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1.
Mol Oral Microbiol ; 32(6): 517-525, 2017 12.
Article in English | MEDLINE | ID: mdl-28744965

ABSTRACT

Our previous studies showed that brpA in Streptococcus mutans, which encodes a member of the LytR-CpsA-Psr family of proteins, can be co-transcribed with brpB upstream as a bicistronic operon, and the intergenic region also has strong promoter activity. To elucidate how brpA expression is regulated, the promoter regions were analyzed using polymerase chain reaction-based deletions and site-directed mutagenesis and a promoterless luciferase gene as a reporter. Allelic exchange mutagenesis was also used to examine genes encoding putative trans-acting factors, and the impact of such mutations on brpA expression was analyzed by reporter assays. Multiple elements in the short brpA promoter (nucleotide -1 to -344 relative to start cordon ATG) were shown to have a major impact on brpA expression, including an FNR-box, for a putative binding site of an FNR-type of transcriptional regulator. When compared with the intact brpA promoter, mutations of the highly conserved nucleotides in FNR-box from TTGATgtttAcCtt to TTACAgaaaGtTac resulted in 1362-fold increases of luciferase activity (P < .001), indicative of the FNR-box-mediated repression as a major mechanism in regulation of brpA expression. When luciferase reporter was fused to the upstream brpBA promoter (nucleotides -784 to -1144), luciferase activity was decreased by 4.5-fold (P < .001) in the brpA mutant, TW14D, and by 67.7-fold (P < .001) in the brpB mutant, JB409, compared with the wild-type, UA159. However, no such effects were observed when the reporter gene was fused to the short brpA promoter and its derivatives. These results also suggest that brpA expression in S. mutans is auto-regulated through the upstream brpBA promoter.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Streptococcus mutans/genetics , Streptococcus mutans/metabolism , Binding Sites , DNA, Bacterial/genetics , Gene Expression Profiling , Luciferases/genetics , Luciferases/metabolism , Mutagenesis, Site-Directed , Operon , Promoter Regions, Genetic , Transcription, Genetic
2.
Mol Oral Microbiol ; 31(2): 115-24, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26172563

ABSTRACT

The NAD(+) and NADH-sensing transcriptional regulator Rex is widely conserved across gram-positive bacteria. Rex monitors cellular redox poise and controls the expression of genes/operons involved in diverse pathways including alternative fermentation, oxidative stress responses, and biofilm formation. The oral cavity undergoes frequent and drastic fluctuations in nutrient availability, pH, temperature, oxygen tension, saliva, and shear forces. The oral streptococci are major colonizers of oral mucosa and tooth surfaces and include commensals as well as opportunistic pathogens, including the primary etiological agent of dental caries, Streptococcus mutans. Current understanding of the Rex regulon in oral bacteria is mostly based on studies in S. mutans and endodontic pathogen Enterococcus faecalis. Indeed, other oral bacteria encode homologs of the Rex protein and much is to be gleaned from more in-depth studies. Our current understanding has Rex positioned at the interface of oxygen and energy metabolism. In biofilms, heterogeneous oxygen tension influences the ratio of intracellular NADH and NAD(+) , which is finely tuned through glycolysis and fermentation. In S. mutans, Rex regulates the expression of glycolytic enzyme NAD(+) -dependent glyceraldehyde 3-phosphate dehydrogenase, and NADH-dependent fermentation enzymes/complexes lactate dehydrogenase, pyruvate dehydrogenase, alcohol-acetaldehyde dehydrogenase, and fumarate reductase. In addition, Rex controls the expression of NADH oxidase, a major enzyme used to eliminate oxidative stress and regenerate NAD(+) . Here, we summarize recent studies carried out on the Rex regulators in S. mutans and E. faecalis. This research has important implications for understanding how Rex monitors redox balance and optimizes fermentation pathways for survival and subsequent pathogenicity.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Dental Caries/microbiology , Gram-Positive Bacteria/physiology , Transcription Factors/physiology , Biofilms/drug effects , Biofilms/growth & development , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Gram-Positive Bacteria/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Mol Oral Microbiol ; 30(4): 255-68, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25421565

ABSTRACT

Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA-binding N-terminal domain and a C-terminal domain highly homologous to the pyridoxal phosphate-dependent aspartate aminotransferases. A pdxR-deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR-deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real-time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B6 biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR-deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B6 . Further studies revealed that although S. mutans is known to require vitamin B6 to grow in defined medium, B6 vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B6 metabolism, acid tolerance response and biofilm formation.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Streptococcus mutans/genetics , Streptococcus mutans/metabolism , Transcription Factors/genetics , Vitamin B 6/metabolism , Adhesins, Bacterial/genetics , Amino Acids/metabolism , Biofilms/drug effects , Culture Media/chemistry , Gene Expression Regulation, Bacterial , Glucosyltransferases/genetics , Humans , Mutation , Operon , Pyridoxal/pharmacology , Pyridoxal Kinase/genetics , Real-Time Polymerase Chain Reaction , Streptococcus mutans/drug effects , Streptococcus mutans/growth & development , Stress, Physiological/genetics , Transaminases/genetics , Transcription Factors/metabolism , Vitamin B 6/biosynthesis , Vitamin B 6/genetics
4.
J Bacteriol ; 196(12): 2166-77, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24682329

ABSTRACT

NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD(+). The critical nature of Nox is 2-fold: it serves to regenerate NAD(+), a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD(+) have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD(+) affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress.


