scholarly journals Riboswitch RS thiT as a molecular tool in Lactococcus lactis

2021 ◽  
Author(s):  
Eduardo P. Hernández-Ortega ◽  
Sjoerd van der Meulen ◽  
Lucas J. Kuijpers ◽  
Jan Kok
Keyword(s):  
Lactococcus Lactis ◽  
Dairy Products ◽  
Fluorescent Protein ◽  
Molecular Genetic ◽  
Genetic Defect ◽  
Control Device ◽  
Thiamine Pyrophosphate ◽  
Gene Encoding ◽  
Expression Of Genes ◽  
Green Fluorescent

Previous RNA sequencing has allowed identifying 129 long 5'-UTRs in the L. lactis MG1363 transcriptome. These sequences potentially harbor cis -acting riboswitches. One of the identified extended 5'-UTRs is a putative thiamine pyrophosphate (TPP) riboswitch. It is located immediately upstream of the thiamine transporter gene thiT ( llmg_0334 ). To confirm this assumption, the 5'-UTR sequence was placed upstream of the gene encoding the super folder green fluorescent protein, sfgfp , allowing examining the expression of sfGFP in the presence or absence of thiamine in the medium. The results show that this sequence indeed represents a thiamine-responsive TPP riboswitch. This RNA-based genetic control device was used to successfully restore the mutant phenotype of an L. lactis strain lacking the major autolysin gene acmA . The L. lactis thiT TPP riboswitch (RS thiT ) is a useful molecular genetic tool enabling to gradually downregulate the expression of genes under its control by adjusting thiamine concentration. Importance The capacity of microbes with biotechnological importance to adapt and survive under quickly changing industrial conditions depends on their ability to adequately control gene expression. Riboswitches are important RNA-based elements involved in rapid and precise gene regulation. Here we present the identification of a natural thiamine-responsive riboswitch of Lactococcus lactis , a bacterium used worldwide in the production of dairy products. We used it to restore a genetic defect in an L. lactis mutant and show that it is a valuable addition to the ever-expanding L. lactis genetic toolbox.

scholarly journals Microplate Bioassay for Nisin in Foods, Based on Nisin-Induced Green Fluorescent Protein Fluorescence

2003 ◽  
Vol 69 (7) ◽  
pp. 4214-4218 ◽  
Author(s):  
J. Reunanen ◽  
P. E. J. Saris
Keyword(s):  
Green Fluorescent Protein ◽  
Lactococcus Lactis ◽  
Culture Supernatant ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Regulatory Proteins ◽  
Protein Fluorescence ◽  
Gene Encoding ◽  
Two Component ◽  
Green Fluorescent

ABSTRACT A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 μg of nisin in cheese, and 1 μg of nisin per ml in salad dressings.

scholarly journals A Propionate-Inducible Expression System for Enteric Bacteria

2005 ◽  
Vol 71 (11) ◽  
pp. 6856-6862 ◽  
Author(s):  
Sung Kuk Lee ◽  
Jay D. Keasling
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
Enteric Bacteria ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Basal Expression ◽  
Expression Of Genes ◽  
Catabolic Genes ◽  
Wide Range ◽  
Green Fluorescent

ABSTRACT A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P prpB ) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P prpB system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P prpB of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P prpB system exhibited negligible basal expression by addition of glucose to the medium.

scholarly journals The Great Capacity on Promoting Melanogenesis of Three Compatible Components in Vernonia anthelmintica (L.) Willd.

2021 ◽  
Vol 22 (8) ◽  
pp. 4073
Author(s):  
Yifan Lai ◽  
Qingyuan Feng ◽  
Rui Zhang ◽  
Jing Shang ◽  
Hui Zhong
Keyword(s):  
Herbal Medicine ◽  
Caffeic Acid ◽  
Fluorescent Protein ◽  
Traditional Chinese Herbal Medicine ◽  
Expression Of Genes ◽  
Proliferation And Differentiation ◽  
Green Fluorescent ◽  
Vernonia Anthelmintica

To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of skin depigmentation disorder, we have made use of Vernonia anthelmintica (L.) Willd., a traditional Chinese herbal medicine that has been proven to be effective in treating vitiligo. Here, we report that the extract of Vernonia anthelmintica (L.) Willd. effectively enhances melanogenesis responses in B16F10. In its compound library, we found three ingredients (butin, caffeic acid and luteolin) also have the activity of promoting melanogenesis in vivo and in vitro. They can reduce the accumulation of ROS induced by hydrogen peroxide and inflammatory response induced by sublethal concentrations of copper sulfate in wild type and green fluorescent protein (GFP)-labeled leukocytes zebrafish larvae. The overall objective of the present study aims to identify which compatibility proportions of the medicines may be more effective in promoting pigmentation. We utilized the D-optimal response surface methodology to optimize the ratio among three molecules. Combining three indicators of promoting melanogenesis, anti-inflammatory and antioxidant capacities, we get the best effect of butin, caffeic acid and luteolin at the ratio (butin:caffeic acid:luteolin = 7.38:28.30:64.32) on zebrafish. Moreover, the effect of melanin content recovery in the best combination is stronger than that of the monomer, which suggests that the three compounds have a synergistic effect on inducing melanogenesis. After simply verifying the result, we performed in situ hybridization on whole-mount zebrafish embryos to further explore the effects of multi-drugs combination on the proliferation and differentiation of melanocytes and the expression of genes (tyr, mitfa, dct, kit) related to melanin synthesis. In conclusion, the above three compatible compounds can significantly enhance melanogenesis and improve depigmentation in vivo.

