Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture

ABSTRACT The arabinose-inducible promoter PBAD is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, thearaE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-β-d-thiogalactopyranoside-induciblePtac and Ptaclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independentaraE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (PBAD ) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGHmutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.

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First report of a tetracycline-inducible gene expression system for mollicutes

Microbiology ◽

10.1099/mic.0.034074-0 ◽

2010 ◽

Vol 156 (1) ◽

pp. 198-205 ◽

Cited By ~ 8

Author(s):

Marc Breton ◽

Evelyne Sagné ◽

Sybille Duret ◽

Laure Béven ◽

Christine Citti ◽

...

Keyword(s):

Gene Expression ◽

Expression System ◽

Inducible Promoter ◽

High Level Expression ◽

Spiroplasma Citri ◽

High Level ◽

Tetr Repressor ◽

Promoter System ◽

Inducible Gene

Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO2 from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO2 tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO2 promoter. Adding tetracycline (>50 ng ml−1) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO2 system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.

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Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4985-4988.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4985-4988 ◽

Cited By ~ 48

Author(s):

Laurence Girbal ◽

Isabelle Mortier-Barrière ◽

Frédéric Raynaud ◽

Céline Rouanet ◽

Christian Croux ◽

...

Keyword(s):

Gene Expression ◽

Clostridium Acetobutylicum ◽

Time Course ◽

Specific Activity ◽

Expression System ◽

Regulatory System ◽

Inducible Promoter ◽

Reporter System ◽

Inducible Gene

ABSTRACT A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of β-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system.

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260. Controlled Nonviral Gene Delivery and Expression Using Stable Neural Stem Cell Line Transfected with a Hypoxia-Inducible Gene Expression System

Molecular Therapy ◽

10.1016/s1525-0016(16)37701-2 ◽

2010 ◽

Vol 18 ◽

pp. S99

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Gene Delivery ◽

Cell Line ◽

Expression System ◽

Nonviral Gene Delivery ◽

Stem Cell Line ◽

Gene Expression System ◽

Neural Stem Cell Line ◽

Inducible Gene

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High efficiency inducible gene expression system based on activation of a chimeric transcription factor in transgenic pine

Plant Cell Reports ◽

10.1007/s00299-005-0009-1 ◽

2005 ◽

Vol 24 (10) ◽

pp. 619-628 ◽

Cited By ~ 5

Author(s):

Wei Tang ◽

Ronald J. Newton ◽

Thomas M. Charles

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

High Efficiency ◽

Expression System ◽

Inducible Gene Expression ◽

Gene Expression System ◽

Chimeric Transcription Factor ◽

Inducible Gene

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Generation of Xylose-inducible promoter tools for Pseudomonas species and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

10.1101/2020.01.31.929794 ◽

2020 ◽

Author(s):

Jake D. Callaghan ◽

Nicholas A. Stella ◽

Kara M. Lehner ◽

Benjamin R. Treat ◽

Kimberly M. Brothers ◽

...

Keyword(s):

Gene Expression ◽

Wound Closure ◽

Inducible Promoter ◽

Secretion System ◽

Type Ii Secretion ◽

Type Ii ◽

Corneal Epithelial ◽

E Coli ◽

Type Ii Secretion System ◽

Promoter System

ABSTRACTTunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and P. fluorescens. The Pxut promoter derived from the P. flurorescens xut operon was incorporated into a broad host-range pBBR1-based plasmid and compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. GFP-fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate cloning of toxic genes using E. coli to generate plasmids. The Pxut promoter was expressed at a lower inducer concentration than PBAD in P. fluorescens and higher gfp levels were achieved using Pxut. Flow cytometry analysis indicated that Pxut was more leaky than PBAD in the tested Pseudomonas species, but was expressed in a higher proportion of cells when induced. D-xylose did not support growth of P. aeruginosa or P. fluorescens as a sole carbon source and is less expensive than many other commonly used inducers which could facilitate large scale applications. The efficacy of this system aided in demonstrating a role for the P. aeruginosa type II secretion system gene from xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species.ImportancePseudomonas species are enormously important in human infections, biotechnology, and as a model system for interrogating basic science questions. In this study we have developed a xylose-inducible promoter system and evaluated it in P. aeruginosa and P. fluorescens and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type 2 secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

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Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis

Journal of Bacteriology ◽

10.1128/jb.180.3.680-689.1998 ◽

1998 ◽

Vol 180 (3) ◽

pp. 680-689 ◽

Cited By ~ 80

Author(s):

Reinhold Horlacher ◽

Karina B. Xavier ◽

Helena Santos ◽

Jocelyne DiRuggiero ◽

Marina Kossmann ◽

...

Keyword(s):

Transport System ◽

Signal Sequence ◽

Binding Protein ◽

Inducible Promoter ◽

Transport Systems ◽

Thermococcus Litoralis ◽

Hyperthermophilic Archaeon ◽

E Coli ◽

Transmembrane Segments ◽

Maltose Transport

ABSTRACT We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeonThermococcus litoralis that is homologous to themalEFG cluster encoding the Escherichia colimaltose transport system. The deduced amino acid sequence of themalE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with aKd of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

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Development of cDNA normalization system and preliminary transcription analysis of KCS genes in apple tissues

Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis ◽

10.11118/actaun201159030009 ◽

2011 ◽

Vol 59 (3) ◽

pp. 9-12 ◽

Cited By ~ 3

Author(s):

Zsolt Albert ◽

Cs. Deák ◽

A. Miskó ◽

M. Tóth ◽

I. Papp

Keyword(s):

Gene Expression ◽

Expression System ◽

Expression Patterns ◽

Housekeeping Genes ◽

Apple Fruit ◽

Rt Pcr ◽

Specific Primers ◽

Specific Expression ◽

Transcription Analysis ◽

Tissue Specific

Wax production is an important aspect of apple (Malus domestica Borkh.) fruit development from both theoretical and practical point of views. The complex molecular mechanism that controls wax biosynthesis is still widely unknown but many studies focused on this topic. We aimed to develop further the experimental framework of these efforts with a description of an improved reference genes expression system. Results in the literature show that similarities exist among the expression of some housekeeping genes of different plant species. Based on these considerations and on gene expression data from Arabidopsis thaliana, some genes in apple were assigned for analysis. EST sequences of apple were used to design specific primers for RT-PCR experiments. Isolation of intact RNA from different apple tissues and performing RT-PCR reaction were also key point in obtaining expression patterns. To monitor DNA contamination of the RNA samples, specific primers were used that amplify intron-containing sequences from the cDNA. We found that actin primers can be used for the detection of intron containing genomic DNA, and tubulin primers are good internal controls in RT-PCR experiments. We were able to make a difference between tissue-specific and tissue-independent gene-expression, furthermore we found tissue specific differences between the expression patterns of candidate genes, that are potentially involved in wax-biosynthesis. Our results show that KCS1 and KCS4 are overexpressed in the skin tissue, this could mean that these genes have skin-specific expression in apple fruit.

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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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