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 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 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 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 BEM46 Shows Eisosomal Localization and Association with Tryptophan-Derived Auxin Pathway in Neurospora crassa

2014 ◽  
Vol 13 (8) ◽  
pp. 1051-1063 ◽  
Author(s):  
K. Kollath-Leiß ◽  
C. Bönniger ◽  
P. Sardar ◽  
F. Kempken
Keyword(s):  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Yellow Fluorescent Protein ◽  
Content Type ◽  
Bioinformatic Tools ◽  
Ascospore Germination ◽  
Alternatively Spliced ◽  
Indole Production

ABSTRACTBEM46 proteins are evolutionarily conserved, but their functions remain elusive. We reported previously that the BEM46 protein inNeurospora crassais targeted to the endoplasmic reticulum (ER) and is essential for ascospore germination. In the present study, we established abem46knockout strain ofN. crassa. This Δbem46mutant exhibited a level of ascospore germination lower than that of the wild type but much higher than those of the previously characterizedbem46-overexpressing and RNA interference (RNAi) lines. Reinvestigation of the RNAi transformants revealed two types of alternatively splicedbem46mRNA; expression of either type led to a loss of ascospore germination. Our results indicated that the phenotype was not due tobem46mRNA downregulation or loss but was caused by the alternatively spliced mRNAs and the peptides they encoded. Using theN. crassaortholog of the eisosomal protein PILA fromAspergillus nidulans, we further demonstrated the colocalization of BEM46 with eisosomes. Employing the yeast two-hybrid system, we identified a single interaction partner: anthranilate synthase component II (encoded bytrp-1). This interaction was confirmedin vivoby a split-YFP (yellow fluorescent protein) approach. The Δtrp-1mutant showed reduced ascospore germination and increased indole production, and we used bioinformatic tools to identify a putative auxin biosynthetic pathway. The genes involved exhibited various levels of transcriptional regulation in the differentbem46transformant and mutant strains. We also investigated the indole production of the strains in different developmental stages. Our findings suggested that the regulation of indole biosynthesis genes was influenced bybem46overexpression. Furthermore, we uncovered evidence of colocalization of BEM46 with the neutral amino acid transporter MTR.

scholarly journals Proteins with CHADs (Conserved Histidine α-Helical Domains) Are Attached to Polyphosphate Granules In Vivo and Constitute a Novel Family of Polyphosphate-Associated Proteins (Phosins)

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Tony Tumlirsch ◽  
Dieter Jendrossek
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Seed Plants ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Oil Seed ◽  
Eukaryotic Microorganisms ◽  
Specific Manner ◽  
Associated Proteins

ABSTRACT On the basis of bioinformatic evidence, we suspected that proteins with a CYTH (CyaB thiamine triphosphatase) domain and/or a CHAD (conserved histidine α-helical domain) motif might represent polyphosphate (polyP) granule-associated proteins. We found no evidence of polyP targeting by proteins with CYTH domains. In contrast, two CHAD motif-containing proteins from Ralstonia eutropha H16 (A0104 and B1017) that were expressed as fusions with enhanced yellow fluorescent protein (eYFP) colocalized with polyP granules. While the expression of B1017 was not detectable, the A0104 protein was specifically identified in an isolated polyP granule fraction by proteome analysis. Moreover, eYFP fusions with the CHAD motif-containing proteins MGMSRV2-1987 from Magnetospirillum gryphiswaldense and PP2307 from Pseudomonas putida also colocalized with polyP granules in a transspecies-specific manner. These data indicated that CHAD-containing proteins are generally attached to polyP granules. Together with the findings from four previously polyP-attached proteins (polyP kinases), the results of this study raised the number of polyP-associated proteins in R. eutropha to six. We suggest designating polyP granule-bound proteins with CHAD motifs as phosins (phosphate), analogous to phasins and oleosins that are specifically bound to the surface of polyhydroxyalkanoate (PHA) granules in PHA-accumulating bacteria and to oil droplets in oil seed plants, respectively. IMPORTANCE The importance of polyphosphate (polyP) for life is evident from the ubiquitous presence of polyP in all species on earth. In unicellular eukaryotic microorganisms, polyP is located in specific membrane-enclosed organelles, called acidocalcisomes. However, in most prokaryotes, polyP is present as insoluble granules that have been designated previously as volutin granules. Almost nothing is known regarding the macromolecular composition of polyP granules. Particularly, the absence or presence of cellular compounds on the surface of polyP granules has not yet been investigated. In this study, we identified a novel class of proteins that are attached to the surface of polyP granules in three model species of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. These proteins are characterized by the presence of a CHAD (conserved histidine α-helical domain) motif that functions as a polyP granule-targeting signal. We suggest designating CHAD motif-containing proteins as phosins [analogous to phasins for poly(3-hydroxybutyrate)-associated proteins and to oleosins for oil droplet-associated proteins in oil seed plants]. The expression of phosins in different species confirmed their polyP-targeting function in a transspecies-specific manner. We postulate that polyP granules in prokaryotic species generally have a complex surface structure that consists of one to several polyP kinases and phosin proteins. We suggest differentiating polyP granules from acidocalcisomes by designating them as polyphosphatosomes.

