TetR-Based Gene Regulation Systems for Francisella tularensis

ABSTRACTThere are a number of genetic tools available for studyingFrancisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems forF. tularensisbased on the Tet repressor, TetR. For the inducible system, atetoperator sequence was cloned into a modifiedF. tularensis groESLpromoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon,luxCDABE, was cloned under the hybridFrancisellatetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association withtetO.Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml−1. In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists ofFTRpregulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with theluxreporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploringF. tularensisprotein function.

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CsrA (BB0184) Is Not Involved in Activation of the RpoN-RpoS Regulatory Pathway in Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.01555-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1511-1522 ◽

Cited By ~ 10

Author(s):

Zhiming Ouyang ◽

Jianli Zhou ◽

Michael V. Norgard

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Animal Studies ◽

Expression System ◽

Regulatory Pathway ◽

Content Type ◽

Mouse Tissues ◽

Differential Gene ◽

Carbon Storage Regulator

ABSTRACTBorrelia burgdorferiencodes a homologue of the bacterial carbon storage regulator A (CsrA). Recently, it was reported that CsrA contributes toB. burgdorferiinfectivity and is required for the activation of the central RpoN-RpoS regulatory pathway. However, many questions concerning the function of CsrA inB. burgdorferigene regulation remain unanswered. In particular, there are conflicting reports concerning the molecular details of how CsrA may modulaterpoSexpression and, thus, how CsrA may influence the RpoN-RpoS pathway inB. burgdorferi. To address these key discrepancies, we examined the role of CsrA in differential gene expression in the Lyme disease spirochete. Upon engineering an induciblecsrAexpression system inB. burgdorferi, controlled hyperexpression of CsrA in a merodiploid strain did not significantly alter the protein and transcript levels ofbosR,rpoS, and RpoS-dependent genes (such asospCanddbpA). In addition, we constructed isogeniccsrAmutants in two widely used infectiousB. burgdorferistrains. When expression ofbosR,rpoS,ospC, anddbpAwas compared between thecsrAmutants and their wild-type counterparts, no detectable differences were observed. Finally, animal studies indicated that thecsrAmutants remained infectious for and virulent in mice. Analyses ofB. burgdorferigene expression in mouse tissues showed comparable levels ofrpoStranscripts by thecsrAmutants and the parental strains. Taken together, these results constitute compelling evidence that CsrA is not involved in activation of the RpoN-RpoS pathway and is dispensable for mammalian infectious processes carried out byB. burgdorferi.

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Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02296-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6795-6802 ◽

Cited By ~ 40

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.

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Construction and Characterization of a Gradually Inducible Expression Vector for Halobacterium salinarum, Based on thekdpPromoter

Applied and Environmental Microbiology ◽

10.1128/aem.07155-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2100-2105 ◽

Cited By ~ 5

Author(s):

Dorthe Kixmüller ◽

Jörg-Christian Greie

Keyword(s):

Gene Expression ◽

Expression Vector ◽

Expression System ◽

Expression Patterns ◽

Inducible Expression ◽

Halobacterium Salinarum ◽

Expression Vectors ◽

Content Type ◽

Inducible Expression System

ABSTRACTGradually inducible expression vectors which are governed by variations of growth conditions are powerful tools for gene expression of conditionally lethal mutants. Furthermore, controlled expression allows monitoring of overproduction of proteins at various stages in their expressing hosts. ForHalobacterium salinarum, which is often used as a paradigm for halophilic archaea, such an inducible expression system is not available to date. Here we show that thekdppromoter (Pkdp), which facilitates gene expression upon K+limitation, can be used to establish such a system for molecular applications. Pkdpfeatures a rather high expression rate, with an approximately 50-fold increase that can be easily varied by K+concentrations in the growth medium. Besides the construction of an expression vector, our work describes the characterization of expression patterns and, thus, offers a gradually inducible expression system to the scientific community.

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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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The Role of miR-210 in the Biological System: A Current Overview

Human Heredity ◽

10.1159/000509280 ◽

2019 ◽

Vol 84 (6) ◽

pp. 233-239

Author(s):

Xu Hui ◽

Hisham Al-Ward ◽

Fahmi Shaher ◽

Chun-Yang Liu ◽

Ning Liu

Keyword(s):

Gene Expression ◽

Regulation Of Gene Expression ◽

Low Oxygen ◽

Cellular Functions ◽

Regulate Gene Expression ◽

System A ◽

Non Coding Rnas ◽

Post Transcriptional Regulation ◽

A Current

Background: MicroRNAs (miRNAs) represent a group of non-coding RNAs measuring 19–23 nucleotides in length and are recognized as powerful molecules that regulate gene expression in eukaryotic cells. miRNAs stimulate the post-transcriptional regulation of gene expression via direct or indirect mechanisms. Summary: miR-210 is highly upregulated in cells under hypoxia, thereby revealing its significance to cell endurance. Induction of this mRNA expression is an important feature of the cellular low-oxygen response and the most consistent and vigorous target of HIF. Key Message: miR-210 is involved in many cellular functions under the effect of HIF-1α, including the cell cycle, DNA repair, immunity and inflammation, angiogenesis, metabolism, and macrophage regulation. It also plays an important regulatory role in T-cell differentiation and stimulation.

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In VivoProgrammed Gene Expression Based on Artificial Quorum Networks

Applied and Environmental Microbiology ◽

10.1128/aem.01113-15 ◽

2015 ◽

Vol 81 (15) ◽

pp. 4984-4992 ◽

Cited By ~ 1

Author(s):

Teng Chu ◽

Yajun Huang ◽

Mingyu Hou ◽

Qiyao Wang ◽

Jingfan Xiao ◽

...

