scholarly journals Markerless Multiple-Gene-Deletion System for Streptococcus mutans

2008 ◽  
Vol 74 (7) ◽  
pp. 2037-2042 ◽  
Author(s):  
Anirban Banerjee ◽  
Indranil Biswas
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Genetic Background ◽  
Double Mutant ◽  
Gene Deletion ◽  
Low Ph ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Modified Method ◽  
Oxidative Agents

ABSTRACT Inactivation or selective modification is essential to elucidate the putative function of a gene. The present study describes an improved Cre-loxP-based method for markerless multiple gene deletion in Streptococcus mutans, the principal etiological agent of dental caries. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. The effectiveness of this modified strategy was demonstrated by the construction of both single and double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans. HtrA and ClpP play key roles in the processing and maturation of several important proteins, including many virulence factors. Deletion of these genes resulted in reducing the organism's ability to withstand exposure to low pH and oxidative agents. The method described here is simple and efficient and can be easily implemented for multiple gene deletions with S. mutans and other streptococci.

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2020 ◽  
Vol 48 (8) ◽  
pp. 4585-4600
Author(s):  
Gabriel A Suárez ◽  
Kyle R Dugan ◽  
Brian A Renda ◽  
Sean P Leonard ◽  
Lakshmi Suryateja Gangavarapu ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Transposon Insertion ◽  
A Genome ◽  
Acinetobacter Baylyi Adp1 ◽  
Transposon Insertion Sequencing ◽  
Improved Methods

Abstract One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Regulation of Exoprotein Gene Expression by the Staphylococcus aureus cvfB Gene

2007 ◽  
Vol 75 (4) ◽  
pp. 1964-1972 ◽  
Author(s):  
Yasuhiko Matsumoto ◽  
Chikara Kaito ◽  
Daisuke Morishita ◽  
Kenji Kurokawa ◽  
Kazuhisa Sekimizu
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Genetic Background ◽  
Promoter Activity ◽  
Double Mutant ◽  
Gene Deletion ◽  
Protein A ◽  
Protease Production ◽  
Gene Deletion Mutant

ABSTRACT We previously reported that the cvfB gene (SA1223) of Staphylococcus aureus is responsible for the virulence of this pathogenic bacterium. We show here that the cvfB gene regulates exoprotein gene expression. In a cvfB gene deletion mutant, hemolysin, DNase, and protease production were decreased, whereas protein A expression was increased. The amount of RNAIII, the transcript from the P3 promoter in the agr locus that regulates the expression of various virulence factors, was also reduced in the cvfB mutant. In addition, P2 and P3 promoter activity in the agr locus was decreased in the mutant. Under the genetic background of the agr-null mutation, cvfB gene disruption decreased the production levels of DNase and protease. Moreover, the cvfB and agr double mutant was less virulent than the agr mutant in silkworms. These results suggest that the cvfB gene product contributes to the expression of virulence factors and to pathogenicity via both agr-dependent and agr-independent pathways.

Cell Wall Ultrastructure in Three Strains of a Cariogenic Streptococcus

1971 ◽  
Vol 29 ◽  
pp. 338-339
Author(s):  
M. J. Kramer ◽  
Alan L. Coykendall
Keyword(s):  
Cell Wall ◽  
Dental Caries ◽  
Streptococcus Mutans ◽  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Genetic Heterogeneity ◽  
Morphological Characteristics ◽  
Electron Microscopic ◽  
Dna Reassociation ◽  
Wall Ultrastructure

During the almost 50 years since Streptococcus mutans was first suggested as a factor in the etiology of dental caries, a multitude of studies have confirmed the cariogenic potential of this organism. Streptococci have been isolated from human and animal caries on numerous occasions and, with few exceptions, they are not typable by the Lancefield technique but are relatively homogeneous in their biochemical reactions. An analysis of the guanine-cytosine (G-C) composition of the DNA from strains K-1-R, NCTC 10449, and FA-1 by one of us (ALC) revealed significant differences and DNA-DNA reassociation experiments indicated that genetic heterogeneity existed among the three strains. The present electron microscopic study had as its objective the elucidation of any distinguishing morphological characteristics which might further characterize the respective strains.

