scholarly journals Repression of the TreR transcriptional regulator in Streptococcus mutans by the global regulator, CcpA

2021 ◽  
Vol 368 (3) ◽  
Author(s):  
E L Lindsay ◽  
R C Faustoferri ◽  
R G Quivey
Keyword(s):  
Streptococcus Mutans ◽  
Sugar Metabolism ◽  
Regulatory Elements ◽  
Etiologic Agent ◽  
Oral Microbiome ◽  
Global Regulator ◽  
Mobility Shift ◽  
Elevated Expression ◽  
Tolerance Response ◽  
Local Regulator

ABSTRACT Streptococcus mutans, the etiologic agent of dental caries in humans, is considered a dominating force in the oral microbiome due to its highly-evolved propensity for survival. The oral pathogen encodes an elaborate array of regulatory elements, including the carbon catabolite-responsive regulator, CcpA, a global regulator key in the control of sugar metabolism and in stress tolerance response mechanisms. The recently characterized trehalose utilization operon, integral for the catabolism of the disaccharide trehalose, is controlled by a local regulator, TreR, which has been implicated in a number of cellular functions outside of trehalose catabolism. Electrophoretic mobility shift assays demonstrated that CcpA bound a putative cre site in the treR promoter. Loss of ccpA resulted in elevated expression of treR in cultures of the organism grown in glucose or trehalose, indicating that CcpA not only acts as a repressor of trehalose catabolism genes, but also the local regulator. The loss of both CcpA and TreR in S. mutans resulted in an impaired growth rate and fitness response, supporting the hypothesis that these regulators are involved in carbon catabolism control and in induction of components of the organism's stress response.

scholarly journals Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499 ◽  
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

scholarly journals Two different negative regulatory elements control the transcription of T-cell activation gene 3 in activated mast cells

1997 ◽  
Vol 323 (2) ◽  
pp. 511-519 ◽  
Author(s):  
Chad K. OH ◽  
Markus NEURATH ◽  
Jeong-Je CHO ◽  
Tekli SEMERE ◽  
Dean D. METCALFE
Keyword(s):  
Protein Synthesis ◽  
T Cell ◽  
Mast Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Integrin Gene

T-cell activation gene 3 (TCA3) encodes a β-chemokine that is transcriptionally regulated in mast cells; the gene has a functional NF-κB element at positions -194 to -185. The 5´-flanking region of this gene is also known to have a negative regulatory region between -2057 and -1342. To characterize the negative regulatory elements (NREs), this region was sequenced and then digested by HindIII enzyme into two fragments, NRE-1 (-2057 to -1493) and NRE-2 (-1492 to -1342). Both NRE-1 and NRE-2 in the 5´–3´ orientation inhibited chloramphenicol acetyltransferase (CAT)-protein synthesis by a TCA3–CAT construct transfected into mast cells that were then activated. Only NRE-1 inhibited CAT-protein synthesis in the 3´–5´ orientation. Further deletion of the 5´ region of NRE-1 partially abolished the inhibitory activity. Both NRE-1 and NRE-2 inhibited the activity of a CD20–CAT construct independent of cell activation. Electrophoretic mobility shift assays showed DNA–protein complex formation with subsequences (CCCCCATTCT) of NRE-1 (NRE-1a) and (CCATGA) of NRE-2 (NRE-2b). NRE-1a appears to be novel. NRE-2b is identical with a putative silencer motif in the αIIb integrin gene. Site-directed mutagenesis demonstrated that both NRE-1a and NRE-2b are important in the negative regulation of TCA3 promoter activity. In vivo ligation-mediated PCR footprinting of the NRE-2 region revealed protection between -1372 and -1354, which contains NRE-2b. The data thus demonstrate identity of a silencer motif, here termed NRE-2b, in both the αIIb integrin gene and the TCA3, and that this silencer region in mast cells is functional both in vivoand in vitro. Further, evidence is presented that the promoter for TCA3 contains a novel silencer motif, termed NRE-1a, characterized by a CT-rich sequence.

