scholarly journals l-Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

2015 ◽  
Vol 81 (6) ◽  
pp. 2082-2089 ◽  
Author(s):  
Yuki Doi
Keyword(s):  
Enterococcus Faecalis ◽  
Crude Glycerol ◽  
Expression System ◽  
Lactate Production ◽  
Biodiesel Production ◽  
Glycerol Metabolism ◽  
Recycling Process ◽  
Content Type ◽  
Ldh Activity ◽  
Inducible Gene

ABSTRACTBiodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process usingEnterococcus faecaliswas developed through physiological studies. TheE. faecalisstrain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD+ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD600]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of lowl-lactate productivity. TheE. faecalispflBgene disruptant (Δpflmutant) expressing theldhL1LPgene produced 300 mMl-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h−1(1.6 g liter−1h−1). Thus, our study demonstrates that metabolically engineeredE. faecaliscan convert crude glycerol tol-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste.

scholarly journals Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

2013 ◽  
Vol 79 (21) ◽  
pp. 6795-6802 ◽  
Author(s):  
Andreas Kaczmarczyk ◽  
Julia A. Vorholt ◽  
Anne Francez-Charlot
Keyword(s):  
Gene Expression ◽  
Caulobacter Crescentus ◽  
Paracoccus Denitrificans ◽  
Expression System ◽  
Model Organisms ◽  
Inducible Gene Expression ◽  
Gene Expression System ◽  
Content Type ◽  
Wide Range ◽  
Inducible Gene

ABSTRACTTunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of thePseudomonas putidaF1cym/cmtsystem. The resulting promoter, termed PQ5, responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in otherAlphaproteobacteria, such as the model organismsCaulobacter crescentus,Paracoccus denitrificans, andMethylobacterium extorquens. In the noninduced state, expression from PQ5is low enough to allow gene depletion analysis, as demonstrated with the essential genephyPofSphingomonassp. strain Fr1. A set of PQ5-based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.

scholarly journals Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Irina Afonina ◽  
June Ong ◽  
Jerome Chua ◽  
Timothy Lu ◽  
Kimberly A. Kline
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Expression System ◽  
Multidrug Resistant ◽  
Essential Genes ◽  
Host Bacterium ◽  
Content Type ◽  
Wide Range ◽  
Life Threatening

ABSTRACT Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. IMPORTANCE Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

scholarly journals Development and Characterization of a Xylose-Inducible Gene Expression System for Clostridium perfringens

2011 ◽  
Vol 77 (23) ◽  
pp. 8439-8441 ◽  
Author(s):  
Hirofumi Nariya ◽  
Shigeru Miyata ◽  
Tomomi Kuwahara ◽  
Akinobu Okabe
Keyword(s):  
Gene Expression ◽  
Clostridium Perfringens ◽  
Expression System ◽  
Xylose Isomerase ◽  
Inducible Gene Expression ◽  
Content Type ◽  
Regulated Expression ◽  
Operator Sequence ◽  
Inducible Gene

ABSTRACTA xylose-inducible gene expression vector forClostridium perfringenswas developed. Plasmid pXCH contains a chromosomal region fromClostridium difficile(xylR-PxylB):xylR, encoding the xylose repressor,xylO, thexyloperator sequence, and PxylB, the divergent promoter upstream ofxylBAencoding xylulo kinase and xylose isomerase. pXCH allows tightly regulated expression of the chloramphenicol acetyltransferase reporter and the α-toxin genes in response to the inducer concentration. Thus, pXCH could constitute a new valuable genetic tool for study ofC. perfringens.

scholarly journals Digestible energy of crude glycerol for pacu and silver catfish

2014 ◽  
Vol 44 (8) ◽  
pp. 1448-1451 ◽  
Author(s):  
Rafael Ernesto Balen ◽  
Patrick Nereu Tetu ◽  
Robie Allan Bombardelli ◽  
Paulo Cesar Pozza ◽  
Fábio Meurer
Keyword(s):  
Fish Species ◽  
Crude Glycerol ◽  
Energy Content ◽  
Biodiesel Production ◽  
Native Fish ◽  
Neotropical Fish ◽  
Digestible Energy ◽  
Silver Catfish ◽  
Glycerol Utilization ◽  
Good Potential

