scholarly journals Cell-Based Assay To Identify Inhibitors of the Hfq-sRNA Regulatory Pathway

2014 ◽  
Vol 58 (9) ◽  
pp. 5500-5509 ◽  
Author(s):  
Shaima A. El-Mowafi ◽  
John N. Alumasa ◽  
Sarah E. Ades ◽  
Kenneth C. Keiler
Keyword(s):  
Gene Expression ◽  
Cyclic Peptides ◽  
Fluorescent Protein ◽  
Cyclic Peptide ◽  
Yellow Fluorescent Protein ◽  
Mobility Shift ◽  
Content Type ◽  
Antibiotic Development ◽  
Split Intein ◽  
Gel Mobility Shift

ABSTRACTNoncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a genetic screen to identify inhibitors from a library of cyclic peptides produced inEscherichia coliusing split-intein circular ligation of peptides and proteins (SICLOPPS), an intein-based technology. One cyclic peptide identified in this screen, RI20, inhibited Hfq-mediated repression of gene expression in conjunction with both RybB and an unrelated sRNA, MicF. Gel mobility shift assays showed that RI20 inhibited binding of Hfq to RybB and MicF with similarKivalues. These data suggest that RI20 inhibits Hfq activity by blocking interactions with sRNAs and provide a paradigm for inhibiting virulence genes in Gram-negative pathogens.

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

2019 ◽  
Vol 201 (14) ◽  
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.

scholarly journals Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Adam A. Pérez ◽  
John P. Gajewski ◽  
Bryan H. Ferlez ◽  
Marcus Ludwig ◽  
Carol S. Baker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Expression System ◽  
Yellow Fluorescent Protein ◽  
Background Level ◽  
Basic Research ◽  
Industrial Applications ◽  
Inducible Expression ◽  
Content Type ◽  
Expression Platform

ABSTRACT Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn2+-inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn2+ uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA 7942) and its cognate SmtB7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA 6803) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster β-carotene 15,15′-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002. IMPORTANCE This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn2+-inducible system, which is based on the smtA 7942 operator/promoter and smtB 7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

scholarly journals Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

2014 ◽  
Vol 80 (21) ◽  
pp. 6704-6713 ◽  
Author(s):  
Amy T. Ma ◽  
Calvin M. Schmidt ◽  
James W. Golden
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Atmospheric Carbon Dioxide ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Yellow Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Ease Of Use ◽  
Algal Biotechnology ◽  
Content Type

ABSTRACTCyanobacteria are photosynthetic bacteria that are currently being developed as biological production platforms. They derive energy from light and carbon from atmospheric carbon dioxide, and some species can fix atmospheric nitrogen. One advantage of developing cyanobacteria for renewable production of biofuels and other biological products is that they are amenable to genetic manipulation, facilitating bioengineering and synthetic biology. To expand the currently available genetic toolkit, we have demonstrated the utility of synthetic theophylline-responsive riboswitches for effective regulation of gene expression in four diverse species of cyanobacteria, including two recent isolates. We evaluated a set of six riboswitches driving the expression of a yellow fluorescent protein reporter inSynechococcus elongatusPCC 7942,Leptolyngbyasp. strain BL0902,Anabaenasp. strain PCC 7120, andSynechocystissp. strain WHSyn. We demonstrated that riboswitches can offer regulation of gene expression superior to that of the commonly used isopropyl-β-d-thiogalactopyranoside induction of alacIq-Ptrcpromoter system. We also showed that expression of the toxic protein SacB can be effectively regulated, demonstrating utility for riboswitch regulation of proteins that are detrimental to biomass accumulation. Taken together, the results of this work demonstrate the utility and ease of use of riboswitches in the context of genetic engineering and synthetic biology in diverse cyanobacteria, which will facilitate the development of algal biotechnology.

scholarly journals Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Celina Frank ◽  
Dieter Jendrossek
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Eukaryotic Cells ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Polyphosphate Granules

ABSTRACT Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have also been described for Agrobacterium tumefaciens (M. Seufferheld, M. Vieira, A. Ruiz, C. O. Rodrigues, S. Moreno, and R. Docampo, J Biol Chem 278:29971–29978, 2003, https://doi.org/10.1074/jbc.M304548200). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this alphaproteobacterium. Unexpectedly, fluorescence microscopic investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H+-translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located close together, near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1 Δppk2) were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear. IMPORTANCE The uptake of alphaproteobacterium-like cells by ancestors of eukaryotic cells and subsequent conversion of these alphaproteobacterium-like cells to mitochondria are thought to be key steps in the evolution of the first eukaryotic cells. The identification of acidocalcisomes in two alphaproteobacterial species some years ago and the presence of homologs of the vacuolar proton-translocating pyrophosphatase HppA, a marker protein of the acidocalcisome membrane in eukaryotes, in virtually all species within the alphaproteobacteria suggest that eukaryotic acidocalcisomes might also originate from related structures in ancestors of alphaproteobacterial species. Accordingly, alphaproteobacterial acidocalcisomes and eukaryotic acidocalcisomes should have similar features. Since hardly any information is available on bacterial acidocalcisomes, this study aimed at the characterization of organelle-like structures in alphaproteobacterial cells, with A. tumefaciens as an example.