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Cell Density ◽

Protective Antigen ◽

Expression System ◽

Edwardsiella Tarda ◽

Content Type ◽

Genetic Components ◽

Wide Range

ABSTRACTThe quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of theVibrio fischeri luxI-luxRquorum sensing system. In order to achievein vivoprogrammed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter ParaBAD, into the QS system.In vitroexpression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density.In vivoexpression assays confirmed that the araQS system presented anin vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applicationsin vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuatedEdwardsiella tardastrain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expressionin vivoand might have potential uses, including, but not limited to, bacterial vector vaccines.

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Discovery and Characterization of Native Deinococcus radiodurans Promoters for Tunable Gene Expression

Applied and Environmental Microbiology ◽

10.1128/aem.01356-19 ◽

2019 ◽

Vol 85 (21) ◽

Author(s):

Angela Chen ◽

Mark W. Sherman ◽

Cynthia Chu ◽

Natalia Gonzalez ◽

Tulshi Patel ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Engineering ◽

Protein Production ◽

Deinococcus Radiodurans ◽

Promoter Sequence ◽

Current Standard ◽

Fluorescent Reporter ◽

Content Type ◽

Oxidative Stresses

ABSTRACT The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of Deinococcus radiodurans for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in D. radiodurans using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in D. radiodurans. Of these top eight, two promoter regions, PDR_1261 and PrpmB, were stronger and more consistent than the most widely used promoter sequence in D. radiodurans, PgroES. Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in Escherichia coli or were D. radiodurans specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in D. radiodurans. IMPORTANCE The tolerance of the extremophile, Deinococcus radiodurans, to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in D. radiodurans, very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for D. radiodurans via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, PgroES. This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in D. radiodurans. The promoters identified in this study will facilitate metabolic engineering of D. radiodurans and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.

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Complete Genome Sequence of Escherichia coli BL21-AI

Microbiology Resource Announcements ◽

10.1128/mra.00009-20 ◽

2020 ◽

Vol 9 (10) ◽

Author(s):

Niketa Bhawsinghka ◽

Katie F. Glenn ◽

Roel M. Schaaper

Keyword(s):

Escherichia Coli ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Expression System ◽

High Yield ◽

Content Type ◽

System A ◽

Phage T7 ◽

High Level

Escherichia coli BL21-AI is a commercially available strain possessing a phage T7-based protein-expression system. A combination of tight regulation and high yield makes it widely used for high-level expression of toxic recombinant proteins. Here, we present the complete genome sequence of BL21-AI and provide insights on its genome.

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Borrelia Host Adaptation Protein (BadP) Is Required for the Colonization of a Mammalian Host by the Agent of Lyme Disease

Infection and Immunity ◽

10.1128/iai.00057-18 ◽

2018 ◽

Vol 86 (7) ◽

Cited By ~ 2

Author(s):

Trever C. Smith ◽

Sarah M. Helm ◽

Yue Chen ◽

Ying-Han Lin ◽

S. L. Rajasekhar Karna ◽

...

Keyword(s):

Gene Expression ◽

Lyme Disease ◽

Immunoblot Analysis ◽

Growth Defect ◽

Mammalian Host ◽

Content Type ◽

Magnesium Chelatase ◽

Virulence Potential ◽

Genes Encoding

ABSTRACT Borrelia burgdorferi , the agent of Lyme disease (LD), uses host-derived signals to modulate gene expression during the vector and mammalian phases of infection. Microarray analysis of mutants lacking the B orrelia host ad aptation r egulator (BadR) revealed the downregulation of genes encoding enzymes whose role in the pathophysiology of B. burgdorferi is unknown. Immunoblot analysis of the badR mutants confirmed reduced levels of these enzymes, and one of these enzymes, encoded by bb0086 , shares homology to prokaryotic magnesium chelatase and Lon-type proteases. The BB0086 levels in B. burgdorferi were higher under conditions mimicking those in fed ticks. Mutants lacking bb0086 had no apparent in vitro growth defect but were incapable of colonizing immunocompetent C3H/HeN or immunodeficient SCID mice. Immunoblot analysis revealed reduced levels of proteins critical for the adaptation of B. burgdorferi to the mammalian host, such as OspC, DbpA, and BBK32. Both RpoS and BosR, key regulators of gene expression in B. burgdorferi , were downregulated in the bb0086 mutants. Therefore, we designated BB0086 the B orrelia host ad aptation p rotein (BadP). Unlike badP mutants, the control strains established infection in C3H/HeN mice at 4 days postinfection, indicating an early colonization defect in mutants due to reduced levels of the lipoproteins/regulators critical for initial stages of infection. However, badP mutants survived within dialysis membrane chambers (DMCs) implanted within the rat peritoneal cavity but, unlike the control strains, did not display complete switching of OspA to OspC, suggesting incomplete adaptation to the mammalian phase of infection. These findings have opened a novel regulatory mechanism which impacts the virulence potential of B . burgdorferi .

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A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile

Journal of Bacteriology ◽

10.1128/jb.00711-18 ◽

2019 ◽

Vol 201 (14) ◽

Cited By ~ 13

Author(s):

Ute Müh ◽

Anthony G. Pannullo ◽

David S. Weiss ◽

Craig D. Ellermeier

Keyword(s):

Gene Expression ◽

Cell Division ◽

Fluorescent Protein ◽

Expression System ◽

Developed Countries ◽

Inducible Expression ◽

Content Type ◽

Antibiotic Development ◽

Crispr Interference ◽

Clostridioides Difficile

ABSTRACT Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile. The xylose-inducible expression vector allows for ∼100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA, and the peptidoglycan synthase gene pbp-0712. CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis. IMPORTANCE Clostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (Pxyl) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile. Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.

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