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 The Coding sequence of firefly luciferase reporter gene affects specific hyperexpressionArabidopsis thaliana cpl1mutant

2017 ◽  
Author(s):  
Hisashi Koiwa ◽  
Akihito Fukudome
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Coding Region ◽  
Luciferase Reporter Gene ◽  
Coding Sequence ◽  
Firefly Luciferase Reporter ◽  
Endogenous Genes ◽  
Firefly Luciferase Reporter Gene

AbstractForward genetic screening of mutants using firefly luciferase (LUC) reporter gene became a standard practice in plant research. Such screenings frequently identified alleles ofCPL1(Carboxyl-terminal Phosphatase-Like 1) regardless of promoters or pathways studied. Expression of the corresponding endogenous genes often shows the minimal difference between wild type andcpl1.Here we show that theLUCcoding sequence is responsible for the high expression incpl1,using a classicalRD29a-LUC. Deletion of theLUC3’-UTR did not change hyperactivation ofLUCincpl1.However, a codon-modifiedLUC(LUC2) produced similar expression levels both in wild type and incpl1. These results indicate that the coding region ofLUCis responsible for thecpl1-specificLUCoverexpression uncoupled with the expression of the endogenous counterpart.

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

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.

scholarly journals Transcriptional Regulation of MDR1, Encoding a Drug Efflux Determinant, in Fluconazole-Resistant Candida albicans Strains through an Mcm1p Binding Site

2006 ◽  
Vol 5 (12) ◽  
pp. 1957-1968 ◽  
Author(s):  
Perry J. Riggle ◽  
Carol A. Kumamoto
Keyword(s):  
Candida Albicans ◽  
Binding Site ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Drug Efflux ◽  
Dependent Manner ◽  
Wild Type ◽  
High Level Expression ◽  
Mobility Shift ◽  
High Level

ABSTRACT Constitutive, high-level transcription of the gene encoding the drug efflux facilitator Mdr1p is commonly observed in laboratory and clinical strains of Candida albicans that are resistant to the antifungal drug fluconazole (FLC). In five independently isolated FLCR laboratory strains, introduction of a wild-type MDR1 promoter fragment fused to the yeast enhanced green fluorescent protein (yEGFP) reporter gene resulted in high-level expression of GFP, demonstrating that overexpression of MDR1 is dependent on a trans-acting factor. This study identified a 35-bp MDR1 promoter element, termed the MDRE, that mediates high-level MDR1 transcription. When inserted into a heterologous promoter, the MDRE was sufficient to mediate high-level expression of the yEGFP reporter gene specifically in MDR1 trans-activation strains. The MDRE promoted transcription in an orientation-independent and dosage-dependent manner. Deletion of the MDRE in the full-length promoter did not abolish MDR1 trans-activation, indicating that elements upstream of the MDRE also contribute to transcription of MDR1 in these overexpression strains. Analysis of the MDRE sequence indicated that it contains an Mcm1p binding site very similar in organization to the site seen upstream of the Saccharomyces cerevisiae MFA1 and STE2 genes. Electrophoretic mobility shift analysis demonstrated that both wild-type, FLC-sensitive and MDR1 trans-activated, FLC-resistant strains contain a factor that binds the MDRE. Depletion of Mcm1p, by use of a strain in which MCM1 expression is under the control of a regulated promoter (44), resulted in a loss of MDRE binding activity. Thus, the general transcription factor Mcm1p participates in the regulation of MDR1 expression.

scholarly journals Membrane Depolarization and Apoptosis-Like Cell Death in an Alkaline Environment in the Rice Pathogen Burkholderia glumae

2021 ◽  
Vol 12 ◽  
Author(s):  
Yewon Nam ◽  
Eunhye Goo ◽  
Yongsung Kang ◽  
Ingyu Hwang
Keyword(s):  
Cell Death ◽  
Membrane Depolarization ◽  
Luria Bertani ◽  
Alkaline Stress ◽  
Dependent Manner ◽  
Wild Type ◽  
Burkholderia Glumae ◽  
Ribonuclease E ◽  
Alkaline Environment ◽  
Genes Encoding

