Analysis of Expression of a Phenazine Biosynthesis Locus of Pseudomonas aureofaciens PGS12 on Seeds with a Mutant Carrying a Phenazine Biosynthesis Locus-Ice Nucleation Reporter Gene Fusion

ABSTRACT Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensiswild-type isolate by targeted gene deletion. The two URA3alleles were sequentially inactivated using theMPAR -flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that theCdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.

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Cloning of a phenazine biosynthetic locus of Pseudomonas aureofaciens PGS12 and analysis of its expression in vitro with the ice nucleation reporter gene.

Applied and Environmental Microbiology ◽

10.1128/aem.60.8.2931-2938.1994 ◽

1994 ◽

Vol 60 (8) ◽

pp. 2931-2938 ◽

Cited By ~ 29

Author(s):

D G Georgakopoulos ◽

M Hendson ◽

N J Panopoulos ◽

M N Schroth

Keyword(s):

Reporter Gene ◽

Ice Nucleation ◽

Pseudomonas Aureofaciens

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Influence of a Functional sigB Operon on the Global Regulators sar and agr inStaphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.183.17.5171-5179.2001 ◽

2001 ◽

Vol 183 (17) ◽

pp. 5171-5179 ◽

Cited By ~ 152

Author(s):

M. Bischoff ◽

J. M. Entenza ◽

P. Giachino

Keyword(s):

Reporter Gene ◽

Growth Phase ◽

Gene Fusion ◽

Firefly Luciferase ◽

Luria Bertani ◽

Transcriptional Analysis ◽

Dependent Manner ◽

Wild Type ◽

Global Regulators ◽

Reporter Gene Fusion

ABSTRACT The growth phase-dependent activity profile of the alternate transcription factor ςB and its effects on the expression of sar and agr were examined in three differentStaphylococcus aureus strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant ςB activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type rsbU allele, but not in the NCTC8325 derivative BB255, which is defective inrsbU. ςB activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a sarP1-sarP2-sarP3(sarP1-P2-P3)-driven firefly luciferase (luc+) reporter gene fusion demonstrated a strong ςB activity- and growth phase-dependent increase in sar expression that was totally absent in either rsbU or ΔrsbUVWsigB mutants. In contrast, expression of theagr locus, as measured by RNAIII levels and by anhldp::luc+ fusion, was found to be higher in the absence of ςB activity, such as inrsbU or ΔrsbUVWsigB mutants, than in wild-type strains. Overexpression of ςB in BB255 derivatives resulted in a clear increase insarP1-P2-P3::luc+ expression as well as a strong decrease in hldp::luc+ expression. The data presented here suggest that ςBincreases sar expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner.

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Target Validation of sRNA with a GFP Reporter Gene Fusion System

Springer Protocols Handbooks - Yersinia Pestis Protocols ◽

10.1007/978-981-10-7947-4_12 ◽

2018 ◽

pp. 115-120

Author(s):

Xiaofang Gao ◽

Zizhong Liu ◽

Yanping Han

Keyword(s):

Reporter Gene ◽

Gene Fusion ◽

Fusion System ◽

Target Validation ◽

Gfp Reporter ◽

Gfp Reporter Gene ◽

Reporter Gene Fusion

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Identification of Environmental Stress-Regulated Genes in Helicobacter pylori by a lacZ Reporter Gene Fusion System

Helicobacter ◽

10.1046/j.1083-4389.2001.00046.x ◽

2001 ◽

Vol 6 (4) ◽

pp. 300-309 ◽

Cited By ~ 22

Author(s):

Nicolette de Vries ◽

Ernst J. Kuipers ◽

Naomi E. Kramer ◽

Arnoud H. M. van Vliet ◽

Jetta J. E. Bijlsma ◽

...

Keyword(s):

Helicobacter Pylori ◽

Environmental Stress ◽

Reporter Gene ◽

Gene Fusion ◽

Fusion System ◽

Lacz Reporter ◽

Lacz Reporter Gene ◽

Reporter Gene Fusion

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Availability of iron to Pseudomonas fluorescens in rhizosphere and bulk soil evaluated with an ice nucleation reporter gene.

Applied and Environmental Microbiology ◽

10.1128/aem.63.1.99-105.1997 ◽

1997 ◽

Vol 63 (1) ◽

pp. 99-105 ◽

Cited By ~ 105

Author(s):

J E Loper ◽

M D Henkels

Keyword(s):

Pseudomonas Fluorescens ◽

Reporter Gene ◽

Ice Nucleation ◽

Bulk Soil

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Host-Pathogen Interactions in Bacterial Endocarditis: Streptococcal Virulence in the Host

Advances in Dental Research ◽

10.1177/08959374970110011901 ◽

1997 ◽

Vol 11 (1) ◽

pp. 69-74 ◽

Cited By ~ 25

Author(s):

Mark C. Herzberg ◽

Maurice W. Meyer ◽

Ali Kiliç ◽

Lin Tao

Keyword(s):

Positive Selection ◽

Gene Fusion ◽

Southern Hybridization ◽

Amylase Activity ◽

Bacterial Endocarditis ◽

Double Negative ◽

Chromosomal Dna ◽

Dual Reporter ◽

Reporter Gene Fusion

To identify streptococcal genes that are expressed during experimental endocarditis, we developed a promoter-less dual reporter gene-fusion (amy, cat) plasmid, pAK36. Chromosomal DNA from S. gordonii V288 was digested with Sau3Al. The resulting fragments were ligated into pAK36. Following transformation into S. gordonii, the library of random gene fusion clones was inoculated into a rabbit to induce experimental endocarditis. Chloramphenicol treatment effected positive selection. Upon euthanization of the rabbits, the valvular vegetations were excised in a sterile field. Surviving clones were isolated and screened in vitro for chloramphenicol sensitivity and negative amylase activity. From the 48 randomly picked, double-negative clones. DNA was isolated and analyzed by Southern hybridization with labeled pAK36 probe. Different insertion patterns were identified, suggesting that no fewer than 13 S. gordonii genes were induced. Therefore, S. gordonii genes are induced during experimental endocarditis, which may contribute to virulence.

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