Subject(s)
Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Multienzyme Complexes/metabolism , NADH, NADPH Oxidoreductases/metabolism , NAD/metabolism , Oxygen/pharmacology , Streptococcus mutans/enzymology , Gene Deletion , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Multienzyme Complexes/genetics , NADH, NADPH Oxidoreductases/genetics , Promoter Regions, Genetic , Streptococcus mutans/genetics , Streptococcus mutans/metabolism
5.
Appl Environ Microbiol ; 78(8): 2914-22, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22327589

ABSTRACT

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by Streptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of brpA is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a Galleria mellonella (wax worm) model, BrpA deficiency was shown to diminish the virulence of S. mutans OMZ175, which, unlike S. mutans UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of S. mutans.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Streptococcus mutans/physiology , Stress, Physiological , Animals , Anti-Bacterial Agents/pharmacology , Artificial Gene Fusion , Bacterial Proteins/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Deletion , Genes, Reporter , Lepidoptera/microbiology , Luciferases/analysis , Luciferases/genetics , Microarray Analysis , Peptidoglycan/metabolism , Streptococcus mutans/drug effects , Streptococcus mutans/genetics , Streptococcus mutans/pathogenicity , Survival Analysis , Virulence
6.
Mol Oral Microbiol ; 26(1): 2-18, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21214869

ABSTRACT

We previously reported that LuxS in Streptococcus mutans is involved in stress tolerance and biofilm formation. In this study, flowcells and confocal laser scanning microscopy were used to further examine the effects of LuxS-deficiency on biofilm formation. Similar to the wild-type strain (UA159), a strain deficient in LuxS (TW26D) bound efficiently to the flowcells and formed microcolonies 4 h after inoculation. Unlike UA159, which accumulated and formed compact, evenly distributed biofilms after 28 h, TW26D showed only loose, sporadic, thin biofilms. DNA microarray analysis revealed alterations in transcription of more than 60 genes in TW26D biofilms by at least 1.5-fold (P < 0.001). Among the upregulated genes were those for sugar-specific enzymes II of the phosphotransferase (PTS) system and the atp operon, which codes for the proton-pumping F-ATPase. Of the downregulated genes, several encode proteins with putative functions in DNA repair. Mutation of selected genes caused severe defects in the ability of the mutants to tolerate low pH and oxidative stress. These results provide additional proof that LuxS-deficiency causes global alterations in the expression of genes central to biofilm formation and virulence of S. mutans, including those involved in energy metabolism, DNA repair and stress tolerance.


Subject(s)
Bacterial Proteins/genetics , Biofilms/growth & development , Carbon-Sulfur Lyases/genetics , Gene Expression Profiling/methods , Streptococcus mutans/enzymology , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/genetics , Bacteriological Techniques , DNA Repair/genetics , Down-Regulation , Humans , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration , Image Processing, Computer-Assisted , Microscopy, Confocal , Mutation/genetics , Oligonucleotide Array Sequence Analysis , Operon/genetics , Oxidants/pharmacology , Oxidative Stress/genetics , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Proton Pumps/genetics , Transcription, Genetic/genetics , Up-Regulation , Virulence/genetics
7.
Biorheology ; 23(1): 51-61, 1986.
Article in English | MEDLINE | ID: mdl-3719091

ABSTRACT

A theoretical and experimental study concerning two-component fluid pulsating flow through cylindrical ducts having a slight constriction is presented. The model corresponds to blood flows through small diameter vessels (smaller than 400 micron) affected by a singular stenosis. The theoretical approach is based on a asymptotical expansion of the stream function. The physical hypotheses used were based on findings from simultaneous visualization methods. The influence of geometrical, hydrodynamical and structural parameters is systematically examined and related to velocity profiles, hydrostatic pressure, surface stresses.


Subject(s)
Constriction, Pathologic/physiopathology , Microcirculation , Models, Biological , Blood Flow Velocity , Blood Viscosity , Humans , Mathematics
8.
Biorheology Suppl ; 1: 151-3, 1984.
Article in English | MEDLINE | ID: mdl-6591969

ABSTRACT

This text recalls the main sequences of a videofilm in which we have looked at the blood flow through small diameter tubes, varying from 200 to 500 micron diameter, using a video equipment where the camera is fixed to a phase contrast microscope. Flows of two fluid model through converging-diverging small tubes have been studied both theoretically and experimentally, where we have considered the influence of tube diameter, stenosis, flow rate, haematocrit upon the red blood cells repartitions and the thickness of peripheral plasma layer.


Subject(s)
Blood Physiological Phenomena , Rheology , Blood Flow Velocity , Constriction , Hematocrit , In Vitro Techniques , Plasma/physiology
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