VicRK and CovR polymorphisms in Streptococcus mutans strains associated with cardiovascular infections

2021 ◽  
Vol 70 (12) ◽  
Author(s):  
Letícia T. Oliveira ◽  
Lívia A. Alves ◽  
Erika N. Harth-Chu ◽  
Ryota Nomura ◽  
Kazuhiko Nakano ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Type Species ◽  
Virulence Gene ◽  
Common Species ◽  
Oral Microbiome ◽  
Nucleotide Polymorphisms ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Coding Regions ◽  
Link Type

Introduction. Streptococcus mutans , a common species of the oral microbiome, expresses virulence genes promoting cariogenic dental biofilms, persistence in the bloodstream and cardiovascular infections. Gap statement. Virulence gene expression is variable among S. mutans strains and controlled by the transcription regulatory systems VicRK and CovR. Aim. This study investigates polymorphisms in the vicRK and covR loci in S. mutans strains isolated from the oral cavity or from the bloodstream, which were shown to differ in expression of covR, vicRK and downstream genes. Methodology. The transcriptional activities of covR, vicR and vicK were compared by RT-qPCR between blood and oral strains after exposure to human serum. PCR-amplified promoter and/or coding regions of covR and vicRK of 18 strains (11 oral and 7 blood) were sequenced and compared to the reference strain UA159. Results. Serum exposure significantly reduced covR and vicR/K transcript levels in most strains (P<0.05), but reductions were higher in oral than in blood strains. Single-nucleotide polymorphisms (SNPs) were detected in covR regulatory and coding regions, but SNPs affecting the CovR effector domain were only present in two blood strains. Although vicR was highly conserved, vicK showed several SNPs, and SNPs affecting VicK regions important for autokinase activity were found in three blood strains. Conclusions. This study reveals transcriptional and structural diversity in covR and vicR/K, and identifies polymorphisms of functional relevance in blood strains, indicating that covR and vicRK might be important loci for S. mutans adaptation to host selective pressures associated with virulence diversity.

scholarly journals Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

1990 ◽  
Vol 10 (6) ◽  
pp. 2653-2659 ◽  
Author(s):  
D Kardassis ◽  
M Hadzopoulou-Cladaras ◽  
D P Ramji ◽  
R Cortese ◽  
V I Zannis ◽  
...  
Keyword(s):  
Binding Site ◽  
Regulatory Elements ◽  
Apob Gene ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Dna Binding Site ◽  
Gene Definition ◽  
Definition Of ◽  
Electrophoretic Mobility Shift Assays

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

scholarly journals Cariogenic Streptococcus mutans produces strain-specific antibiotics that impair commensal colonization

2019 ◽  
Author(s):  
Xiaoyu Tang ◽  
Yuta Kudo ◽  
Jonathon Baker ◽  
Sandra LaBonte ◽  
Peter A. Jordan ◽  
...  
Keyword(s):  
Small Molecules ◽  
Streptococcus Mutans ◽  
Dental Plaque ◽  
Biosynthetic Pathway ◽  
Etiologic Agent ◽  
Commensal Bacteria ◽  
Tooth Decay ◽  
Tetramic Acid ◽  
Globally Distributed ◽  
Caries Tooth

Streptococcus mutans is a common constituent of dental plaque and an etiologic agent of dental caries (tooth decay). Here we elucidate a biosynthetic pathway, encoded by globally distributed strains of S. mutans, which produces a series of bioactive small molecules including reutericyclin and two N-acyl tetramic acid analogues active against oral commensal bacteria. This pathway may provide S. mutans with a competitive advantage, promoting dysbiosis and caries pathogenesis.

scholarly journals Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Lidia A. Daimiel ◽  
María E. Fernández-Suárez ◽  
Sara Rodríguez-Acebes ◽  
Lorena Crespo ◽  
Miguel A. Lasunción ◽  
...  
Keyword(s):  
Cellular Response ◽  
Binding Protein ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Gene Characterization ◽  
Cis Acting

DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.

scholarly journals Metabolic Modeling of Streptococcus mutans Reveals Complex Nutrient Requirements of an Oral Pathogen

mSystems ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Kenan Jijakli ◽  
Paul A. Jensen
Keyword(s):  
Streptococcus Mutans ◽  
Single Gene ◽  
Metabolic Model ◽  
The United States ◽  
Defined Medium ◽  
Oral Microbiome ◽  
Tooth Decay ◽  
Oral Pathogen ◽  
Content Type ◽  
A Genome

ABSTRACT Streptococcus mutans is a Gram-positive bacterium that thrives under acidic conditions and is a primary cause of tooth decay (dental caries). To better understand the metabolism of S. mutans on a systematic level, we manually constructed a genome-scale metabolic model of the S. mutans type strain UA159. The model, called iSMU, contains 675 reactions involving 429 metabolites and the products of 493 genes. We validated iSMU by comparing simulations with growth experiments in defined medium. The model simulations matched experimental results for 17 of 18 carbon source utilization assays and 47 of 49 nutrient depletion assays. We also simulated the effects of single gene deletions. The model’s predictions agreed with 78.1% and 84.4% of the gene essentiality predictions from two experimental data sets. Our manually curated model is more accurate than S. mutans models generated from automated reconstruction pipelines and more complete than other manually curated models. We used iSMU to generate hypotheses about the S. mutans metabolic network. Subsequent genetic experiments confirmed that (i) S. mutans catabolizes sorbitol via a sorbitol-6-phosphate 2-dehydrogenase (SMU_308) and (ii) the Leloir pathway is required for growth on complex carbohydrates such as raffinose. We believe the iSMU model is an important resource for understanding the metabolism of S. mutans and guiding future experiments. IMPORTANCE Tooth decay is the most prevalent chronic disease in the United States. Decay is caused by the bacterium Streptococcus mutans, an oral pathogen that ferments sugars into tooth-destroying lactic acid. We constructed a complete metabolic model of S. mutans to systematically investigate how the bacterium grows. The model provides a valuable resource for understanding and targeting S. mutans’ ability to outcompete other species in the oral microbiome.

scholarly journals Transcription factor B cell lineage-specific activator protein regulates the gene for human X-box binding protein 1.

1996 ◽  
Vol 183 (2) ◽  
pp. 393-401 ◽  
Author(s):  
A M Reimold ◽  
P D Ponath ◽  
Y S Li ◽  
R R Hardy ◽  
C S David ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cell ◽  
Cell Lines ◽  
Binding Protein ◽  
Cell Lineage ◽  
Regulatory Elements ◽  
Target Sequence ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Activator Protein

The transcription factor human X-box binding protein 1 (hXBP-1) is a basic region-leucine zipper protein implicated in the regulation of major histocompatibility complex class II gene expression as well as in exocrine gland and skeletal development. Multiple regulatory elements in the hXBP-1 promoter lie 3' to the transcription start site, including the hX2 site, whose core sequence is an AP-1-like element identical to the hXBP-1 target sequence in the HLA-DRA promoter. One complex identified by electrophoretic mobility shift assay (EMSA), complex 3, was previously shown to protect the hX2 site and more 3' bases. Sequence analysis now shows that this region contains a consensus binding site for transcription factor BSAP (B cell lineage-specific activator protein). Complex 3 and BSAP have identical cell-type specificities, as they are found only in pre-B and mature B cell lines. In EMSAs, BSAP antibody specifically recognized complex 3, and in vitro translated BSAP could bind to an hXBP promoter fragment. Cotransfections using an hXBP-1 reporter construct indicated that BSAP downregulates the hXBP-1 promoter. The highest levels of hXBP-1 mRNA were found when BSAP was not expressed, in pre-Pro-B cells and in plasma cell lines. In addition, hXBP-1 and BSAP levels were inversely correlated along the early stages of B cell development. In the regulation of the hXBP-1 promoter, a strong positive transcriptional influence at the hX2 site is opposed by the downregulatory actions of BSAP.