scholarly journals PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

2014 ◽  
Vol 82 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Ziyu Sun ◽  
Jing Shi ◽  
Chang Liu ◽  
Yongxin Jin ◽  
Kewei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Colonization ◽  
Mrna Level ◽  
Sos Response ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Chronic Infections ◽  
Mobility Shift ◽  
Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by atacpromoter repressed the endogenousprtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

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 Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus

2013 ◽  
Vol 79 (7) ◽  
pp. 2218-2224 ◽  
Author(s):  
Jeffrey L. Bose ◽  
Paul D. Fey ◽  
Kenneth W. Bayles
Keyword(s):  
Staphylococcus Aureus ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Exchange System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genetic Tools ◽  
Allelic Exchange ◽  
Green Fluorescent

ABSTRACTThebursa aurealistransposon has been used to create transposon insertion libraries ofBacillus anthracisandStaphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessentialS. aureusgenes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study ofS. aureus.

scholarly journals Fluorescence-Based Bacterial Bioreporter for Specific Detection of Methyl Halide Emissions in the Environment

2013 ◽  
Vol 79 (21) ◽  
pp. 6561-6567 ◽  
Author(s):  
Muhammad Farhan Ul Haque ◽  
Thierry Nadalig ◽  
Françoise Bringel ◽  
Hubert Schaller ◽  
Stéphane Vuilleumier
Keyword(s):  
Fluorescent Protein ◽  
Stratospheric Ozone ◽  
Yellow Fluorescent Protein ◽  
Attractive Alternative ◽  
Detailed Knowledge ◽  
Methyl Halides ◽  
Natural Sources ◽  
Content Type ◽  
Degrading Bacteria ◽  
Methyl Halide

ABSTRACTMethyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, chloromethane (CH3Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics ofMethylobacterium extorquensCM4, an aerobic alphaproteobacterium which utilizes chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the chloromethane dehalogenase genecmuAfused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced intoM. extorquensCM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model chloromethane-producing plantArabidopsis thalianain particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions.

scholarly journals Use of Degradation Tags To Control Protein Levels in the Cyanobacterium Synechocystis sp. Strain PCC 6803

2013 ◽  
Vol 79 (8) ◽  
pp. 2833-2835 ◽  
Author(s):  
Brian P. Landry ◽  
Jana Stöckel ◽  
Himadri B. Pakrasi
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Terminal Protein ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Protein Levels ◽  
Steady State Fluorescence ◽  
Tunable Range ◽  
Control Protein ◽  
Synechocystis Sp

ABSTRACTWe generated a collection ofssrA-based C-terminal protein degradation tags with different degradation strengths. The steady-state fluorescence levels of different enhanced yellow fluorescent protein (eYFP) tag variants in aSynechocystissp. indicated a tunable range from 1% to 50% of untagged eYFP.

scholarly journals Regulation of d-Xylose Metabolism in Caulobacter crescentus by a LacI-Type Repressor

2007 ◽  
Vol 189 (24) ◽  
pp. 8828-8834 ◽  
Author(s):  
Craig Stephens ◽  
Beat Christen ◽  
Kelly Watanabe ◽  
Thomas Fuchs ◽  
Urs Jenal
Keyword(s):  
Gene Expression ◽  
Energy Source ◽  
Regulatory Mechanism ◽  
Caulobacter Crescentus ◽  
Heterologous Gene Expression ◽  
Xylose Metabolism ◽  
Mobility Shift ◽  
Inducible Promoters ◽  
Gel Mobility Shift ◽  
Operator Binding

ABSTRACT In the oligotrophic freshwater bacterium Caulobacter crescentus, d-xylose induces expression of over 50 genes, including the xyl operon, which encodes key enzymes for xylose metabolism. The promoter (P xylX ) controlling expression of the xyl operon is widely used as a tool for inducible heterologous gene expression in C. crescentus. We show here that P xylX and at least one other promoter in the xylose regulon (P xylE ) are controlled by the CC3065 (xylR) gene product, a LacI-type repressor. Electrophoretic gel mobility shift assays showed that operator binding by XylR is greatly reduced in the presence of d-xylose. The data support the hypothesis that there is a simple regulatory mechanism in which XylR obstructs xylose-inducible promoters in the absence of the sugar; the repressor is induced to release DNA upon binding d-xylose, thereby freeing the promoter for productive interaction with RNA polymerase. XylR also has an effect on glucose metabolism, as xylR mutants exhibit reduced expression of the Entner-Doudoroff operon and their ability to utilize glucose as a sole carbon and energy source is compromised.

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