The increase in global biodiesel production is originating a glycerol surplus, which has no defined destination. An alternative to overcome this problem is its use as energy source in animal feeding. In Brazil, Pacu (Piaractus mesopotamicus) is one of the most farmed native fish species, whereas Silver catfish (Rhamdia quelen) is suitable for production in subtropical region. Considering little knowledge about crude glycerol utilization in feeds for Neotropical fish species, it was evaluated the apparent digestibility coefficients (ADCs) for energy of crude glycerol for P. mesopotamicus and R. quelen. The digestibility and digestible energy content of crude glycerol can be considered excellent even when compared to energy of common ingredients such as maize and wheat, presenting 0.97 and 0.89 of energy ADCs, and 15.2 and 13.95MJ kg-1 of digestible energy for Pacu and Silver catfish, respectively. In conclusion, crude glycerol is an energetic ingredient with good potential in Brazilian native fish diets.

scholarly journals From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

2015 ◽  
Vol 197 (18) ◽  
pp. 2908-2919 ◽  
Author(s):  
Anthony O. Gaca ◽  
Pavel Kudrin ◽  
Cristina Colomer-Winter ◽  
Jelena Beljantseva ◽  
Kuanqing Liu ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Enterococcus Faecalis ◽  
Second Messengers ◽  
Stringent Response ◽  
Synthetase Activity ◽  
Protective Effects ◽  
Content Type ◽  
Regulatory Effects ◽  
Complex Target

ABSTRACTThe bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. InEnterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolaseE. faecalisRel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation inFirmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEfsynthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEfalso efficiently utilized GMP to form GMP 3′-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity ofE. faecalisenzymes involved in GTP biosynthesis and, to a lesser extent, transcription ofrrnBbyEscherichia coliRNA polymerase. Activation ofE. coliRelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEfwas activated only by ppGpp. Furthermore, enzymatic activity of RelQEfis insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of “long” RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp.IMPORTANCEAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ ofEnterococcus faecalis(RelQEf), we found that, in addition to (p)ppGpp, RelQEfis an efficient producer of pGpp (GMP 3′-diphosphate).In vitroanalysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEfand suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

Cell cycle arrest and astrocytic differentiation resulting from PTEN expression in glioma cells

1999 ◽  
Vol 91 (5) ◽  
pp. 822-830 ◽  
Author(s):  
Jun-ichi Adachi ◽  
Katsumi Ohbayashi ◽  
Tomonari Suzuki ◽  
Tomio Sasaki
Keyword(s):  
Expression System ◽  
Biological Significance ◽  
Glioma Cells ◽  
Genetic Alterations ◽  
Pten Expression ◽  
Human Glioma ◽  
Wild Type ◽  
Content Type ◽  
Astrocytic Differentiation ◽  
Fine Print

Object. Genetic alterations of the PTEN gene (also known as MMAC1 or TEP1) have frequently been identified in high-grade gliomas, indicating that inactivation of PTEN plays a crucial role in human glioma progression. The aim of this study was to assess the biological significance of PTEN inactivation in the development of glioma.Methods. The authors introduced wild-type PTEN complementary DNA into four human glioma cell lines (T98G, U-251MG, U-87MG, and A172) containing endogenous aberrant PTEN alleles. The number of colonies transfected with the wild-type PTEN was reduced to 15 to 32% of those found after transfection of a control vector, suggesting growth suppression by the exogenous PTEN. To analyze phenotypic alterations produced by PTEN expression, T98G-derived clones with inducible PTEN expression were further established using a tetracycline-regulated inducible gene expression system. Induction of PTEN expression suppressed the in vitro growth of T98G cells with accumulation of G1 phase cells. Furthermore, when cells were cultured in the presence of the extracellular matrix (ECM), PTEN expression caused distinct morphological changes, with multiple and elongated cytoplasmic processes similar to those of normal astrocytes. The level of glial fibrillary acidic protein, an intermediate protein specifically expressed in differentiated astrocytes, was upregulated concomitantly.Conclusions. These findings strongly indicate that exogenous PTEN expression inhibits the proliferation of glioma cells by inducing G1 arrest and elicits astrocytic differentiation in the presence of the ECM. Inactivation of PTEN would play an important role in the enhancement of unregulated growth of undifferentiated glioma cells.

scholarly journals Pharmacodynamics of Daptomycin against Enterococcus faecium and Enterococcus faecalis in the Murine Thigh Infection Model