The rice pathogen Burkholderia glumae uses amino acids as a principal carbon source and thus produces ammonia in amino acid-rich culture medium such as Luria–Bertani (LB) broth. To counteract ammonia-mediated environmental alkaline toxicity, the bacterium produces a public good, oxalate, in a quorum sensing (QS)-dependent manner. QS mutants of B. glumae experience alkaline toxicity and may undergo cell death at the stationary phase when grown in LB medium. Here, we show that the cell-death processes of QS mutants due to alkaline environmental conditions are similar to the apoptosis-like cell death reported in other bacteria. Staining QS mutants with bis-(1,3-dibutylbarbituric acid)-trimethine oxonol revealed membrane depolarization. CellROX™ staining showed excessive generation of reactive oxygen species (ROS) in QS mutants. The expression of genes encoding HNH endonuclease (BGLU_1G15690), oligoribonuclease (BGLU_1G09120), ribonuclease E (BGLU_1G09400), and Hu-beta (BGLU_1G13530) was significantly elevated in QS mutants compared to that in wild-type BGR1, consistent with the degradation of cellular materials as observed under transmission electron microscopy (TEM). A homeostatic neutral pH was not attainable by QS mutants grown in LB broth or by wild-type BGR1 grown in an artificially amended alkaline environment. At an artificially adjusted alkaline pH, wild-type BGR1 underwent apoptosis-like cell death similar to that observed in QS mutants. These results show that environmental alkaline stress interferes with homeostatic neutral cellular pH, induces membrane depolarization, and causes apoptosis-like cell death in B. glumae.

scholarly journals Quorum Sensing-Mediated and Growth Phase-Dependent Regulation of Metabolic Pathways in Hafnia alvei H4

2021 ◽  
Vol 12 ◽  
Author(s):  
Congyang Yan ◽  
Xue Li ◽  
Gongliang Zhang ◽  
Yaolei Zhu ◽  
Jingran Bi ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Cell Density ◽  
Growth Phase ◽  
Metabolic Pathways ◽  
High Cell Density ◽  
Dependent Manner ◽  
High Cell ◽  
Wild Type ◽  
Hafnia Alvei ◽  
Density Dependent

Quorum sensing (QS) is a widespread regulatory mechanism in bacteria used to coordinate target gene expression with cell density. Thus far, little is known about the regulatory relationship between QS and cell density in terms of metabolic pathways in Hafnia alvei H4. In this study, transcriptomics analysis was performed under two conditions to address this question. The comparative transcriptome of H. alvei H4 wild-type at high cell density (OD600 = 1.7) relative to low cell density (OD600 = 0.3) was considered as growth phase-dependent manner (GPDM), and the transcriptome profile of luxI/R deletion mutant (ΔluxIR) compared to the wild-type was considered as QS-mediated regulation. In all, we identified 206 differentially expressed genes (DEGs) mainly presented in chemotaxis, TCA cycle, two-component system, ABC transporters and pyruvate metabolism, co-regulated by the both density-dependent regulation, and the results were validated by qPCR and swimming phenotypic assays. Aside from the co-regulated DEGs, we also found that 59 DEGs, mediated by density-independent QS, function in pentose phosphate and histidine metabolism and that 2084 cell-density-dependent DEGs involved in glycolysis/gluconeogenesis and phenylalanine metabolism were influenced only by GPDM from significantly enriched analysis of transcriptome data. The findings provided new information about the interplay between two density-dependent metabolic regulation, which could assist with the formulation of control strategies for this opportunistic pathogen, especially at high cell density.

scholarly journals Phosphate Starvation-Inducible Proteins ofBacillus subtilis: Proteomics and Transcriptional Analysis

2000 ◽  
Vol 182 (16) ◽  
pp. 4478-4490 ◽  
Author(s):  
Haike Antelmann ◽  
Christian Scharf ◽  
Michael Hecker
Keyword(s):  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphate Starvation ◽  
Transcriptional Analysis ◽  
Northern Hybridization ◽  
Dependent Manner ◽  
Wild Type ◽  
Extracellular Medium ◽  
Flight Mass Spectrometry ◽  
Cell Extracts

ABSTRACT The phosphate starvation response in Bacillus subtiliswas analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of ςB, the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and aphoR mutant. By matrix-assisted laser desorption ionization–time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of glpQ and ydhFconfirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that glpQ is transcribed monocistronically from a ςA promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore, ydhF might be cotranscribed withphoB initiating from the phoB promoter. Only a small group of proteins remained phosphate starvation inducible in bothphoR and sigB mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by ςB-dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a ςB-dependent manner and in the sigB mutant probably via ςH. YxiE was induced by phosphate starvation independently of ςB and PhoPR.

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