scholarly journals Controlled Gene Expression in Bifidobacteria by Use of a Bile-Responsive Element

2011 ◽  
Vol 78 (2) ◽  
pp. 581-585 ◽  
Author(s):  
Lorena Ruiz ◽  
Pablo Álvarez-Martín ◽  
Baltasar Mayo ◽  
Clara G. de los Reyes-Gavilán ◽  
Miguel Gueimonde ◽  
...  
Keyword(s):  
Bile Salts ◽  
Promoter Activity ◽  
Bifidobacterium Longum ◽  
Upstream Region ◽  
Surface Acting ◽  
Content Type ◽  
Reporter Activity ◽  
Responsive Element ◽  
Inducible Gene

ABSTRACTThe promoter activity of the upstream region of the bile-inducible genebetAfromBifidobacterium longumsubsp.longumNCC2705 was characterized. DNA fragments were cloned into the reporter vector pMDYAbfB, and the arabinofuranosidase activity was determined under differentin vitroconditions. A segment of 469 bp was found to be the smallest operational unit that retains bile inducibility. The reporter activity was strongly affected by the presence of ox gall, cholate, and conjugated cholate, but not by other bile salts and cell-surface-acting compounds. Remarkably, this bile-inducible system was also active in other bifidobacteria containingbetAhomologs.

scholarly journals Germinant Synergy FacilitatesClostridium difficileSpore Germination under Physiological Conditions

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Travis J. Kochan ◽  
Michelle S. Shoshiev ◽  
Jessica L. Hastie ◽  
Madeline J. Somers ◽  
Yael M. Plotnick ◽  
...  
Keyword(s):  
Amino Acids ◽  
Clostridium Difficile ◽  
Spore Germination ◽  
Bile Salts ◽  
Gi Tract ◽  
Infectious Diarrhea ◽  
Content Type ◽  
Increased Risk ◽  
Calcium Reabsorption

ABSTRACTClostridium difficileis a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host.C. difficileis the leading cause of nosocomial infectious diarrhea worldwide.C. difficileinfection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step inC. difficilepathogenesis is the metabolic reactivation of dormant spores within the gastrointestinal (GI) tract through a process known as germination. In this work, we aim to elucidate the specific conditions and the location within the GI tract that facilitate this process. Our data suggest thatC. difficilegermination occurs through a two-step biochemical process that is regulated by pH and bile salts, amino acids, and calcium present within the GI tract. Maximal germination occurs at a pH ranging from 6.5 to 8.5 in the terminal small intestine prior to bile salt and calcium reabsorption by the host. Germination can be initiated by lower concentrations of germinants when spores are incubated with a combination of bile salts, calcium, and amino acids, and this synergy is dependent on the availability of calcium. The synergy described here allows germination to proceed in the presence of inhibitory bile salts and at physiological concentrations of germinants, effectively decreasing the concentrations of nutrients required to initiate an essential step of pathogenesis.IMPORTANCEClostridium difficileis an anaerobic spore-forming human pathogen that is the leading cause of nosocomial infectious diarrhea worldwide. Germination of infectious spores is the first step in the development of aC. difficileinfection (CDI) after ingestion and passage through the stomach. This study investigates the specific conditions that facilitateC. difficilespore germination, including the following: location within the gastrointestinal (GI) tract, pH, temperature, and germinant concentration. The germinants that have been identified in culture include combinations of bile salts and amino acids or bile salts and calcium, butin vitro, these function at concentrations that far exceed normal physiological ranges normally found in the mammalian GI tract. In this work, we describe and quantify a previously unreported synergy observed when bile salts, calcium, and amino acids are added together. These germinant cocktails improve germination efficiency by decreasing the required concentrations of germinants to physiologically relevant levels. Combinations of multiple germinant types are also able to overcome the effects of inhibitory bile salts. In addition, we propose that the acidic conditions within the GI tract regulateC. difficilespore germination and could provide a biological explanation for why patients taking proton pump inhibitors are associated with increased risk of developing a CDI.

scholarly journals The Surface-Associated Exopolysaccharide of Bifidobacterium longum 35624 Plays an Essential Role in Dampening Host Proinflammatory Responses and Repressing Local TH17 Responses

