scholarly journals cis-Acting Determinant Limiting Expression of Sphingomyelinase Genesph2inLeptospira interrogans, Identified with agfpReporter Plasmid

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
James Matsunaga ◽  
David A. Haake
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fusion Expression ◽  
Leptospira Interrogans ◽  
Reporter Plasmid ◽  
Content Type ◽  
Molecular Determinants ◽  
Upstream Element ◽  
Green Fluorescent

ABSTRACTMany strains of the spirocheteLeptospira interrogansserovar Pomona express the osmotically inducible sphingomyelinase genesph2at much higher levels than strains from other serovars. We developed a new green fluorescent protein (GFP) reporter plasmid to examinesph2gene expression determinants. The vector enables the fusion of the test promoter to the ribosome-binding site and coding region ofgfp. We fused thesph2promoters from theL. interrogansserovar Lai strain 56601 and from theL. interrogansserovar Pomona strain LC82-25 togfpto examine the molecular determinants of differentialsph2expression between the two strains. Similar to what was observed with the nativesph2genes, the introduction of the plasmids into the Lai 56601 strain resulted in near background levels ofgfpexpression from the Laisph2promoter, while the expression from the Pomonasph2promoter was high. The expression of both fusions increased at physiologic levels of osmolarity achieved by adding sodium chloride to the culture medium. We examined the role of a 17-bp upstream element found in allL. interrogansstrains expressing low basal levels ofsph2and missing from Pomona strains that expresssph2at high levels. When the 17-bp sequence present upstream of the Laisph2promoter was deleted or scrambled, the fusion expression increased substantially. Conversely, the insertion of the 17-bp sequence upstream of the Pomonasph2promoter diminished fusion expression. In contrast, the removal of an insertion sequence-like element that is found only in the Pomonasph2upstream sequence had no effect on the expression from the Pomonasph2fusion in the Lai strain. These findings demonstrate the utility of thegfpreporter plasmid in analyzing gene expression inL. interrogans.IMPORTANCEGenetic tools are needed to examine gene expression in the pathogenLeptospira interrogans. We developed a reporter plasmid that replicates inL. interroganswith green fluorescent protein (GFP) as the readout of promoter activity. We demonstrated an application of the new reporter plasmid by identifying an upstream element responsible for the poor basal expression of thesph2sphingomyelinase gene in anL. interrogansserovar Lai strain. This new tool is useful for the discovery of the molecular determinants ofL. interrogansgene expression.

scholarly journals Identification of a cis-acting determinant limiting expression of sphingomyelinase genesph2inLeptospira interroganswith agfpreporter plasmid

2018 ◽  
Author(s):  
James Matsunaga ◽  
David A. Haake
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fusion Expression ◽  
Upstream Sequence ◽  
Leptospira Interrogans ◽  
Reporter Plasmid ◽  
Molecular Determinants ◽  
Upstream Element ◽  
Green Fluorescent

ABSTRACTMany Pomona strains of the spirocheteLeptospira interrogansexpress the osmotically-inducible sphingomyelinase gene,sph2, at much higher levels than non-Pomona strains. We developed a new green fluorescent protein (GFP) reporter plasmid to examinesph2gene expression determinants. The vector allows fusion of the test promoter to the ribosome-binding site and coding region ofgfp. We fused thesph2promoters from the serovar Lai strain 56601 and from the serovar Pomona strain LC82-25 togfpto examine the molecular determinants of differentialsph2expression between the two strains. Similar to what is observed with the nativesph2genes, introduction of the plasmids into the Lai 56601 strain resulted in near background levels ofgfpexpression from the Laisph2promoter, while expression from the Pomonasph2promoter was high. Expression from both fusions increased at physiologic levels of osmolarity achieved by addition of sodium chloride to the culture medium. We examined the role of a 17 bp upstream element found in allL. interrogansstrains expressing low basal levels ofsph2and missing from Pomona strains that expresssph2at high levels. When the 17 bp sequence present upstream of the Laisph2promoter was deleted or scrambled, fusion expression increased substantially. Conversely, insertion of the 17 bp sequence upstream of the Pomonasph2promoter diminished fusion expression. In contrast, removal of an insertion sequence-like element that is found only in the Pomonasph2upstream sequence had no effect on expression from the Pomonasph2fusion in the Lai strain. These findings demonstrate the utility of thegfpreporter plasmid in analyzing gene expression inL. interrogans.IMPORTANCEGenetic tools are needed to examine gene expression in the pathogenLeptospira interrogans. We developed a reporter plasmid that replicates inL. interroganswith green fluorescent protein (GFP) as the read out of promoter activity. We demonstrated an application of the new reporter plasmid by identifying an upstream element responsible for the poor basal expression of thesph2sphingomyelinase gene in a serovar Lai strain ofL. interrogans. This new tool is useful for discovery of the molecular determinants ofL. interrogansgene expression.

scholarly journals PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

2013 ◽  
Vol 80 (4) ◽  
pp. 1477-1481 ◽  
Author(s):  
Karina Klevanskaa ◽  
Nadja Bier ◽  
Kerstin Stingl ◽  
Eckhard Strauch ◽  
Stefan Hertwig
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent Protein ◽  
Vibrio Vulnificus ◽  
Shuttle Vector ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACTAn efficient electroporation procedure forVibrio vulnificuswas designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 106transformants per μg DNA were achieved. The vector stably replicated in bothV. vulnificusandEscherichia coliand was also successfully introduced intoVibrio parahaemolyticusandVibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and thevvhBAhemolysin operon were inserted into the vector and functionally expressed inVibrioandE. coli.

Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

2003 ◽  
pp. 245-260
Author(s):  
Laura E. Via ◽  
Subramanian Dhandayuthapani ◽  
Dusanka Deretic ◽  
V. Deretic
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent

Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cryptococcus Neoformans ◽  
Differential Gene Expression ◽  
Fluorescent Protein ◽  
Differential Gene ◽  
Green Fluorescent

scholarly journals The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium

2001 ◽  
Vol 67 (2) ◽  
pp. 948-955 ◽  
Author(s):  
Biao Ma ◽  
Mary B. Mayfield ◽  
Michael H. Gold
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Phanerochaete Chrysosporium ◽  
Fluorescent Protein ◽  
Schizophyllum Commune ◽  
Extracellular Fluid ◽  
Gene Promoter ◽  
Green Fluorescent Protein Gene ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfpcoding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of theegfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly withegfp, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of theegfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of theegfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studyingcis-acting elements in the mnp1 gene promoter.

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.

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