scholarly journals Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology

2013 ◽  
Vol 79 (19) ◽  
pp. 6006-6015 ◽  
Author(s):  
Meike Baumgart ◽  
Simon Unthan ◽  
Christian Rückert ◽  
Jasintha Sivalingam ◽  
Alexander Grünberger ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Stress Conditions ◽  
Phenotypic Characterization ◽  
Industrial Biotechnology ◽  
Plasmid Copy ◽  
Enhanced Yellow Fluorescent Protein ◽  
Modification System ◽  
Content Type

ABSTRACTThe activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producerCorynebacterium glutamicumATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant ofC. glutamicumATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of aC. glutamicumchassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology.

scholarly journals Vitamin K3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
P. Blanco ◽  
F. Corona ◽  
M. B. Sánchez ◽  
J. L. Martínez
Keyword(s):  
Antibiotic Resistance ◽  
Vitamin K ◽  
Stenotrophomonas Maltophilia ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Yellow Fluorescent Protein ◽  
Efflux Pumps ◽  
Fluorescence Signal ◽  
Multidrug Efflux ◽  
Content Type

ABSTRACT Stenotrophomonas maltophilia is an opportunistic pathogen with increasing prevalence, which is able to cause infections in immunocompromised patients or in those with a previous pathology. The treatment of the infections caused by this bacterium is often complicated due to the several intrinsic antibiotic resistance mechanisms that it presents. Multidrug efflux pumps are among the best-studied mechanisms of S. maltophilia antibiotic resistance. Some of these efflux pumps have a basal expression level but, in general, their expression is often low and only reaches high levels when the local regulator is mutated or bacteria are in the presence of an effector. In the current work, we have developed a yellow fluorescent protein (YFP)-based sensor with the aim to identify effectors able to trigger the expression of SmeVWX, an efflux pump that confers resistance to quinolones, chloramphenicol, and tetracycline when it is expressed at high levels. With this purpose in mind, we tested a variety of different compounds and analyzed the fluorescence signal given by the expression of YFP under the control of the smeVWX promoter. Among the tested compounds, vitamin K3, which is a compound belonging to the 2-methyl-1,4-naphthoquinone family, is produced by plants in defense against infection, and has increasing importance in human therapy, was able to induce the expression of the SmeVWX efflux pump. In addition, a decrease in the susceptibility of S. maltophilia to ofloxacin and chloramphenicol was observed in the presence of vitamin K3, in both wild-type and smeW-deficient strains.

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 Structural Study of MPN387, an Essential Protein for Gliding Motility of a Human-Pathogenic Bacterium, Mycoplasma pneumoniae

2016 ◽  
Vol 198 (17) ◽  
pp. 2352-2359 ◽  
Author(s):  
Yoshito Kawakita ◽  
Miki Kinoshita ◽  
Yukio Furukawa ◽  
Isil Tulum ◽  
Yuhei O. Tahara ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Coiled Coil ◽  
Core Structure ◽  
Gliding Motility ◽  
Pathogenic Bacterium ◽  
Predicted Amino Acid Sequence ◽  
Content Type ◽  
Gliding Mechanism

ABSTRACTMycoplasma pneumoniaeis a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed.IMPORTANCEHuman mycoplasma pneumonia is caused by a pathogenic bacterium,Mycoplasma pneumoniae. This tiny, 2-μm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma “gliding motility” is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is directly involved in the gliding mechanism.

scholarly journals Dynamics of Mitochondrial RNA-Binding Protein Complex in Trypanosoma brucei and Its Petite Mutant under Optimized Immobilization Conditions

2014 ◽  
Vol 13 (9) ◽  
pp. 1232-1240 ◽  
Author(s):  
Zhenqiu Huang ◽  
Sabine Kaltenbrunner ◽  
Eva Šimková ◽  
David Stanĕk ◽  
Julius Lukeš ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Yellow Fluorescent Protein ◽  
Rna Binding Protein ◽  
Mitochondrial Rna ◽  
Content Type ◽  
Petite Mutant

ABSTRACT There are a variety of complex metabolic processes ongoing simultaneously in the single, large mitochondrion of Trypanosoma brucei . Understanding the organellar environment and dynamics of mitochondrial proteins requires quantitative measurement in vivo . In this study, we have validated a method for immobilizing both procyclic stage (PS) and bloodstream stage (BS) T. brucei brucei with a high level of cell viability over several hours and verified its suitability for undertaking fluorescence recovery after photobleaching (FRAP), with mitochondrion-targeted yellow fluorescent protein (YFP). Next, we used this method for comparative analysis of the translational diffusion of mitochondrial RNA-binding protein 1 (MRP1) in the BS and in T. b. evansi . The latter flagellate is like petite mutant Saccharomyces cerevisiae because it lacks organelle-encoded nucleic acids. FRAP measurement of YFP-tagged MRP1 in both cell lines illuminated from a new perspective how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism. This work represents the first attempt to examine this process in live trypanosomes.

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