Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range

ABSTRACT Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the PQ pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin (TA) loci present in E. faecalis, par pAD1 of the pAD1 plasmid and par EF0409 located on the E. faecalis chromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalis. IMPORTANCE E. faecalis is an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the PQ pheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function in E. faecalis and to further elucidate its potential roles in cell physiology.

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An Archaeal Fluoride-Responsive Riboswitch Provides an Inducible Expression System for Hyperthermophiles

Applied and Environmental Microbiology ◽

10.1128/aem.02306-17 ◽

2018 ◽

Vol 84 (7) ◽

Cited By ~ 15

Author(s):

Michael Clayton Speed ◽

Brett W. Burkhart ◽

Jonathan W. Picking ◽

Thomas J. Santangelo

Keyword(s):

Gene Expression ◽

Transcription Initiation ◽

Expression System ◽

Regulatory Control ◽

Cell Physiology ◽

Content Type ◽

Thermococcus Kodakarensis ◽

Export Pathway ◽

Regulated Expression ◽

Regulated Gene Expression

ABSTRACT Robust genetic systems for the hyperthermophilic Thermococcales have facilitated the overexpression of native genes, enabled the addition of sequences encoding secretion signals, epitope, and affinity tags to coding regions, and aided the introduction of sequences encoding new proteins in these fast-growing fermentative heterotrophs. However, tightly controlled and easily manipulated systems facilitating regulated gene expression are limited for these hosts. Here, we describe an alternative method for regulatory control reliant on a cis -encoded functional riboswitch in the model archaeon Thermococcus kodakarensis . Despite the hyperthermophilic growth temperatures, the proposed structure of the riboswitch conforms to a fluoride-responsive riboswitch encoded in many bacteria and similarly functions to regulate a component-conserved fluoride export pathway. Deleting components of the fluoride export pathway generates T. kodakarensis strains with increased fluoride sensitivity. The mechanism underlying regulated expression suggested that the riboswitch-encoding sequences could be utilized as a tunable expression cassette. When appended to a reporter gene, the riboswitch-mediated control system provides fluoride-dependent tunable regulatory potential, offering an alternative system for regulating gene expression. Riboswitch-regulated expression is thus ubiquitous in extant life and can be exploited to generate regulated expression systems for hyperthermophiles. IMPORTANCE Gene expression is controlled by a myriad of interconnected mechanisms that interpret metabolic states and environmental cues to balance cell physiology. Transcription regulation in Archaea is known to employ both typical repressors-operators and transcription activators to regulate transcription initiation in addition to the regulation afforded by chromatin structure. It was perhaps surprising that the presumed ancient mechanism of riboswitch-mediated regulation is found in Bacteria and Eukarya , but seemingly absent in Archaea . We demonstrate here that a fluoride-responsive riboswitch functions to regulate a detoxification pathway in the hyperthermophilic archaeon Thermococcus kodakarensis . The results obtained define a universal role for riboswitch-mediated regulation, adumbrate the presence of several riboswitch-regulated genes in Thermococcus kodakarensis , demonstrate the utility of RNA-based regulation at high temperatures, and provide a novel riboswitch-regulated expression system to employ in hyperthermophiles.

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Engineering Cyanobacterial Cell Morphology for Enhanced Recovery and Processing of Biomass

Applied and Environmental Microbiology ◽

10.1128/aem.00053-17 ◽

2017 ◽

Vol 83 (9) ◽

Cited By ~ 15

Author(s):

Adam Jordan ◽

Jenna Chandler ◽

Joshua S. MacCready ◽

Jingcheng Huang ◽

Katherine W. Osteryoung ◽

...

Keyword(s):

Cell Division ◽

Cell Morphology ◽

Dynamic Range ◽

Enhanced Recovery ◽

Downstream Processing ◽

Crop Species ◽

Content Type ◽

Alternative Crop ◽

Cell Harvesting ◽

Broad Dynamic Range

ABSTRACT Cyanobacteria are emerging as alternative crop species for the production of fuels, chemicals, and biomass. Yet, the success of these microbes depends on the development of cost-effective technologies that permit scaled cultivation and cell harvesting. Here, we investigate the feasibility of engineering cell morphology to improve biomass recovery and decrease energetic costs associated with lysing cyanobacterial cells. Specifically, we modify the levels of Min system proteins in Synechococcus elongatus PCC 7942. The Min system has established functions in controlling cell division by regulating the assembly of FtsZ, a tubulin-like protein required for defining the bacterial division plane. We show that altering the expression of two FtsZ-regulatory proteins, MinC and Cdv3, enables control over cell morphology by disrupting FtsZ localization and cell division without preventing continued cell growth. By varying the expression of these proteins, we can tune the lengths of cyanobacterial cells across a broad dynamic range, anywhere from an ∼20% increased length (relative to the wild type) to near-millimeter lengths. Highly elongated cells exhibit increased rates of sedimentation under low centrifugal forces or by gravity-assisted settling. Furthermore, hyperelongated cells are also more susceptible to lysis through the application of mild physical stress. Collectively, these results demonstrate a novel approach toward decreasing harvesting and processing costs associated with mass cyanobacterial cultivation by altering morphology at the cellular level. IMPORTANCE We show that the cell length of a model cyanobacterial species can be programmed by rationally manipulating the expression of protein factors that suppress cell division. In some instances, we can increase the size of these cells to near-millimeter lengths with this approach. The resulting elongated cells have favorable properties with regard to cell harvesting and lysis. Furthermore, cells treated in this manner continue to grow rapidly at time scales similar to those of uninduced controls. To our knowledge, this is the first reported example of engineering the cell morphology of cyanobacteria or algae to make them more compatible with downstream processing steps that present economic barriers to their use as alternative crop species. Therefore, our results are a promising proof-of-principle for the use of morphology engineering to increase the cost-effectiveness of the mass cultivation of cyanobacteria for various sustainability initiatives.