2016 ◽  
Vol 82 (24) ◽  
pp. 7185-7196 ◽  
Author(s):  
Elisa Schiavi ◽  
Marita Gleinser ◽  
Evelyn Molloy ◽  
David Groeger ◽  
Remo Frei ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune Cells ◽  
Essential Role ◽  
Inflammatory Responses ◽  
Bifidobacterium Longum ◽  
Molecular Characteristics ◽  
Interleukin 17 ◽  
Content Type ◽  
Exopolysaccharide Production

ABSTRACTThe immune-modulating properties of certain bifidobacterial strains, such asBifidobacterium longumsubsp.longum35624 (B. longum35624), have been well described, although the strain-specific molecular characteristics associated with such immune-regulatory activity are not well defined. It has previously been demonstrated thatB. longum35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response.B. longum35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPSneg) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPSnegby genetic complementation. Administration ofB. longum35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPSnegdid not protect against the development of colitis, with associated enhanced recruitment of IL-17+lymphocytes to the gut. Moreover, intranasal administration of sEPSnegalso resulted in enhanced recruitment of IL-17+lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced byB. longum35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local TH17 responses.IMPORTANCEParticular gut commensals, such asB. longum35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensal-host interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cellsin vitro. In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surface-associated exopolysaccharide of theB. longum35624 cell envelope in the prevention of aberrant inflammatory responses.

scholarly journals GntR Family Regulator DasR Controls Acetate Assimilation by Directly Repressing the acsA Gene in Saccharopolyspora erythraea

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Di You ◽  
Bai-Qing Zhang ◽  
Bang-Ce Ye
Keyword(s):  
Environmental Changes ◽  
Carbon Sources ◽  
Saccharopolyspora Erythraea ◽  
Upstream Region ◽  
Acetate Metabolism ◽  
Acetyl Coa ◽  
Content Type ◽  
Acetate Assimilation ◽  
Responsive Element ◽  
Gntr Family

ABSTRACT The GntR family regulator DasR controls the transcription of genes involved in chitin and N -acetylglucosamine (GlcNAc) metabolism in actinobacteria. GlcNAc is catabolized to ammonia, fructose-6-phosphate (Fru-6P), and acetate, which are nitrogen and carbon sources. In this work, a DasR-responsive element ( dre ) was observed in the upstream region of acsA1 in Saccharopolyspora erythraea . This gene encodes acetyl coenzyme A (acetyl-CoA) synthetase (Acs), an enzyme that catalyzes the conversion of acetate into acetyl-CoA. We found that DasR repressed the transcription of acsA1 in response to carbon availability, especially with GlcNAc. Growth inhibition was observed in a dasR -deleted mutant (Δ dasR ) in the presence of GlcNAc in minimal medium containing 10 mM acetate, a condition under which Acs activity is critical to growth. These results demonstrate that DasR controls acetate assimilation by directly repressing the transcription of the acsA1 gene and performs regulatory roles in the production of intracellular acetyl-CoA in response to GlcNAc. IMPORTANCE Our work has identified the DasR GlcNAc-sensing regulator that represses the generation of acetyl-CoA by controlling the expression of acetyl-CoA synthetase, an enzyme responsible for acetate assimilation in S. erythraea . The finding provides the first insights into the importance of DasR in the regulation of acetate metabolism, which encompasses the regulatory network between nitrogen and carbon metabolism in actinobacteria, in response to environmental changes.

scholarly journals The MarR Family Regulator BmrR Is Involved in Bile Tolerance ofBifidobacterium longumBBMN68 via Controlling the Expression of an ABC Transporter

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Qi Xu ◽  
Zhengyuan Zhai ◽  
Haoran An ◽  
Yang Yang ◽  
Jia Yin ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Stress Response ◽  
Heterologous Expression ◽  
Bile Salts ◽  
Intestinal Tract ◽  
Bifidobacterium Longum ◽  
Transcriptional Regulator ◽  
Mobility Shift ◽  
Content Type ◽  
Bile Tolerance

