scholarly journals Isogenic Strain Construction and Gene Targeting inCandida dubliniensis

2001 ◽  
Vol 183 (9) ◽  
pp. 2859-2865 ◽  
Author(s):  
Peter Staib ◽  
Gary P. Moran ◽  
Derek J. Sullivan ◽  
David C. Coleman ◽  
Joachim Morschhäuser
Keyword(s):  
Reporter Gene ◽  
Gene Fusion ◽  
Recombinant Dna ◽  
Efflux Pump ◽  
Acid Resistance ◽  
Selection Marker ◽  
Targeted Gene Deletion ◽  
Yeast Extract Peptone Dextrose ◽  
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.

Streptococcal Reporter Gene-Fusion Vector for Identification of in Vivo Expressed Genes

Plasmid ◽  
1999 ◽  
Vol 42 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Ali O. Kiliç ◽  
Mark C. Herzberg ◽  
Maurice W. Meyer ◽  
Xuemei Zhao ◽  
Lin Tao
Keyword(s):  
Reporter Gene ◽  
Gene Fusion ◽  
Reporter Gene Fusion

Environmentally regulated genes of Helicobacter pylori identified by a random lacZ reporter gene fusion system

1999 ◽  
Vol 11 (12) ◽  
pp. A98
Author(s):  
N. de Vries ◽  
E. J. Kuipers ◽  
A. H.M. van Vliet ◽  
N. E. Kramer ◽  
S. Bereswill ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Reporter Gene ◽  
Gene Fusion ◽  
Fusion System ◽  
Lacz Reporter ◽  
Lacz Reporter Gene ◽  
Reporter Gene Fusion

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

1997 ◽  
Vol 11 (1) ◽  
pp. 69-74 ◽  
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.

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

2001 ◽  
Vol 183 (17) ◽  
pp. 5171-5179 ◽  
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.

scholarly journals MDR1-Mediated Drug Resistance inCandida dubliniensis

2001 ◽  
Vol 45 (12) ◽  
pp. 3416-3421 ◽  
Author(s):  
Stephanie Wirsching ◽  
Gary P. Moran ◽  
Derek J. Sullivan ◽  
David C. Coleman ◽  
Joachim Morschhäuser
Keyword(s):  
Drug Resistance ◽  
Efflux Pump ◽  
Brefeldin A ◽  
Acid Resistance ◽  
Amino Acid Sequences ◽  
Candida Dubliniensis ◽  
Major Facilitator Superfamily ◽  
Multidrug Efflux Pump ◽  
Major Facilitator

ABSTRACT Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role ofCdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by theMPA R-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression ofCdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1inactivation was identical to that seen in two similarly constructedC. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 andCdMDR1 in clinical C. albicans andC. dubliniensis strains seems to confer the same drug resistance profile in both species.

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

Helicobacter ◽  
2001 ◽  
Vol 6 (4) ◽  
pp. 300-309 ◽  
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

scholarly journals Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria

2021 ◽  
Vol 22 (4) ◽  
pp. 2062
Author(s):  
Aneta Kaczor ◽  
Karolina Witek ◽  
Sabina Podlewska ◽  
Veronique Sinou ◽  
Joanna Czekajewska ◽  
...  
Keyword(s):  
Molecular Modelling ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Potential Mechanism ◽  
Multidrug Efflux ◽  
Aromatic Rings ◽  
Allosteric Site ◽  
Multidrug Efflux Pump ◽  
Dual Action

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

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