat caries studies.

The Keyes method for scoring dental caries in the rat animal model has been used as the basis for a computer-assisted scoring system on the Apple Macintosh personal computer that is described here. The program provides the ability to draw on diagrams of the rat molar dentition and to enter enamel lesion scores (L, E) and dentinal lesion scores (Ds, Dm, and Dx) for all aspects of the molars. The scores and drawings may be changed at any time. Total carious teeth and totals of L, E, Ds, Dm, and Dx are automatically calculated and presented upon user command. Error trapping for typographical errors is provided. In addition, the program provides automatic storage of the collected information, searching and sorting on any of the experimental parameters and on-disk help files that can be accessed from any place within the program. The capacity to obtain printed copy of any or all of the stored information is provided in the program. The program also enables the creation of external data files containing totals for carious teeth, in addition to L, E, Ds, Dm, and Dx totals for each animal of each group represented in the data base. These files are suitable for the transfer of data to commercially available programs for graphical and statistical analyses. The programming technique we describe could be extended to other model systems with standardized scoring for oral disease.

Animals↗
PubMed1988

Transposon mutagenesis and physiological analysis of strains containing inactivated form I and form II ribulose bisphosphate carboxylase/oxygenase genes in Rhodobacter sphaeroides.

Strains of Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) were constructed such that either the gene encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC-O) or the gene encoding form II RuBPC-O was inactivated. Both strains were capable of photoheterotrophic growth with malate as the electron donor, with only slight differences in growth rate and overall carboxylase specific activity compared with the wild-type strain. Photolithotrophic growth with 1.5% CO2 in hydrogen was also possible for R. sphaeroides strains containing only one of the two RuBPC-O enzyme forms, although the differences in growth rates between wild-type and carboxylase mutant strains were greater under these conditions. These results indicate that the two forms of RuBPC-O are independently regulated. In addition, the regulatory system governing RuBPC-O synthesis may, in some cases, compensate for the lack of the missing enzyme.

Carbon Dioxide↗
PubMed1984

Cloning and expression in Escherichia coli of the form II ribulose 1,5-bisphosphate carboxylase/oxygenase gene from Rhodopseudomonas sphaeroides.

The gene encoding the form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) from Rhodopseudomonas (R.) sphaeroides has been identified on a 3-kb EcoRI fragment and cloned into a broad-host-range, high-copy-number plasmid, using the gene from Rhodospirillum (Rs.) rubrum as a hybridization probe. Subclones of the gene from R. sphaeroides in pBR322 and pUC8 show substantial levels of expression and enzymatic activity in whole cells and crude cell extracts of Escherichia coli. This enzymatic activity has been shown to be similar in many respects to that of the protein purified from R. sphaeroides.

Cloning, Molecular↗