ABSTRACTIn order to colonize the human gastrointestinal tract and exert their beneficial effects, bifidobacteria must effectively cope with toxic bile salts in the intestine; however, the molecular mechanism underlying bile tolerance is poorly understood. In this study, heterologous expression of a MarR family transcriptional regulator, BmrR, significantly reduced the ox bile resistance ofLactococcus lactisNZ9000, suggesting that BmrR might play a role in the bile stress response.In silicoanalysis combined with reverse transcription-PCR assays demonstrated thatbmrRwas cotranscribed withbmrAandbmrB, which encoded multidrug resistance (MDR) ABC transporters. Promoter prediction and electrophoretic mobility shift assays revealed that BmrR could autoregulate thebmrRABoperon by binding to thebmrbox (ATTGTTG-6nt-CAACAAT) in the promoter region. Moreover, heterologous expression ofbmrAandbmrBinL. lactisyielded 20.77-fold higher tolerance to 0.10% ox bile, compared to the wild-type strain. In addition, ox bile could disrupt the DNA binding activity of BmrR as a ligand. Taken together, our findings indicate that thebmrRABoperon is autoregulated by the transcriptional regulator BmrR and ox bile serves as an inducer to activate the bile efflux transporter BmrAB in response to bile stress inBifidobacterium longumBBMN68.IMPORTANCEBifidobacteria are natural inhabitants of the human intestinal tract. Some bifidobacterial strains are used as probiotics in fermented dairy production because of their health-promoting effects. Following consumption, bifidobacteria colonize the lower intestinal tract, where the concentrations of bile salts remain nearly 0.05% to 2.0%. Bile salts, as detergent-like antimicrobial compounds, can cause cellular membrane disruption, protein misfolding, and DNA damage. Therefore, tolerance to physiological bile stress is indeed essential for bifidobacteria to survive and to exert probiotic effects in the gastrointestinal tract. InB. longumBBMN68, the MarR-type regulator BmrR was involved in the bile stress response by autoregulating thebmrRABoperon, and ox bile as an inducer could increase the expression of the BmrAB transporter to enhance the bile tolerance of BBMN68. Our study represents a functional analysis of thebmrRABoperon in the bile stress response, which will provide new insights into bile tolerance mechanisms inBifidobacteriumand other bacteria.

Localization of a phytochrome-responsive element within the upstream region of pea rbcS-3A

1990 ◽  
Vol 10 (10) ◽  
pp. 5565-5568
Author(s):  
P M Gilmartin ◽  
N H Chua
Keyword(s):  
Binding Sites ◽  
Promoter Activity ◽  
Red Light ◽  
Upstream Region ◽  
Etiolated Seedlings ◽  
Responsive Element

The pea rbcS-3A promoter with a 5' deletion to -166 (-166 rbcS-3A) contains two GT-1-binding sites. Mutational analyses demonstrated that a decrease in affinity for GT-1 correlates with reduced promoter activity. Transcription of -166 rbcS-3A in transgenic etiolated seedlings is induced by red light and suppressed by far-red light, indicating that it contains a phytochrome-responsive element.

scholarly journals CR6-Interacting Factor 1 Interacts with Orphan Nuclear Receptor Nur77 and Inhibits Its Transactivation

2005 ◽  
Vol 19 (1) ◽  
pp. 12-24 ◽  
Author(s):  
Ki Cheol Park ◽  
Kwang-Hoon Song ◽  
Hyo Kyun Chung ◽  
Ho Kim ◽  
Dong Wook Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Receptor ◽  
Promoter Activity ◽  
Trichostatin A ◽  
In Vivo Studies ◽  
Orphan Nuclear Receptor ◽  
Histone Deacetylase Inhibitor Trichostatin ◽  
Responsive Element