Recruitment of the LIM protein hic-5 to focal contacts of human platelets

1998 ◽  
Vol 111 (15) ◽  
pp. 2181-2188 ◽  
Author(s):  
J. Hagmann ◽  
M. Grob ◽  
A. Welman ◽  
G. van Willigen ◽  
M.M. Burger
Keyword(s):  
Fluorescent Protein ◽  
Human Platelets ◽  
Gene Encoding ◽  
Chain Reactions ◽  
Amino Terminal ◽  
Culture Cells ◽  
Lim Domains ◽  
Focal Contacts ◽  
Green Fluorescent ◽  
Carboxy Terminal

Platelets are anuclear, membrane-bounded fragments derived from megakaryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts. The focal contacts resemble those of tissue culture cells. However, they lack paxillin, a conspicuous component of these organelles. We found that instead of paxillin, platelets contain a related protein with a molecular mass of 55 kDa that crossreacts with a monoclonal antibody against paxillin. The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned out to be identical to a recently discovered gene encoding hic-5. Like paxillin, hic-5 is a cytoskeletal protein containing four carboxy-terminal LIM domains and LD motifs in the amino-terminal half. The LIM domains of both hic-5 and paxillin are capable of targetting green fluorescent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125(FAK) and talin from platelet extracts. Only trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell line, and in megakaryocytes cultured from CD34+ cells obtained from umbilical cord blood. However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers specific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 expression must occur late in the maturation of megakaryocytes into platelets.

Laser-induced gene expression in specific cells of transgenic zebrafish

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1953-1960 ◽  
Author(s):  
M.C. Halloran ◽  
M. Sato-Maeda ◽  
J.T. Warren ◽  
F. Su ◽  
Z. Lele ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Transgenic Zebrafish ◽  
Hsp70 Promoter ◽  
Expression Of Genes ◽  
Transgenic Lines ◽  
Transgenic Embryos ◽  
Green Fluorescent

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

scholarly journals Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Catherine P. Chia ◽  
Noriko Inoguchi ◽  
Kyle C. Varon ◽  
Bradley M. Bartholomai ◽  
Hideaki Moriyama
Keyword(s):  
Dictyostelium Discoideum ◽  
Active Sites ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Unicellular Eukaryote ◽  
Gene Encoding ◽  
Conserved Motifs ◽  
N Terminus ◽  
Key Enzyme ◽  
Green Fluorescent

Abstract Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

scholarly journals Use of Green Fluorescent Protein To Monitor Cell Envelope Stress in Lactococcus lactis

2009 ◽  
Vol 76 (3) ◽  
pp. 978-981 ◽  
Author(s):  
Ana Belén Campelo ◽  
Ana Rodríguez ◽  
Beatriz Martínez
Keyword(s):  
Green Fluorescent Protein ◽  
Lactococcus Lactis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Dot Blot ◽  
Reporter System ◽  
Fast Detection ◽  
Envelope Stress ◽  
Dot Blot Assay ◽  
Green Fluorescent

ABSTRACT A Lactococcus lactis reporter system suitable to detect cell envelope stress in high-throughput settings was developed by fusing the CesR-regulated promoter of llmg0169 to the gfpuv gene. A dot blot assay allowed fast detection of green fluorescent protein (GFP) fluorescence even at low production levels. Unexpectedly, this promoter was also induced by mitomycin C via CesR.

scholarly journals Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E

2007 ◽  
Vol 74 (3) ◽  
pp. 653-659 ◽  
Author(s):  
Harry B. Hines ◽  
Alexander D. Kim ◽  
Robert G. Stafford ◽  
Shirin S. Badie ◽  
Ernst E. Brueggeman ◽  
...  
Keyword(s):  
Natural Product ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Sulfhydryl Group ◽  
Cleavage Sites ◽  
Gene Encoding ◽  
Fluorescent Substrate ◽  
C Terminus ◽  
Protease Cleavage Sites ◽  
Green Fluorescent

ABSTRACT The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.

scholarly journals Identification and Characterization of a Novel Gene Involved in the trans-Specific Nematicidal Activity of Photorhabdus luminescens LN2

2009 ◽  
Vol 75 (12) ◽  
pp. 4221-4223 ◽  
Author(s):  
Xuehong Qiu ◽  
Richou Han ◽  
Xun Yan ◽  
Mingxing Liu ◽  
Li Cao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transposon Mutagenesis ◽  
Nematicidal Activity ◽  
Photorhabdus Luminescens ◽  
Gene Encoding ◽  
Heterorhabditis Indica ◽  
Green Fluorescent ◽  
Identification And Characterization

ABSTRACT Photorhabdus luminescens subsp. akhurstii LN2 from Heterorhabditis indica LN2 showed nematicidal activity against axenic Heterorhabditis bacteriophora H06 infective juveniles (IJs). Transposon mutagenesis identified an LN2 mutant that supports the growth of H06 nematodes. Tn5 disrupted the namA gene, encoding a novel 364-residue protein and involving the nematicidal activity. The green fluorescent protein-labeled namA mutant was unable to colonize the intestines of H06 IJs.

scholarly journals A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts

2002 ◽  
Vol 68 (10) ◽  
pp. 5051-5056 ◽  
Author(s):  
Jacob Glenting ◽  
Søren M. Madsen ◽  
Astrid Vrang ◽  
Anders Fomsgaard ◽  
Hans Israelsen
Keyword(s):  
Lactococcus Lactis ◽  
Fluorescent Protein ◽  
Human Kidney ◽  
Heterologous Proteins ◽  
Selection System ◽  
Erythromycin Resistance ◽  
Homoserine Dehydrogenase ◽  
Mammalian Expression ◽  
Genes Encoding ◽  
Green Fluorescent

ABSTRACT We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic Lactococcus lactis MG1363Δthr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the hom-thrB operon) as a selective marker. Using strain MG1363Δthr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in L. lactis. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.

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