scholarly journals Improved dynamic range of a rhamnose-inducible promoter for gene expression in Burkholderia

2021 ◽  
Author(s):  
Andrew M Hogan ◽  
Kevin R Jeffers ◽  
Armando Palacios ◽  
Silvia T Cardona
Keyword(s):  
Gene Expression ◽  
Related Species ◽  
Dynamic Range ◽  
Inducible Promoter ◽  
Maximum Activity ◽  
Essential Genes ◽  
Heterologous Protein Expression ◽  
Bacterial Physiology ◽  
Promoter System ◽  
Genetic Toolkit

A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of this toolkit. In Burkholderia and related species, the L-rhamnose-inducible promoter is among the first choices due to its tight control and the lack of viable alternatives. To improve upon its maximum activity and dynamic range, we explored the effect of promoter system modifications in B. cenocepacia with a LacZ-based reporter. By combining the bacteriophage T7 gene 10 stem loop and engineered rhaI transcription factor-binding sites, we obtained a rhamnose-inducible system with a 6.5-fold and 3.0-fold increase in maximum activity and dynamic range, respectively, compared to the native promoter. We then added the modified promoter system to pSCrhaB2 and pSC201, common genetic tools used for plasmid-based and chromosome-based gene expression, respectively, in Burkholderia, creating pSCrhaB2plus and pSC201plus. We demonstrated the utility of pSCrhaB2plus for gene expression in B. thailandensis , B. multivorans and B. vietnamiensis and used pSC201plus to control highly expressed essential genes from the chromosome of B. cenocepacia . The utility of the modified system was demonstrated as we recovered viable mutants to control ftsZ , rpoBC , and rpsF , whereas the unmodified promoter was unable to control rpsF . The modified expression system allowed control of an essential gene depletion phenotype at lower levels of L-rhamnose, the inducer. pSCRhaB2plus and pSC201plus are expected to be valuable additions to the genetic toolkit for Burkholderia and related species. Importance Species of Burkholderia are dually recognized as being of attractive biotechnological potential but also opportunistic pathogens for immunocompromised individuals. Understanding the genotype-phenotype relationship is critical for synthetic biology approaches in Burkholderia to disentangle pathogenic from beneficial traits. A diverse genetic toolkit, including inducible promoters, is the foundation for these investigations. We thus sought to improve on the commonly used rhamnose-inducible promoter system. Our modifications resulted in both higher levels of heterologous protein expression and broader control over highly-expressed essential genes in B. cenocepacia . The significance of our work is in expanding the genetic toolkit to enable more comprehensive studies into Burkholderia and related bacteria.

scholarly journals An Estradiol-Inducible Promoter Enables Fast, Graduated Control of Gene Expression in Fission Yeast

2016 ◽  
Author(s):  
Makoto J. Ohira ◽  
David G. Hendrickson ◽  
R. Scott McIsaac ◽  
Nicholas Rhind
Keyword(s):  
Gene Expression ◽  
Fission Yeast ◽  
Dose Response ◽  
Dynamic Range ◽  
Inducible Promoter ◽  
Experimental Manipulation ◽  
Control Of Gene Expression ◽  
Inducible Promoters ◽  
Linear Dose ◽  
Promoter System

ABSTRACTThe fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β-estradiol-regulated function of the human estrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3EV, turns on quickly, can reach a maximal induction of 20 fold, and exhibits a linear dose response over its entire induction range, with few off target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast.

scholarly journals 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

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.

scholarly journals Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Biology Open ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. bio055343 ◽  
Author(s):  
Daniel Chu ◽  
An Nguyen ◽  
Spenser S. Smith ◽  
Zuzana Vavrušová ◽  
Richard A. Schneider
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Inducible Promoter ◽  
In Ovo ◽  
Control Of Gene Expression ◽  
Promoter Construct ◽  
Avian Development ◽  
Spatiotemporal Control ◽  
Promoter System

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

scholarly journals Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression inClostridium perfringens

2010 ◽  
Vol 77 (2) ◽  
pp. 471-478 ◽  
Author(s):  
Andrea H. Hartman ◽  
Hualan Liu ◽  
Stephen B. Melville
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Shuttle Vector ◽  
Yellow Fluorescent Protein ◽  
Inducible Promoter ◽  
Gliding Motility ◽  
Glucuronidase Activity ◽  
Atpase Gene ◽  
Regulated Gene Expression ◽  
Promoter System

ABSTRACTClostridium perfringensis a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working withC. perfringens, a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is anEscherichia coli-C. perfringensshuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of genebgaL(bgaR-PbgaL). To measure transcription at thebgaLpromoter in pKRAH1, theE. colireporter genegusA, encoding β-glucuronidase, was placed downstream of the PbgaLpromoter to make plasmid pAH2. When transformed into three strains ofC. perfringens, pAH2 exhibited lactose-inducible expression.C. perfringensstrain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated genebglR, encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express theyfp-pilBgene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility inC. perfringens. Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in apilCmutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.

