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

2018 ◽  
Vol 84 (7) ◽  
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.

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

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Keith E. Weaver ◽  
Yuqing Chen ◽  
Elly M. Miiller ◽  
Jake N. Johnson ◽  
Alex A. Dangler ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Expression Vector ◽  
Dynamic Range ◽  
Cell Physiology ◽  
System Function ◽  
High Background ◽  
Content Type ◽  
Broad Dynamic Range ◽  
Regulated Expression ◽  
Regulated Gene Expression

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.

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

2014 ◽  
Vol 82 (4) ◽  
pp. 1511-1522 ◽  
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.

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

2012 ◽  
Vol 78 (7) ◽  
pp. 2100-2105 ◽  
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.

scholarly journals σIfromBacillus subtilis: Impact on Gene Expression and Characterization of σI-Dependent Transcription That Requires New Types of Promoters with Extended −35 and −10 Elements

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Olga Ramaniuk ◽  
Martin Převorovský ◽  
Jiří Pospíšil ◽  
Dragana Vítovská ◽  
Olga Kofroňová ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Iron Metabolism ◽  
Transcription Initiation ◽  
Iron Homeostasis ◽  
Model Organism ◽  
Content Type ◽  
Σ Factor

ABSTRACTThe σIsigma factor fromBacillus subtilisis a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI. Further analysis revealed that the majority of these genes were affected indirectly by σI. The σIregulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (thedhbandykuoperons) are involved in iron metabolism. The involvement of σIin iron metabolism was confirmed phenotypically. Next, we set up anin vitrotranscription system and defined and experimentally validated the promoter sequence logo that, in addition to −35 and −10 regions, also contains extended −35 and −10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organismB. subtilis.IMPORTANCEIn bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σIregulon from the industrially important model Gram-positive bacteriumBacillus subtilis. We reveal that σIaffects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of theB. subtilistranscription machinery.

scholarly journals Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

2004 ◽  
Vol 279 (18) ◽  
pp. 18776-18782 ◽  
Author(s):  
Atze T. Das ◽  
Xue Zhou ◽  
Monique Vink ◽  
Bep Klaver ◽  
Koen Verhoef ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Evolution ◽  
Expression System ◽  
Gene Expression System ◽  
Regulated Gene Expression

scholarly journals In VivoProgrammed Gene Expression Based on Artificial Quorum Networks

2015 ◽  
Vol 81 (15) ◽  
pp. 4984-4992 ◽  
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.

scholarly journals Pirated Siderophores Promote Sporulation in Bacillus subtilis

2017 ◽  
Vol 83 (10) ◽  
Author(s):  
Gabrielle M. Grandchamp ◽  
Lews Caro ◽  
Elizabeth A. Shank
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Iron Acquisition ◽  
Microbial Interactions ◽  
Mineral Nutrient ◽  
Cell Physiology ◽  
Content Type ◽  
E Coli ◽  
Esterase Gene

ABSTRACT In microbial communities, bacteria chemically and physically interact with one another. Some of these interactions are mediated by secreted specialized metabolites that act as either intraspecies or interspecies signals to alter gene expression and to change cell physiology. Bacillus subtilis is a well-characterized soil microbe that can differentiate into multiple cell types, including metabolically dormant endospores. We were interested in identifying microbial interactions that affected sporulation in B. subtilis. Using a fluorescent transcriptional reporter, we observed that coculturing B. subtilis with Escherichia coli promoted sporulation gene expression via a secreted metabolite. To identify the active compound, we screened the E. coli Keio Collection and identified the sporulation-accelerating cue as the siderophore enterobactin. B. subtilis has multiple iron acquisition systems that are used to take up the B. subtilis-produced siderophore bacillibactin, as well as to pirate exogenous siderophores such as enterobactin. While B. subtilis uses a single substrate binding protein (FeuA) to take up both bacillibactin and enterobactin, we discovered that it requires two distinct genes to sporulate in response to these siderophores (the esterase gene besA for bacillibactin and a putative esterase gene, ybbA, for enterobactin). In addition, we found that siderophores from a variety of other microbial species also promote sporulation in B. subtilis. Our results thus demonstrate that siderophores can act not only as bacterial iron acquisition systems but also as interspecies cues that alter cellular development and accelerate sporulation in B. subtilis. IMPORTANCE While much is known about the genetic regulation of Bacillus subtilis sporulation, little is understood about how other bacteria influence this process. This work describes an interaction between Escherichia coli and B. subtilis that accelerates sporulation in B. subtilis. The interaction is mediated by the E. coli siderophore enterobactin; we show that other species' siderophores also promote sporulation gene expression in B. subtilis. These results suggest that siderophores not only may supply bacteria with the mineral nutrient iron but also may play a role in bacterial interspecies signaling, providing a cue for sporulation. Siderophores are produced by many bacterial species and thus potentially play important roles in altering bacterial cell physiology in diverse environments.

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