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Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Biology ◽

10.3390/biology9120430 ◽

2020 ◽

Vol 9 (12) ◽

pp. 430

Author(s):

Amreen ◽

Hayssam M. Ali ◽

Mohammad Ahmad ◽

Mohamed Z. M. Salem ◽

Altaf Ahmad

Keyword(s):

Hydrogen Peroxide ◽

Real Time ◽

Mammalian Cells ◽

Dynamic Range ◽

Resonance Energy ◽

Living Cells ◽

Stress Conditions ◽

Live Cells ◽

Cell Physiology ◽

Broad Dynamic Range

Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.

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Tools for Functional Postgenomic Analysis of Listeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.00314-08 ◽

2008 ◽

Vol 74 (13) ◽

pp. 3921-3934 ◽

Cited By ~ 132

Author(s):

Ian R. Monk ◽

Cormac G. M. Gahan ◽

Colin Hill

Keyword(s):

Listeria Monocytogenes ◽

Dynamic Range ◽

Fold Increase ◽

Plasmid Transfer ◽

Stable Gene ◽

Food Borne ◽

Regulated Expression ◽

Regulated Gene Expression ◽

Chromosomal Mutations ◽

Internalin A

ABSTRACT We describe the development of genetic tools for regulated gene expression, the introduction of chromosomal mutations, and improved plasmid transfer by electroporation in the food-borne pathogen Listeria monocytogenes. pIMK, a kanamycin-resistant, site-specific, integrative listeriophage vector was constructed and then modified for overexpression (pIMK2) or for isopropyl-β-d-thiogalactopyranoside (IPTG)-regulated expression (pIMK3 and pIMK4). The dynamic range of promoters was assessed by determining luciferase activity, P60 secretion, and internalin A-mediated invasion. These analyses demonstrated that pIMK4 and pIMK3 have a stringently controlled dynamic range of 540-fold. Stable gene overexpression was achieved with pIMK2, giving a range of expression for the three vectors of 1,350-fold. The lactococcal pORI280 system was optimized for the generation of chromosomal mutations and used to create five new prfA star mutants. The combination of pIMK4 and pORI280 allowed streamlined creation of “IPTG-dependent” mutants. This was exemplified by creation of a clean deletion mutant with deletion of the universally essential secA gene, and this mutant exhibited a rapid loss of viability upon withdrawal of IPTG. We also improved plasmid transfer by electroporation into three commonly used laboratory strains of L. monocytogenes. A 125-fold increase in transformation efficiency for EGDe compared with the widely used protocol of Park and Stewart (S. F. Park and G. S. Stewart, Gene 94:129-132, 1990) was observed. Maximal transformation efficiencies of 5.7 × 106 and 6.7 × 106 CFU per μg were achieved for EGDe and 10403S, respectively, with a replicating plasmid. An efficiency of 2 × 107 CFU per μg is the highest efficiency reported thus far for L. monocytogenes F2365.

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Multiplex Assays of Luminex for Identification of COVID-19 Antibodies in Patient Serum: Performance Evaluation and Comparison to ELISA

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqaa161.201 ◽

2020 ◽

Vol 154 (Supplement_1) ◽

pp. S92-S92

Author(s):

M S Shapiro ◽

X Wang ◽

D R Mendu ◽

A Firpo

Keyword(s):