scholarly journals Transcriptome analysis reveals that ClpXP proteolysis controls key virulence properties of Streptococcus mutans

Microbiology ◽  
2011 ◽  
Vol 157 (10) ◽  
pp. 2880-2890 ◽  
Author(s):  
Jessica K. Kajfasz ◽  
Jacqueline Abranches ◽  
José A. Lemos
Keyword(s):  
Streptococcus Mutans ◽  
Molecular Mechanisms ◽  
Transformation Efficiency ◽  
Slow Growth ◽  
Malolactic Fermentation ◽  
Global Regulator ◽  
Enhanced Expression ◽  
Upregulated Genes ◽  
Virulence Properties

The ClpXP proteolytic complex is critical for maintaining cellular homeostasis, as well as expression of virulence properties. However, with the exception of the Spx global regulator, the molecular mechanisms by which the ClpXP complex exerts its influence in Streptococcus mutans are not well understood. Here, microarray analysis was used to provide novel insights into the scope of ClpXP proteolysis in S. mutans. In a ΔclpP strain, 288 genes showed significant changes in relative transcript amounts (P≤0.001, twofold cut-off) as compared with the parent. Similarly, 242 genes were differentially expressed by a ΔclpX strain, 113 (47 %) of which also appeared in the ΔclpP microarrays. Several genes associated with cell growth were downregulated in both mutants, consistent with the slow-growth phenotype of the Δclp strains. Among the upregulated genes were those encoding enzymes required for the biosynthesis of intracellular polysaccharides (glg genes) and malolactic fermentation (mle genes). Enhanced expression of glg and mle genes in ΔclpP and ΔclpX strains correlated with increased storage of intracellular polysaccharide and enhanced malolactic fermentation activity, respectively. Expression of several genes known or predicted to be involved in competence and mutacin production was downregulated in the Δclp strains. Follow-up transformation efficiency and deferred antagonism assays validated the microarray data by showing that competence and mutacin production were dramatically impaired in the Δclp strains. Collectively, our results reveal the broad scope of ClpXP regulation in S. mutans homeostasis and identify several virulence-related traits that are influenced by ClpXP proteolysis.

Identification and characterization of the regulatory elements of the inducible acetamidase operon from Mycobacterium smegmatis

2007 ◽  
Vol 53 (5) ◽  
pp. 599-606 ◽  
Author(s):  
Selvakumar Subbian ◽  
Sujatha Narayanan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Reporter System ◽  
Mobility Shift ◽  
Crude Cell Lysate ◽  
Multiple Promoters ◽  
Promoter Probe ◽  
Mobility Shift Assay

The highly inducible acetamidase promoter from Mycobacterium smegmatis has been used as a tool in the study of mycobacterial genetics. The 4.2 kb acetamidase operon contains four putative open reading frames (ORFs) (amiC, amiA, amiD, and amiS) upstream of the 1.2 kb acetamidase ORF (amiE). In this article, using electrophoretic mobility shift assay and promoter probe analyses with a lacZ reporter system, we show the position of three putative operators within the acetamidase operon in M. smegmatis. Results from these studies reinforce previous findings about the involvement of multiple promoters in the regulation of acetamidase gene expression. Each of the identified operators are positioned upstream of the respective promoter reported in previous studies. We also found that the crude cell lysate of M. smegmatis containing potential regulators, obtained from bacteria grown under inducing or noninducing conditions, binds to specific operators. The binding affinity of each operator with its cognate regulator is significantly different from the other. This supports not only the previous model of acetamidase gene regulation in M. smegmatis but also explains the role of these operators in controlling the expression of respective promoters under different growth conditions.

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