scholarly journals Xylose-Inducible Promoter Tools for Pseudomonas Species and Their Use in Implicating a Role for the Type II Secretion System Protein XcpQ in the Inhibition of Corneal Epithelial Wound Closure

2020 ◽  
Vol 86 (14) ◽  
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 ◽  
Content Type ◽  
Corneal Epithelial ◽  
Type Ii Secretion System ◽  
Promoter System

ABSTRACT Tunable 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 Pseudomonas fluorescens. The Pxut promoter, derived from the P. fluorescens xut operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. Green fluorescent protein (GFP) fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate the cloning of genes toxic to E. coli to generate plasmids. The Pxut promoter was activated 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 leakier than PBAD in the Pseudomonas species tested but was expressed in a higher proportion of cells when induced. d-Xylose as a sole carbon source did not support the growth of P. aeruginosa or P. fluorescens and is less expensive than many other commonly used inducers, which could facilitate large-scale applications. The efficacy of this system was demonstrated by its use to reveal a role for the P. aeruginosa type II secretion system gene 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. IMPORTANCE Pseudomonas species are enormously important in human infections, in biotechnology, and as model systems for investigating basic science questions. In this study, we have developed a xylose-inducible promoter system, 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 II secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

An Engineered B. subtilis Inducible Promoter System with over 10 000-Fold Dynamic Range

2019 ◽  
Vol 8 (7) ◽  
pp. 1673-1678 ◽  
Author(s):  
Sebastian M. Castillo-Hair ◽  
Masaya Fujita ◽  
Oleg A. Igoshin ◽  
Jeffrey J. Tabor
Keyword(s):  
Dynamic Range ◽  
Inducible Promoter ◽  
Promoter System

scholarly journals AtetRA-based promoter system for the generation of conditional knockouts inCampylobacter jejuni

2019 ◽  
Author(s):  
Eli J. Cohen ◽  
Rui Tong Quek ◽  
Morgan Beeby
Keyword(s):  
Gene Expression ◽  
Human Health ◽  
Campylobacter Jejuni ◽  
Target Gene ◽  
Tetracycline Resistance ◽  
Future Research ◽  
Human Pathogen ◽  
Target Gene Expression ◽  
Promoter System ◽  
Genetic Toolkit

AbstractCampylobacter jejuniis responsible for tens of millions of cases of gastroenteritis each year. Despite its prevalence and impact on human health, the repertoire of genetic tools available for researchers to studyC. jejuniremains limited. In order to expand upon the genetic toolkit in this species, we have engineered a system for generating conditional knockouts based on thetetRAtetracycline-resistance cassette. This system exhibits tight repressibility and titratability of target-gene expression and will be useful for future research on this important human pathogen.

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

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 198-205 ◽  
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.

scholarly journals The Escherichia colirhaSR-PrhaBADInducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

2016 ◽  
Vol 82 (22) ◽  
pp. 6715-6727 ◽  
Author(s):  
Jeffrey Meisner ◽  
Joanna B. Goldberg
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Catabolite Repression ◽  
Inducible Promoter ◽  
Translational Initiation ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Promoter System

ABSTRACTThearaC-ParaBADinducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range inEscherichia coli, which has led to its widespread use in other bacteria. Although anecdotal evidence suggests thataraC-ParaBADis leaky inPseudomonas aeruginosa, neither a thorough analysis of this inducible promoter system inP. aeruginosanor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of thearaC-ParaBADsystem inP. aeruginosa. Using transcriptional fusions to alacZreporter gene, we determined that the noninduced expression fromaraC-ParaBADis high and cannot be reduced by carbon catabolite repression as it can inE. coli. Modulating translational initiation by altering ribosome-binding site strength reduced the noninduced activity but also decreased the maximal induced activity and narrowed the induction range. Integrating the inducible promoter system into the posttranscriptional regulatory network that controls catabolite repression inP. aeruginosasignificantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of thearaC-ParaBADsystem, we found that thelacIq-PtacandrhaSR-PrhaBADinducible promoter systems had significantly lower noninduced expression and were inducible over a broader range thanaraC-ParaBAD. We demonstrated that noninduced expression from thearaC-ParaBADsystem supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis inP. aeruginosa, problems that were avoided withrhaSR-PrhaBAD. rhaSR-PrhaBADis tightly controlled, allows gene expression over a wide range, and represents a significant improvement overaraC-ParaBADinP. aeruginosa.IMPORTANCEWe report the shortcomings of the commonly usedEscherichia coli araC-ParaBADinducible promoter system inPseudomonas aeruginosa, successfully reengineered it to improve its functionality, and show that theE. colirhaSR-PrhaBADsystem is tightly controlled and allows inducible gene expression over a wide range inP. aeruginosa.

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