Dynamic Range ◽

High Titer ◽

Mount Sinai Hospital ◽

Antibody Testing ◽

Luminex Assay ◽

Multiplex Assays ◽

Negative Results ◽

Broad Dynamic Range ◽

Circulating Antibodies ◽

Mount Sinai

Abstract Introduction/Objective Mount Sinai Hospital has received emergency use authorization (EUA) from the FDA for Coronavirus Disease 2019 (COVID-19) antibody testing using ELISA. This serological assay detects and titrates the presence of circulating antibodies to COVID-19. Other platforms have aimed to achieve the credentials of the ELISA instrument, including the multiplex assays of Luminex. The platform is known to have a greater throughput (384 wells vs. 96 wells per microplate) and faster processing speed (8 hours vs. 17 hours). Methods Luminex utilizes beads that couple to the same COVID-19 antigens (mRBD and mSpike) which were utilized for the ELISA assay. The beads are read determining the mean fluorescence intensity (MFI). In order to compare the two methods, our study included 61 patients with COVID-19 at Mount Sinai Hospital, to screen and titrate their sera using Luminex, and to correspond the MFI values with the ELISA titers. Results The Luminex assay has achieved the same level of confidence as ELISA. The 61 patients, representing 30 negatives and 31 positives, are consistently identified as such on both platforms. Our data highlights 32% of patients with a low titer (<1:160), 42% of patients with a high titer (1:160 ~ 1:320), and 26% of patients with a very high titer level (>1:320). These titers correlated well with the MFI values. Based on a cutoff of 80,000 MFI, the sensitivity and specificity of the assay is 98% and 85%, respectively, with no overlapping of MFI between positive and negative results. Conclusion Overall, the study has demonstrated that the Luminex is a strong alternative for the ELISA platform. The Luminex highlights the broad dynamic range with no overlapping between positives and negatives. Migration from ELISA to Luminex, a platform with faster and greater throughput, is therefore, highly desirable.

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Automatic Modulation Classification Based on Deep Feature Fusion for High Noise Level and Large Dynamic Input

Sensors ◽

10.3390/s21062117 ◽

2021 ◽

Vol 21 (6) ◽

pp. 2117

Author(s):

Hui Han ◽

Zhiyuan Ren ◽

Lin Li ◽

Zhigang Zhu

Keyword(s):

Neural Network ◽

Frequency Domain ◽

Noise Level ◽

Background Noise ◽

Dynamic Range ◽

Feature Fusion ◽

Superior Performance ◽

Modulation Classification ◽

High Background ◽

Automatic Modulation Classification

Automatic modulation classification (AMC) is playing an increasingly important role in spectrum monitoring and cognitive radio. As communication and electronic technologies develop, the electromagnetic environment becomes increasingly complex. The high background noise level and large dynamic input have become the key problems for AMC. This paper proposes a feature fusion scheme based on deep learning, which attempts to fuse features from different domains of the input signal to obtain a more stable and efficient representation of the signal modulation types. We consider the complementarity among features that can be used to suppress the influence of the background noise interference and large dynamic range of the received (intercepted) signals. Specifically, the time-series signals are transformed into the frequency domain by Fast Fourier transform (FFT) and Welch power spectrum analysis, followed by the convolutional neural network (CNN) and stacked auto-encoder (SAE), respectively, for detailed and stable frequency-domain feature representations. Considering the complementary information in the time domain, the instantaneous amplitude (phase) statistics and higher-order cumulants (HOC) are extracted as the statistical features for fusion. Based on the fused features, a probabilistic neural network (PNN) is designed for automatic modulation classification. The simulation results demonstrate the superior performance of the proposed method. It is worth noting that the classification accuracy can reach 99.8% in the case when signal-to-noise ratio (SNR) is 0 dB.

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A tandem giant magnetoresistance assay for one-shot quantification of clinically relevant concentrations of N-terminal pro-B-type natriuretic peptide in human blood

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03227-5 ◽

2021 ◽

Author(s):

Fanda Meng ◽

Weisong Huo ◽

Jie Lian ◽

Lei Zhang ◽

Xizeng Shi ◽

...

Keyword(s):

Detection Limit ◽

Giant Magnetoresistance ◽

Dynamic Range ◽

Point Of Care ◽

Sample Volume ◽

Small Sample ◽

Broad Dynamic Range ◽

Gmr Sensors ◽

Gmr Sensor ◽

Sample Volume Requirement

AbstractWe report a microfluidic sandwich immunoassay constructed around a dual-giant magnetoresistance (GMR) sensor array to quantify the heart failure biomarker NT-proBNP in human plasma at the clinically relevant concentration levels between 15 pg/mL and 40 ng/mL. The broad dynamic range was achieved by differential coating of two identical GMR sensors operated in tandem, and combining two standard curves. The detection limit was determined as 5 pg/mL. The assay, involving 53 plasma samples from patients with different cardiovascular diseases, was validated against the Roche Cobas e411 analyzer. The salient features of this system are its wide concentration range, low detection limit, small sample volume requirement (50 μL), and the need for a short measurement time of 15 min, making it a prospective candidate for practical use in point of care analysis.

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Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range

Nature Communications ◽

10.1038/ncomms1477 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 166

Author(s):

Scott M. Tabakman ◽

Lana Lau ◽

Joshua T. Robinson ◽

Jordan Price ◽

Sarah P. Sherlock ◽

...

Keyword(s):

Dynamic Range ◽

Protein Microarrays ◽

Broad Dynamic Range

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

Journal of Bacteriology ◽

10.1128/jb.00324-15 ◽

2015 ◽

Vol 197 (18) ◽

pp. 2908-2919 ◽

Cited By ~ 58

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00506-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 9

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.

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