scholarly journals Evaluation of Clinical, Biochemical and Microbiological Markers Related to Dental Caries

2021 ◽  
Vol 18 (11) ◽  
pp. 6049
Author(s):  
Maria D Ferrer ◽  
Salvadora Pérez ◽  
Aránzazu López Lopez ◽  
José Luis Sanz ◽  
Maria Melo ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Oral Health ◽  
Dental Caries ◽  
Biochemical Parameters ◽  
Salivary Flow ◽  
Low Ph ◽  
Tooth Brushing ◽  
Sugar Solution ◽  
Before And After ◽  
Caries Lesions

Our aim was to evaluate clinical, biochemical and microbiological markers related to dental caries in adults. A sample that consisted of 75 volunteers was utilized. The presence of caries and the presence of plaque and gingival indices were determined. Unstimulated salivary flow, pH, lactate, Streptococcus mutans and Streptococcus dentisani were measured in the participants’ plaque and saliva samples before and after rinsing with a sugar solution. Lactate in plaque was found to be significantly related to age, gender, tooth-brushing frequency, the presence of cavitated caries lesions and plaque and gingival indices (p < 0.05). The levels of S. dentisani in plaque increased significantly with tooth-brushing frequency (p = 0.03). Normalized plaque S. dentisani values and the percentage of S. dentisani were slightly higher in patients with basal lactic acid levels ≤ 50 mg/L. After rinsing with a sugary solution, the percentage of S. mutans levels in plaque were higher in patients with lactic acid levels > 350 mg/L (p = 0.03). Tooth-brushing frequency was the factor which was most associated with oral health. Women reflected better clinical and biochemical parameters than men. Low pH and high lactic acid levels tended to be associated with high caries rates. No association was found between bacteria levels and caries indices.

Streptococcus Mutans Strains in Mother-Child Pairs of Children with Early Childhood Caries

2019 ◽  
Vol 43 (4) ◽  
pp. 252-256 ◽  
Author(s):  
Priya Subramaniam ◽  
Revathy Suresh
Keyword(s):  
Early Childhood ◽  
Dental Caries ◽  
Streptococcus Mutans ◽  
Early Childhood Caries ◽  
Mutans Streptococci ◽  
Similar Distribution ◽  
Healthy Children ◽  
Only Children ◽  
Polymerase Chain ◽  
Childhood Caries

Objective: Dental caries is both an infectious and transmissible disease. Maternal transfer of Mutans Streptococci occurs at an early age and is important in the initiation of dental caries in children. The aim of this study was to identify certain strains of Streptococcus mutans in mother-child pairs, of children with early childhood caries. Study design: Sixty mother-child pairs of healthy children aged 18–36 months were selected. Mothers with high levels of Streptococcus mutans in their saliva and only children with ECC were included. Dental plaque samples were collected from mother-child pairs. The plaque samples were stored, transferred to the laboratory and analyzed for Streptococcus mutans strains c, f, e and k, present in mother-child pairs using Real time Polymerase Chain Reaction (PCR) technique. Data obtained was subjected to statistical analysis for level of similarity in Streptococcus mutans strains present in mother-child pairs. Results: A similar distribution of Streptococcus mutans strains c, f and k was identified in 28 mother-child pairs. Streptococcus mutans strain e was seen in 18 pairs. Conclusion: Less than 50% of mother-child pairs showed similarity in distribution of Streptococcus mutans strains.

scholarly journals Oxidative Stressors Modify the Response of Streptococcus mutans to Its Competence Signal Peptides

2017 ◽  
Vol 83 (22) ◽  
Author(s):  
Matthew De Furio ◽  
Sang Joon Ahn ◽  
Robert A. Burne ◽  
Stephen J. Hagen
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Dental Caries ◽  
Signaling Pathway ◽  
Streptococcus Mutans ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Oral Biofilm ◽  
Genetic Competence ◽  
Content Type