Abstract CR6-interacting factor 1 (CRIF1) was recently identified as a nuclear protein that interacts with the Gadd45 (growth arrest and DNA damage inducible 45) family of proteins and participates in the regulation of the G1/S phase of the cell cycle. However, the nuclear action of CRIF1 is largely unknown. In this study, we demonstrate that CRIF1 acts as a novel coregulator of transactivation of the orphan nuclear receptor Nur77. Both in vitro and in vivo studies show that CRIF1 interacts with Nur77 via the Nur77 AB domain and that it dramatically inhibits the AB domain-mediated transactivation of Nur77. Transient transfection assays demonstrate that CRIF1 inhibits steroid receptor coactivator-2-mediated Nur77 transactivation, and silencing of endogenous CRIF1 by small interfering RNA relieves this repression. CRIF1 possesses intrinsic repressor activities that are not affected by the histone deacetylase inhibitor Trichostatin A. In addition, overexpression of CRIF1 inhibits TSH/protein kinase A-induced Nur-responsive element promoter activity. CRIF1 inhibited Nur77-dependent induction of E2F1 promoter activity, mRNA expression, and Nur77-mediated G1/S progression in cell cycle. These results suggest that CRIF1 acts as a repressor of the orphan nuclear receptor Nur77 by inhibiting AB domain-mediated transcriptional activity.

scholarly journals An In Vitro Enrichment Strategy for Formulating Synergistic Synbiotics

2019 ◽  
Vol 85 (16) ◽  
Author(s):  
Car Reen Kok ◽  
David Fabian Gomez Quintero ◽  
Clement Niyirora ◽  
Devin Rose ◽  
Amanda Li ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Synergistic Effects ◽  
Bifidobacterium Longum ◽  
High Rate ◽  
Host Responses ◽  
Ecological Niches ◽  
Bacterial Populations ◽  
Content Type

ABSTRACT Research on the role of diet on gut and systemic health has led to considerable interest toward identifying novel therapeutic modulators of the gut microbiome, including the use of prebiotics and probiotics. However, various host responses have often been reported among many clinical trials. This is in part due to competitive exclusion as a result of the absence of ecological niches as well as host-mediated constraints via colonization resistance. In this research, we developed a novel in vitro enrichment (IVE) method for isolating autochthonous strains that can function as synergistic synbiotics and overcome these constraints. The method relied on stepwise in vitro fecal fermentations to enrich for and isolate Bifidobacterium strains that ferment the prebiotic xylooligosaccharide (XOS). We subsequently isolated Bifidobacterium longum subsp. longum CR15 and then tested its establishment in 20 unique fecal samples with or without XOS. The strain was established in up to 18 samples but only in the presence of XOS. Our findings revealed that the IVE method is suitable for isolating potential synergistic probiotic strains that possess the genetic and biochemical ability to ferment specific prebiotic substrates. The IVE method can be used as an initial high-throughput screen for probiotic selection and isolation prior to further characterization and in vivo tests. IMPORTANCE This study describes an in vitro enrichment method to formulate synergistic synbiotics that have potential for establishing autochthonous strains across multiple individuals. The rationale for this approach—that the chance of survival of a bacterial strain is improved by providing it with its required resources—is based on classic ecological theory. From these experiments, a human-derived strain, Bifidobacterium longum subsp. longum CR15, was identified as a xylooligosaccharide (XOS) fermenter in fecal environments and displayed synergistic effects in vitro. The high rate of strain establishment observed in this study provides a basis for using synergistic synbiotics to overcome the responder/nonresponder phenomenon that occurs frequently in clinical trials with probiotic and prebiotic interventions. In addition, this approach can be applied in other protocols that require enrichment of specific bacterial populations prior to strain isolation.

scholarly journals Localization of a phytochrome-responsive element within the upstream region of pea rbcS-3A.

1990 ◽  
Vol 10 (10) ◽  
pp. 5565-5568 ◽  
Author(s):  
P M Gilmartin ◽  
N H Chua
Keyword(s):  
Binding Sites ◽  
Promoter Activity ◽  
Red Light ◽  
Upstream Region ◽  
Etiolated Seedlings ◽  
Responsive Element

The pea rbcS-3A promoter with a 5' deletion to -166 (-166 rbcS-3A) contains two GT-1-binding sites. Mutational analyses demonstrated that a decrease in affinity for GT-1 correlates with reduced promoter activity. Transcription of -166 rbcS-3A in transgenic etiolated seedlings is induced by red light and suppressed by far-red light, indicating that it contains a phytochrome-responsive element.