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
James M. Kidd ◽  
Kamilia Abdelraouf ◽  
Tomefa E. Asempa ◽  
Romney M. Humphries ◽  
David P. Nicolau
Keyword(s):  
Enterococcus Faecalis ◽  
Enterococcus Faecium ◽  
Hill Equation ◽  
Infection Model ◽  
Free Drug Concentration ◽  
Time Curve ◽  
Content Type ◽  
Concentration Time ◽  
The Hill ◽  
Susceptibility Breakpoint

ABSTRACT The Clinical and Laboratory Standards Institute (CLSI) daptomycin MIC susceptibility breakpoint for the treatment of enterococcal infections is ≤4 μg/ml. However, patients receiving daptomycin for the treatment of infections caused by enterococci with MICs of ≤4 μg/ml may experience treatment failures. We assessed the pharmacodynamics of daptomycin against enterococci in a neutropenic murine thigh infection model and determined the exposures necessary for bacteriostasis and a 1-log10-CFU reduction of Enterococcus faecalis and Enterococcus faecium. We further characterized daptomycin efficacy at clinically achievable exposures. Six E. faecium and 6 E. faecalis isolates (daptomycin MICs, 0.5 to 32 μg/ml) were studied. Daptomycin was administered at various doses over 24 h to achieve area under the free drug concentration-time curve-to-MIC ratios (fAUC0–24/MIC) ranging from 1 to 148. Daptomycin regimens that simulate mean human exposures following doses of 6, 8, and 10 mg/kg of body weight/day were also studied. Efficacy was assessed by the differences in the number of log10 CFU per thigh at 24 h. The Hill equation was used to estimate the fAUC0–24/MIC required to achieve bacteriostasis and a 1-log10-CFU reduction. For E. faecium, a 1-log10-CFU reduction required an fAUC0–24/MIC of 12.9 (R2 = 0.71). For E. faecalis, a 1-log10-CFU reduction was not achieved, while the fAUC0–24/MIC required for stasis was 7.2 (R2 = 0.8). With a human-simulated regimen of 6 mg/kg/day, a 1-log10-CFU reduction was observed in 3/3 E. faecium isolates with MICs of <4 μg/ml and 0/3 E. faecium isolates with MICs of ≥4 μg/ml; however, a 1-log10-CFU reduction was not achieved for any of the 6 E. faecalis isolates. These results, alongside clinical data, prompt a reevaluation of the current breakpoint.

The use of the risk assessment in the life cycle assessment framework

2015 ◽  
Vol 26 (3) ◽  
pp. 389-406 ◽  
Author(s):  
Maria Francesca Milazzo ◽  
Francesco Spina
Keyword(s):  
Risk Assessment ◽  
Life Cycle Assessment ◽  
Life Cycle ◽  
Human Health ◽  
Biodiesel Production ◽  
Health Impacts ◽  
Complete Analysis ◽  
Transfer Factors ◽  
Content Type ◽  
Human Health Impacts

Purpose – The purpose of this paper is to quantify the human health impacts of soy-biodiesel production with the aim to discuss about its environmental sustainability. Design/methodology/approach – The integrated use of two current approaches, risk assessment (RA) and life cycle assessment (LCA), has allowed improvement of the potentialities of both in obtaining a more complete analysis. The implementation of a life cycle indicator for the assessment of the impacts on the human health, integrating the features of both approaches, is the main focus of this paper. Findings – It has been found that, although the biodiesel is a green fuel, it has some criticalities in its life cycle, which cannot be disregarded. In fact, even if biodiesel is essentially a clean fuel there are some phases, prior to the industrial phase, that can cause negative effects on human health and ecosystems. Practical implications – Results suggest some measures which can be adopted to substantially reduce human health impacts. Further alternative could be analysed in future to gain more insight about the use of biodiesel fuels. Originality/value – The estimation of the impacts of a process producing biodiesel has been made by using a novel approach. The novelty is associated with the calculation of the impacts on human health by using the transfer factors applied in RA. The use of such factors, properly modified in order to estimate the impacts on a wider scale than a site-dimension, allows defining a holistic approach, as LCA and RA are used as complete units but at the same time can be related to each other.

scholarly journals CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Valerie J. Price ◽  
Wenwen Huo ◽  
Ardalan Sharifi ◽  
Kelli L. Palmer
Keyword(s):  
Antibiotic Resistance ◽  
High Risk ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Treatment Options ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Content Type ◽  
New Genes ◽  
Restriction Modification

ABSTRACT Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

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