ABSTRACTThe dental caries pathogenStreptococcus mutansis continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence, and virulence ofS. mutans. Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence inS. mutans. Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (H2O2), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction ofcomXin a progressive and cumulative fashion, whereas the response to H2O2displayed a strong threshold behavior. Low concentrations of H2O2had little effect on induction ofcomXor the bacteriocin genecipB, but expression of these genes declined sharply if extracellular H2O2exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to H2O2, depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and H2O2affect theS. mutanscompetence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others.IMPORTANCEStreptococcus mutansinhabits the oral biofilm, where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth ofS. mutansand its important virulence-associated behaviors, such as genetic competence.S. mutanscompetence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influenceS. mutanscompetence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study, we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single-cell level. Our data show that different reactive oxygen species have different effects onS. mutanscompetence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others.

scholarly journals Essential Roles of thesppRAFructose-Phosphate Phosphohydrolase Operon in Carbohydrate Metabolism and Virulence Expression byStreptococcus mutans

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Lin Zeng ◽  
Robert A. Burne
Keyword(s):  
Dental Caries ◽  
Organic Acids ◽  
Streptococcus Mutans ◽  
Biochemical Characterization ◽  
Constitutive Expression ◽  
Extracellular Dna ◽  
Tooth Surface ◽  
Oral Diseases ◽  
Sugar Phosphate ◽  
Content Type

ABSTRACTThe dental caries pathogenStreptococcus mutanscan ferment a variety of sugars to produce organic acids. Exposure ofS. mutansto certain nonmetabolizable carbohydrates, such as xylitol, impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress inS. mutanswas demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here, we studied an operon inS. mutans,sppRA, which was highly expressed in thefruKmutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg2+and Mn2+but inhibited by NaF. SppR, a DeoR family regulator, repressed the expression of thesppRAoperon to minimum levels in the absence of the fructose-derived metabolite F-1-P and likely also F-6-P. The accumulation of F-1-P, as a result of growth on fructose, not only inducedsppAexpression, but it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression ofsppA, via a plasmid or by deletingsppR, greatly alleviated fructose-induced stress in afruKmutant, enhanced resistance to xylitol, and reversed the effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show thatS. mutansis capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors.IMPORTANCEStreptococcus mutansis a major etiologic agent for dental caries, primarily due to its ability to form biofilms on the tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon inS. mutansthat regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways that fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases.

scholarly journals Antimicrobial activity of Cymbopogon citratus (Poaceae) essential oil on Streptococcus mutans biofilm and cytotoxic effect on keratinocytes and fibroblasts

2018 ◽  
Vol 66 (4) ◽  
pp. 1519
Author(s):  
Mailen Ortega Cuadros ◽  
Adriana Patricia Tofiño Rivera ◽  
Luciano Jose Merini ◽  
Maria Cecilia Martinez Pabon
Keyword(s):  
Antimicrobial Activity ◽  
Dental Caries ◽  
Essential Oil ◽  
Streptococcus Mutans ◽  
Human Fibroblasts ◽  
Positive Control ◽  
Negative Control ◽  
Cymbopogon Citratus ◽  
Significant Difference ◽  
Efficient Alternative

Dental caries is a pathology of multifactorial origin and currently natural products are an efficient alternative treatment; The work sought to evaluate the antimicrobial activity of the Cymbopogon citratus essential oil and the citral and myrcene components against Streptococcus mutans ATCC UA159, as well as their cytotoxicity on keratinocytes and human fibroblasts. The viability effect against Streptococcus mutans on biofilms was evaluated through exposure to the three substances by using the MBEC technique-high-throughput at concentrations of 1, 0.1, and 0.01 µg/mL and chlorhexidine as positive control. The cytotoxicity of the compounds was evaluated on keratinocytes and fibroblasts through the MTT reduction technique, using 0.5 mM H2O2 as cell-death control (negative control) and ethanol 1% as vehicle control (positive control). The three substances evaluated had effects on the viability of Streptococcus mutans with mortality between 74% and 96%, without significant difference among them (p > 0.393); additionally, no cytotoxicity was evident on keratinocytes and fibroblasts in a 24-h treatment. The substances evaluated showed significant antimicrobial effects; hence, these should be studied further as potential co-adjuvants to prevent dental caries that cause minor adverse effects

Sign in / Sign up

Export Citation Format

Share Document