scholarly journals Involvement of Scratch2 in GalR1-mediated depression-like behaviors in the rat ventral periaqueductal gray

2021 ◽  
Vol 118 (24) ◽  
pp. e1922586118
Author(s):  
Yutao Yang ◽  
Yueting Li ◽  
Bo Liu ◽  
Chenchen Li ◽  
Zijin Liu ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Chronic Mild Stress ◽  
Periaqueductal Gray ◽  
Upstream Region ◽  
Mild Stress ◽  
Behavioral Experiments ◽  
E Box ◽  
Elevated Expression

Galanin receptor1 (GalR1) transcript levels are elevated in the rat ventral periaqueductal gray (vPAG) after chronic mild stress (CMS) and are related to depression-like behavior. To explore the mechanisms underlying the elevated GalR1 expression, we carried out molecular biological experiments in vitro and in animal behavioral experiments in vivo. It was found that a restricted upstream region of the GalR1 gene, from −250 to −220, harbors an E-box and plays a negative role in the GalR1 promoter activity. The transcription factor Scratch2 bound to the E-box to down-regulate GalR1 promoter activity and lower expression levels of the GalR1 gene. The expression of Scratch2 was significantly decreased in the vPAG of CMS rats. Importantly, local knockdown of Scratch2 in the vPAG caused elevated expression of GalR1 in the same region, as well as depression-like behaviors. RNAscope analysis revealed that GalR1 mRNA is expressed together with Scratch2 in both GABA and glutamate neurons. Taking these data together, our study further supports the involvement of GalR1 in mood control and suggests a role for Scratch2 as a regulator of depression-like behavior by repressing the GalR1 gene in the vPAG.

scholarly journals Antifungal Susceptibility Profiles and Drug Resistance Mechanisms of Clinical Lomentospora prolificans Isolates

2020 ◽  
Vol 64 (11) ◽  
Author(s):  
Yongqin Wu ◽  
Nina Grossman ◽  
Marissa Totten ◽  
Warda Memon ◽  
Anna Fitzgerald ◽  
...  
Keyword(s):  
Amino Acid ◽  
Tandem Repeats ◽  
Hot Spot ◽  
Antifungal Susceptibility ◽  
Antifungal Drugs ◽  
Upstream Region ◽  
Amino Acid Residues ◽  
Content Type ◽  
Echinocandin Resistance

ABSTRACT Lomentospora prolificans is an opportunistic fungal pathogen with low susceptibility to current antifungal drugs. Here, we tested the in vitro susceptibility of 8 drugs against 42 clinical L. prolificans isolates. All isolates showed high MICs to voriconazole (MIC90>16 μg/ml), itraconazole (MIC90>16 μg/ml), posaconazole (MIC90>16 μg/ml), isavuconazole (MIC90>16 μg/ml), amphotericin B (MIC90>16 μg/ml), and terbinafine (MIC90>64 μg/ml) and high minimum effective concentrations (MECs) to micafungin (MEC90>8 μg/ml), with the exception of miltefosine showing an MIC90 value of 4 μg/ml. We examined six different in vitro drug combinations and found that the combination of voriconazole and terbinafine achieved the most synergistic effort against L. prolificans. We then annotated the L. prolificans whole genome and located its Cyp51 and Fks1 genes. We completely sequenced the two genes to determine if any mutation would be related to azole and echinocandin resistance in L. prolificans. We found no amino acid changes in Cyp51 protein and no tandem repeats in the 5′ upstream region of the Cyp51 gene. However, we identified three intrinsic amino acid residues (G138S, M220I, and T289A) in the Cyp51 protein that were linked to azole resistance. Likewise, two intrinsic amino acid residues (F639Y, W695F) that have reported to confer echinocandin resistance were found in Fks1 hot spot regions. In addition, three new amino acid alterations (D440A, S634R, and H1245R) were found outside Fks1 hot spot regions, and their contributions to echinocandin resistance need future investigation. Overall, our findings support the notion that L. prolificans is intrinsically resistant to azoles and echinocandins.

Sign in / Sign up

Export Citation Format

Share Document